KR100892866B1 - Fixation method of biological materials on implant surface - Google Patents

Abstract

A method for fixing biological materials on a surface of an implant is provided to secure a stable oxide layer by fixing the biological materials after forming a surface of the oxide layer and an intermediate reaction layer. A method for fixing biological materials on a surface of an implant comprises the following steps of: forming a chemical oxide layer on a surface of the implant; activating an anodized oxide layer by treating the surface of the implant using a liquid-phase reaction such as piranha or a gas-phase reaction such as H2O-Plasma; and forming an intermediate reaction layer on a surface of the anodized oxide layer using an intermediate reaction material having a silane group.

Description

Fixation Method of Biological Materials on Implant Surfaces

The present invention relates to a method of immobilizing a bioactive material on an implant surface.

Light and strong titanium metals are of increasing importance in the fields of aviation, high-strength sports equipment, bio implants and the like. In particular, in the field of bio metals, titanium is widely used as a metal that does not cause an inflammatory reaction or other immune response when placed in the body.

Dental implants are divided into fixtures (called implants or fixtures) that act as artificial tooth roots in the missing tooth area, abutments that connect the fixture to the crown, and crowns. The implant is largely composed of a connection to the abutment and an implant surface in direct contact with the alveolar bone. In dental implants, the surface is very important as it determines the duration of bone adhesion and future function.

Dental implants are parts that carry the same sense and function as the original teeth by implanting a titanium artificial root into the alveolar bone without damaging the surrounding teeth at the site where the tooth is missing. Dental implants are implanted into the alveolar bone, and after a certain period of time, the alveolar bone and bone adhesion, and after the bone adhesion is connected to the abutment and then to the artificial tooth (crown). After the artificial teeth are connected to the implant, they usually function normally, and after 15-20 years of bone adhesion, osteoadhesion should be maintained semipermanently. The pre-fatigue life test conditions for this are mainly 5 million cycles with 250 N load.

One of the key performance indicators of dental implants is to shorten the period of bone adhesion. In other words, if bone adhesion does not occur within a short period of time, it is difficult to form a dental implant market as the treatment period becomes longer, and it is absolutely required to shorten the initial bone adhesion period because the normal chewing function of the implant is not smooth in the future. It is becoming.

Bioactive substances such as growth factor (TGF-β) and Insulin-like growth factor-1 (IGF-1) or bone morphogenetic protein (BMP) that promote osteoblast proliferation and differentiation Since it is impossible to coat the dental implants, in order to significantly shorten the procedure period, a method of applying directly to the surface of the implant or distributing it around the surgical site is used. However, the method of applying the bioactive substance to the surface of the implant before the implantation procedure is difficult because it requires a large amount of material and does not remain effectively during implantation, and the application of the bioactive substance to the treatment site is extremely limited because the applied area is extremely limited. There is a disadvantage that the cost of the procedure is increased without effective bone regeneration and bone adhesion on the entire implant surface. Moreover, since the standardization of the procedure is not made and the treatment method and the management of the patient are not efficient, it is practically impossible to be adopted as a standardized procedure.

Dental implants are classified into design technology and surface modification technology. Surface modification technology is an important factor in determining the initial bone adhesion period and the life of the implant, so it is divided into 1 to 4 generations according to the development of the technology.

First-generation implants are machined implants developed by Branemark, Sweden, in 1965. As the first generation implant requires 4-8 months of bone adhesion, various problems have been raised, and a second generation implant has been developed to increase the surface area of the implant for the purpose of shortening the bone adhesion period. As the surface modification method of second generation implants, acid treatment, sand blasting, acid treatment after sand blasting, and sintering were developed, and the bone adhesion period of second generation implant was reduced to 3-6 months. Third-generation implants include HA (hydroxylapatite) coatings, anodization, fluorinated surfaces, and hydrophilic surfaces. In the case of HA coating, it is included in the second generation at the time of development, but in the classification of technical progress, it is in the third generation, and anodization promotes bone adhesion by making the surface porous and thickening the oxide layer. The fluorinated surface was developed by ASTRA, Sweden, to promote bone adhesion by adding fluorine to the implant surface. The surface with improved hydrophilicity was developed by ITI, Switzerland, to shorten the period of bone adhesion by enhancing surface energy and increasing hydrophilicity. It is. The third generation of implants reduced bone adhesion time to 2-4 months by adding bone-inducing substances, such as fluorine, calcium and phosphorus, and by making the surface porous and forming a thick oxide layer. However, there is a limitation in that the third generation of implants takes more than three months to recover the missing teeth, and the overall success rate is slightly better than the second generation of implants, but the alveolar bone is poor. The success rate is still low, but in case of bad tooth quality such as elderly patients, the situation is limited. That is, the third generation of surface modification technology is limited to shorten the bone adhesion period within three months, so the development of the fourth generation of implants is urgently required.

Therefore, the mechanism of alveolar bone formation to shorten the period of bone adhesion to 2 months has been revealed by some researchers, BMP belonging to the TGF group has been reported to play an important role. After the publication of the first study on BMP, many researchers have conducted many experiments with animals. Later, it was announced that humans as well as animals can induce bone regeneration and strengthen bone tissue. However, when the BMP was administered directly to the subject, it disappeared within a few minutes, and it was confirmed that the enzyme was present in the body to be degraded in a short time, thereby losing its function. As a result, in order to effectively induce new bone formation, it is necessary to administer the implant at a fairly high concentration within a short time. That is, as many problems are faced in terms of stability, pharmacokinetics, and cost, many researchers have developed methods to produce large amounts of BMP to promote bone growth by gene therapy. The method of producing the target protein by increasing the number of cells by applying the genetic recombination has been applied, and recently, using the peptide synthesizer (peptide synthesizer) to prepare the oligopeptides by organic synthesis method rather than genetic manipulation of the desired amino acid sequence to these materials Various researches are being actively conducted.

Production of a target product by genetic engineering is mainly used to introduce a gene that produces the same protein as a native protein into a gene of a microorganism or an animal cell and to increase the number of cells to mass produce a target recombinant protein. In order to introduce the gene, the virus vector, cell-mediate and non-viral vector, plasmid, lipofection, etc. are used. In sexual and humoral immune responses, the expression level of transgenic genes is remarkably reduced.

On the other hand, the production of the target substance incorporating a peptide synthesizer does not require a series of genetic manipulations and is not necessarily the whole amino acid sequence of the native protein, but is particularly essential for the functioning of the protein. It is a very effective way to develop and produce oligopeptides. After purification, HPLC is performed to purify the synthesized material, which is much simpler and easier to purify than the purification step of the desired product by genetic engineering. In addition, the use of a synthesizer allows modification of some amino acids in the amino acid sequence of the oligopeptide (eg, acetylation, amidation, phosphorylation, etc.) according to the purpose, so that oligopeptides that are partially different from the structure of the amino acid sequence of the natural protein are artificial. It has the advantage that it can be manufactured by transforming it into. In particular, when the amino acid sequence is a very short sequence compared to the protein, organic synthesis using a synthesizer is the development of a stable cell line (PCR, cloning, screening of clones, etc.) that produces the target substance by genetic recombination, and then mass culture to isolate the target substance. Compared to the refinement process, development time and costs can be reduced.

Recently, attempts have been made to find amino acid sequences that play a key role among the total amino acid sequences of BMPs and extracellular matrix involved in bone formation. These amino acid sequences are synthesized using a synthesizer and evaluated for their effectiveness. Research is ongoing.

Various surface treatment techniques have been developed so far, but there is no research on analyzing the mechanism of bone adhesion reaction between bone and implant. Recently, research on reactions at the interface between bone and implants has been conducted, and studies on cells producing bones have been carried out, and cell recognition promoting bone adhesion has been analyzed.

As a method for shortening the bone adhesion period of the implant, methods of immobilizing a bioactive substance such as RGD on the implant surface have been used. At this time, there is a method of forming a silane compound that is well immobilized on the titanium surface as an intermediate layer to fix the bioactive material. However, in the titanium natural oxide film, since only 15 to 20% of the hydroxyl groups for stabilizing the intermediate layer are present on the titanium surface, the silane film does not secure sufficient stability. On this surface, the silane layer is continuously leaked from the body fluid in the human body, and thus, the bioactive substance functions effectively during implantation.

For example, Korean Patent Publication No. 2006-0110190 (Dental implant coated with a bioactive material and its coating method), 2006-110189, 2006-0082060, 2006-0101019 and the like are known as related applications. The contents disclosed in these patents are incorporated by reference of the present invention.

Accordingly, the present inventors developed and studied a titanium surface treatment technology for immobilizing bioactive substances for the purpose of implant surface modification, and an intermediate coating for coating a bioactive substance on the surface of a living implant implant made of titanium or titanium alloy. The present invention has been completed by developing a synthetic peptide immobilization method to impart chemical and physical stability of the membrane.

An object of the present invention is to provide a substrate treatment method for stabilizing when the silane compound is immobilized on the titanium surface and a method for immobilizing a physiologically active substance on the immobilized silane compound.

In order to achieve the above object, the present invention comprises the steps of: (2) forming a chemical oxide film on the surface of the implant (1) for living implantation; Ii) activating (3) the anodic oxide film by treating the implant surface using a piranha, O ₃ or H ₂ O-Plasma activation reaction; Iii) forming an intermediate reactor layer of a reactor-forming material having a silane group (4) on the surface of the anodic oxide film; Iii) immobilizing a linker connecting the intermediate reaction layer and the bioactive material formed on the substrate, including the silane group; And iii) immobilizing the bioactive material 6 for promoting bone adhesion to the implant on which the intermediate reactor layer is formed.

In the method of immobilizing a physiologically active substance on the surface of an implant of the present invention, the implant is preferably composed of titanium as a main component, and the chemical oxide film of step (iii) is a phosphoric acid / sulfuric acid solution (that is, an electrolyte solution in which sulfuric acid and phosphoric acid are mixed). It is preferably formed using an anodization method in the).

Further, in the immobilization method of a physiologically active substance on the implant surface of the present invention, the intermediate material is formed in the reactor R ₂ - made of a structural formula of _{(CH 2) n -Si- (R} 1) 3, wherein R ₁ is - Cl, -OCH ₃ , -OCH ₂ CH ₃ is selected from the group consisting of R ₂ is -NH ₂ (amine), -COOH (carboxyl), -CHO (aldehyde), epoxy group and -CH = CH ₂ (alkyne) It is selected from the group consisting of), It is preferable that the said n is 3-18. The intermediate reactor forming material is more preferably an APTES (3-aminopropyltriethoxysilane) compound or TSBA (triethoxysilylbutanaldehyde) compound.

In addition, in the method of immobilizing a physiologically active substance on the surface of the implant of the present invention, the linker material connecting the silane group and the physiologically active substance in step iii) is a sock that can chemically covalently bond to the functional group of the silane and the bioactive substance. However, a functional group material should be used, which is preferably a BMOE (Bis-maleimidoethane) compound or SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) compound.

In addition, in the method of immobilizing a bioactive material on the surface of the implant of the present invention, the bioactive material of step iii) is preferably a synthetic peptide, a natural protein composed of amino acids, or a material having a biomimetic structure in the same manner It can be done.

In addition, in the method of immobilizing a bioactive material on the surface of the implant of the present invention, in order to maximize the active characteristics of the bioactive material and to control the surface concentration of the bioactive material in the step of immobilizing the bioactive material in the step iii), Preferably, the matrix material 6 is introduced together, wherein the matrix material 7 is in the form of a single molecule or polymer similar in size to the bioactive material, and at one end is -NH ₂ , -COOH, -COH, and- More preferably, the functional group selected from the group consisting of SH has a functional group which is not reactive with bioactive substances such as -OH and -CH ₃ at the other end, and most preferably PLL (Poly-L-Lysine hydrochloride). Do.

In addition, referring to Figure 22, the implant surface treatment method of the present invention is to form a chemical oxide film using an anodizing method in a phosphoric acid / sulfuric acid mixed solution on the surface of the implant (1) for biological implants (T) as the main component (2) And a surface treatment using piranha, O ₃ , H ₂ O-Plasma, etc. to activate the anodic oxide film (3), and to form an intermediate reactor layer having a silane group (4); (5) forming a linker layer capable of immobilizing the silane group and the bioactive material, and immobilizing the bioactive material for promoting bone adhesion to the formed implant (6), maximizing the active properties of the bioactive material In order to control the surface concentration, the introduction of the matrix material (7) was added in the step of introducing the bioactive material after the intermediate reaction layer forming step at a suitable concentration ratio with the bioactive material. (Fig. 22).

 By such a configuration, the present invention has a thick oxide surface and rough surface to form a more stable oxide film for use in implants, and promotes bone formation as well as the effect that cells are better adsorbed and growth rate is increased during bone formation. By fixing the synthetic peptide (bio-like substance) to a constant concentration on the implant surface it can shorten the period of bone adhesion and in particular increase the success rate of patients with low bone mass or lack of bone mass.

A detailed description of the steps according to the invention is as follows.

i) anodization

Anodization is performed using an electrolyte solution containing sulfuric acid and phosphoric acid. The anodic oxidation system consists of a positive electrode, a negative electrode, sulfuric acid solution in a reactor used as an electrolyte, and a direct current electrostatic device, and uses a stirrer to remove bubbles generated during the anodic oxidation process. (disc) was ultrasonically washed with ethanol and distilled water, and then the disk was fixed to the positive electrode, platinum was fixed to the negative electrode, and a solution containing sulfuric acid (H ₂ SO ₄ ) / phosphate (H ₃ PO ₄ ) electrolyte was added to the reaction vessel at 300V. As a result of anodizing using a current of 0.53A at a voltage, an oxide layer having a porous structure with an inner diameter of about 2 to 5um is formed.

Ii) treating the implant surface with piranha, O ₃ and H ₂ O-Plasma to activate (3) the anodic oxide film, thereby modifying the surface to stably fix the silane film. Dry substrates are treated with UV / O treatment equipment (UVO-cleaner, Jelight, USA) for 10 minutes or 10 minutes to 1 hour oxidation in a piranha solution mixed with 30% hydrogen peroxide and 70% sulfuric acid. (Macromol. Symp. 253 (2007), p115-121)

 Iii) forming an intermediate reactor layer with an intermediate reactor forming material having a silane group (4) on the surface of the anodic oxide film; In order to immobilize the bioactive material covalently and stably on the surface of the anodic oxide, a silane material having three desorption functional groups on the surface of the activated anodic oxide film and having a functional group for immobilizing the bioactive material may be prepared in an organic solvent such as ethanol. After reaction for more than 1 hour with a solution of ~ 10% concentration level, the membrane is stabilized at a high temperature of more than 100 ℃ by washing. Surface Science 458 (2000), p 80-90]

Iii) immobilizing (5) the bioactive material for promoting bone adhesion to the implant in which the intermediate reactor layer is formed; In order to stably fix the bioactive material that promotes bone adhesion on the surface of the anodized film, a surface functional group formed through the silane film and a functional group capable of chemical covalent bonding are introduced into the bioactive material and the substrate is immersed in the dissolved solution. Allow spontaneous association in a simple form. (Mat.Sci. & Eng.C 23 (2003) 395)

The inventors of the present invention confirmed that the silane coating film was immobilized on the titanium anodized surface of APTES (Aminopropyltriethoxysilane), which is one of the silane compounds, through XPS (Table 1. Before coating the silane layer on the anodized surface). After XPS elemental analysis data).

Elements (XPS) Anodized surface Anodized surface after silane layer coating  At% Ratio  At% Ratio C 28.98 3.00 30.09 4.07 O 49.9 5.17 48.25 6.53 Ti 9.66 1.00 7.39 1.00 P 9.93 1.03 11.53 1.56 N 1.54 0.16 0.98 0.13 Si 0 0.00 1.75 4.22

In addition, in order to confirm the stability of the immobilized silane material, the fluorescent material FITC (Fluorescence isothiocyanate) on the titanium anodized surface on which APTES was fixed was reacted with the amine group of APTES and fixed on the surface. The elution of the fluorescent material with time was confirmed in the state in which the substrate to which the fluorescent material was immobilized was immersed in Hank's Balanced salt solution, which is a bio-like solution (FIG. 6). The control group was a titanium natural oxide film surface was confirmed a difference from the anodized surface of the present invention and it was found that the anodized surface is present more stably than the natural oxide surface (Fig. 23).

According to what is known so far ( Langmuir 2003 , 19, 200-204; United States Patent, 6,645,644), the surface treatment with phosphoric acid on the surface of titanium increased the hydroxyl group of the surface to increase the stability of the silane coating film. It is. In the present invention, after the titanium surface was treated by anodization and confirmed by XPS, it was confirmed that the phosphorus and oxygen atoms on the anodized surface increased based on the Ti element, and the phosphoric acid used during anodization was present between the crystals of the titanium oxide. (Table 2. XPS elemental analysis data of titanium natural oxide surface and anodized surface).

Elements (XPS) Natural oxide surface Anodized surface  At% Ratio  At% Ratio  O 79.57 4.07 75.19 5.69  P 0.89 0.05 11.59 0.88 Ti 19.53 1.00 13.22 1.00

Unlike the conventional method, the immobilization method of the present invention forms an oxide film surface through anodization and then forms an intermediate reactor layer with an intermediate reactor-forming material having a silane group (4), thereby immobilizing the bioactive material. Its thick and rough surface forms a more stable oxide film for use in implants, and by fixing a synthetic peptide (bio-like substance) that promotes bone formation at a constant concentration on the implant surface as well as showing the effect of growing cells better during bone formation. It can shorten the period of bone adhesion and increase the success rate of procedure, especially in patients with low bone mass or lack of bone mass.

The method according to the invention can also be used for dental or other fixation of bone grafts.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples and may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art.

Example 1 Anodic Oxidation to Titanium and Its Activation

The technique for activating the biotransplantation anodization membrane is to clean the surface of the anodized implant with neutral detergent, high purity ethanol and ultrapure distilled water, and then dry it for 10 minutes in UV / O treatment equipment (UVO-cleaner, Jelight, USA). Under the condition of maintaining current and voltage, or the same washing and drying process, the implant was subjected to oxidation reaction for 10 minutes to 1 hour in piranha solution mixed with 30% hydrogen peroxide and 70% sulfuric acid solution, and high-purity ethanol and ultrapure distilled water. This is done through a process of washing and drying. (Macromol. Symp. 253 (2007) 115-121)

Example 2 Fixing of Silane and PLL

<2-1> Fixing Silane (APTES)

The inventors carried out the immobilization reaction by determining the reaction conditions of the APTES compound based on the silane group fixation based on 20 specimens (CellNest). Therefore, the volume and amount of each solution are adjusted according to the number of substrates. In other words, if the number of implants to be treated is changed, the total volume of the reaction solution must be changed and the amount of silane compound used therein is also changed. However, the concentration of the reaction solution is prepared to have a constant value regardless of the volume of the reaction solution.

Each specimen was washed for 10 minutes at room temperature using an ultrasonic cleaner (Cole-Palmer, 55 kHz) in 200 ml of ethanol and 200 ml of tertiary distilled water before use, and dried under nitrogen, followed by UV-O3 cleaner (Jelight UVO-cleaner 42-). 220) was used to remove surface residual organics.

A silane compound of APTES (3-aminopropyltriethoxysilane) (Aldrich, Cat No. 440 140) was used to form a functional group by silane coating the specimen. The washed specimens were placed in a petri dish, a petri dish was placed in the insertion portion of the glove box, and the pressure was reduced for 20 minutes. 10 ml of ethanol (Ethanol anhydrous, Aldrich, Cat No. 277649) were placed in a 15 ml Falcon tube and 250 µl (prepared 2.5% APTES solution) was added using a 1 ml pipette. Mix the solution vigorously. The prepared solution was poured into the petri dish with the side to be reacted up, and then the lid was covered and the reaction was performed at room temperature for 90 minutes. After the reaction, 50 ml of EtOH was charged in a 100 ml beaker and 20 ml of ethanol was charged in a petri dish. After 90 minutes, the finished substrate was washed by shaking 8-10 times using 50 ml of EtOH in a beaker and then transferred to a petri dish containing 20 ml of ethanol. After washing all 20 sheets, the substrate was removed from the glove box with the petri dish lid and transferred to the air. Before nitrogen drying, the resultant was washed by shaking 8-10 times with 50 ml of ethanol in the beaker once again in air and dried immediately with nitrogen. The dried substrate was placed in a glass petri dish in an oven at 110 ° C. and baked for 1 hour with a lid closed.

<2-2> PLL fixing method

We prepared PLL fixation based on 20 specimens. Therefore, the volume and amount of each solution vary depending on the number of substrates.

Each specimen was washed for 10 minutes at room temperature using an ultrasonic cleaner (Cole-Palmer, 55 kHz) in 200 ml of ethanol and 200 ml of tertiary distilled water before use, and dried under nitrogen, followed by UV-O3 cleaner (Jelight UVO-cleaner 42-). 220) was used to remove surface residual organics.

A silane compound of triethoxysilylbutanaldehyde (TSBA) (Gelest. Inc, USA) was used for silane coating the specimen. The washed specimen was placed in a petri dish and decompressed in a glove box for 20 minutes, and then the specimen was placed. 10 ml of anhydrous ethanol (Ethanol anhydrous, aldrich, Cat. No. 277649) was added to a 15 ml Falcon tube and TSBA was added using a 1 ml pipette to add 250 µl (preparation of 2.5% TSBC solution). The solution was mixed in turn. The prepared solution was poured into the petri dish with the side to be reacted up, and then the lid was reacted at room temperature for 90 minutes. After the reaction, 50 ml of EtOH was charged in a 50 ml or 100 ml beaker and 20 ml of ethanol was charged in a petri dish. After 90 minutes, the finished substrate was washed by shaking 8-10 times using 50 ml of EtOH contained in a beaker, and then transferred to a petri dish containing 20 ml ethanol. After all 20 sheets had been cleaned, the substrate was transferred to the air with the Petri dish lid closed. Before nitrogen drying, the substrate was dried by using nitrogen gas immediately after shaking 8-10 times with 50 ml of ethanol contained in a beaker in air. The dried substrate was placed in a glass petri dish in an oven at 110 ° C. and baked for 1 hour while covering the lid.

The TSBA immobilized substrate was placed in a petri dish with the reaction surface facing up. Then, 2 mg of Poly-L-Lysine hydrochloride (PLL) (Aldrich, USA, Cat No. P2658) polymer was dissolved in 10 ml of pH 9.0 bicarbonate buffer (NaHCO ₃ / Na ₂ CO ₃ ), and reversible aldehyde and amine groups were added thereto. Reaction solution was added by adding 10 µl of NaCNBH ₃ (SODIUM CYANOBOROHYDRIDE, 5.0M SOLUTION IN AQUEOUS CA.1M SODIUM HYDROXIDE, Aldrich, Cat. Was prepared. The petri dish containing the substrate was allowed to react for 1 hour. Four 100 ml beakers were prepared for washing, two beakers were filled with 50 ml pH 9.0 bicarbonate buffer and the other two with 50 ml tertiary distilled water. After the reaction, the mixture was washed twice by shaking 8-10 times in a pH 9.0 bicarbonate buffer, and then washed twice by shaking 8-10 times in distilled water, followed by drying the substrate using nitrogen gas or clean air.

<2-3> APTES, PLL Fixed Results

1) Check for reaction

Fixation of APTES and PLL thin films on each titanium oxide layer was confirmed using XPS (X-ray photonic spectrometry, SIGMA PROBE ESCA, ThermoVG, UK) and fluorescence microscope (Axiovert 200, ZEISS, Germany) (FIG. 1 to 3). .

Specifically, after the reaction of the APTES and TSBA-PLL to the anodized titanium oxide film was confirmed through the surface element analysis through XPS to determine whether or not fixed. As shown in FIG. 1, after the APTES reaction, the silicon (Si) element, which was not observed on the existing anodized titanium substrate, was increased, and the amount of the carbon element was increased, and the carboxyl functional groups on the surface were reduced after the silane reaction. It was. After the TSBA reaction, as shown in FIG. 2, the increase of the silicon element and the amount of the carbon element were increased, and the carboxyl functional groups on the surface were reduced after the silane reaction, and the carbonyl group was further increased. In addition, as the PLL is fixed to TSBA, the amount of nitrogen (N) element is increased as shown in FIG. 3, and as the PLL is fixed at 420 eV, the binding energy of ammonium salt form in TSBA state, It was confirmed that the binding energy of the organic matrix) was changed to 400 eV.

As a result of measuring the fluorescence image of each step using FITC, a fluorescent substance reacting selectively with the amine, as shown in Fig. 4, after the APTES and PLL were fixed, the fluorescence was observed evenly over the entire area, and the APTES and PLL were evenly fixed throughout the entire area. Seems to have been. On the other hand, weak fluorescence was observed in some regions of the anodized titanium surface and TSBA in a fixed state, which is considered to be fluorescence immobilized on physical adsorption or surface impurities. It is also used to provide the simplest Q.C. Can be used in a manner (FIG. 4).

2) fixed quantity quantification

 Bromophenol Blue Sodium Salt (BPB) (Aldrich, USA) is a substance that selectively ionically adsorbs amines and is not dissociated by water when the bond is formed, but dissociated by a piperidine solution. In addition, it has a high molar extinction coefficient of 60,000 at 600 nm and can be quantified even at low amine concentrations. Using this property of BPB, the concentration of amine in AET (2-aminoethanthiol) thin film formed by self-assembly thin film on Au substrate was quantified by SPR instrument and the concentration of amine immobilized on other substrate was quantified based on this. .

As shown in FIG. 5, the anodized titanium substrate of each step was placed in a 25 ° C. thermostat for 12 hours in an aqueous BPB solution (1 mg / ml) to allow adsorption to occur. Thereafter, each substrate was washed vigorously with tertiary distilled water three times, and then placed in a 25 ° C. thermostat for 1 hour in tertiary distilled water to wash the simply adsorbed BPB. Thereafter, each substrate was washed vigorously with tertiary distilled water three times, dried with nitrogen gas, and then immersed in 1 ml of 20% piperidine aqueous solution for 1 hour so that BPB fixed on the substrate was dissociated by piperidine. The solution was then measured for absorbance at 500-700 nm using a UV spectrometer (JASCO 550, JASCO, USA). The measured absorbance graph was subjected to data processing of absorbance at 600 nm on the basis of 525 nm and 650 nm.

As a result of quantitative determination of surface amine concentration by BPB method, the fixed concentration of AET thin film on Au substrate was 3.4 / nm ² (0.56 nmol / cm ² ), and the APTES concentration on the naturally oxidized titanium oxide substrate and anodized titanium oxide substrate It was confirmed that the fixed concentrations had concentrations of 4.0 pieces / nm ² (0.66 nmol / cm ² ) and 10.8 pieces / nm ² (1.8 nmol / cm ² ), respectively. The fixed concentration of APTES in anodized titanium oxide substrate is 2.84 times that of APSM in the monocrystalline silicon wafer, 3.8 / nm ² (0.63 nmol / cm ² ), which is higher than the actual surface area 2.4 calculated by CLSM. However, considering that the resolution of the CLSM picture is 200 nm it was confirmed that the similar value was shown.

3) stability evaluation

① hydrolysis stability evaluation (Fig. 6)

In order to evaluate the hydrolytic stability of APTES and PLL formed in each titanium oxide film in the physiological environment, FITC (Flourescein isothiocyanate) (Aldrich, 90%) was reacted with an amine (AP) reactor of APTES. FITC was dissolved in a pH 9.0 carbonate / bicarbonate buffer at a concentration of 0.5 mg / ml, and the substrate was fixed in APTES and allowed to react at room temperature for 1 hour. After the reaction, the substrate was removed from the solution, washed twice in pH 9.0 carbonate / bicarbonate buffer, twice in tertiary distilled water, and dried with nitrogen. Before the experiment, the titration curve was drawn by dissolving FITC in Hank's balanced salt solution (10x concentrate, without Ca ²⁺ , Mg ²⁺ or HCO ^3- , Aldrich) with 1.0-50 nm concentration. A linear titration curve was confirmed at the concentration (FIG. 7).

FITC-attached specimens were placed in a cuvette containing Hank's balanced salt solution, and the fluorescence intensity of the supernatant was measured up to 30 days (FIG. 8).

② Physical stability assessment

The ASTM F-1044 method is approved by the Food and Drug Administration (FDA) for evaluating the shear force on coated thin films of dental implants with porous surfaces. In the present invention, the shear strength in the static state of the APTES and PLL thin films fixed on the anodized titanium oxide surface using the above method was evaluated using a universal testing machine (EHF-EB20-40L, Shimatsu, Japan). It was. This shear strength test held the substrate under the same conditions as in FIG. 9 and exerted a force at a rate of 1 mm / min to the strength required for separation of the interface between the coated thin film and the substrate. As a result, both APTES and PLL showed values of 37 MPa and 30 MPa, respectively, exceeding 20 MPa, which are required by the FDA (FIG. 10).

In addition, the degree of damage due to the shear force applied when the dental implant fixture is placed on the bone was confirmed through a test of directly placing on the calf bone of the pig. The calf bones of pigs had their tendons removed before testing. APTES and PLL thin films were formed on the fixtures provided by Osstem (4 mm X 10 mm, avana, GSII type, Osstem, Korea) in the same manner as in the above specimen, and the FITC was reacted with the amine groups of each thin film. In order to prevent fluorescence quenching, the experiment was conducted while the surface of the fixture was not exposed to light. The fixture was placed using Hanaro standard kit (Osstem, Korea). In the same way as the general implant procedure, the guide drill, 2.0 twist drill, 2.0 / 3.0 pilot drill, 3.0 twist drill, and 3.6 mm twist drill are drilled in sequence and then torque gauge meter (DTDK-20EX, BESTTOOL-KANON). , Japan) was used to measure the shear force applied to the 4.0 fixture. After implantation, the bone in which the fixture was placed was cut using a file saw so as not to damage the surface of the fixture. Fixtures removed from the cut bones were removed by ultrasonic cleaning for 10 minutes on a 1 wt% triton-X 100 solution, and the degree of peeling of the fixed APTES and PLL thin films was measured using a fluorescence microscope. The fluorescence microscopy was measured by dividing the head, body and tail into three parts for each fixture, and selecting ten random locations for each part. According to the peeling degree of each measured site | part, it evaluated by dividing into large (40% or more peeling), medium (10-40% peeling), and small (10% or less peeling).

11 shows the results of measuring the shear force applied to the 4.0 fixture using the pig calf bone. It took 45-55 Ncm of force to be placed on the pig bone, which is 3 ~ 4 times the normal force on the surface, considering that the shear force required when placing a general dental implant is about 15 Ncm. It can be seen that the condition. As shown in the SEM image (Fig. 12) before and after implantation, the porous structure of the anodized titanium surface was worn and worn only at the end portion of the porous structure while maintaining the structure at the shear force of 45 Ncm, rather than collapse of the porous structure itself. It was confirmed that bone was filled between the structures. In other words, even after a portion of the silane thin film is worn and disappeared, the structure of the anodized titanium oxide film is maintained even after implantation using dense pork bone, so that the silane thin film inside and outside the hole is maintained and remains on the implant surface to interact with biological tissue. You can expect it.

13 to 15 are evaluation data for 90 fluorescence data obtained by measuring three sections at three sites for three samples using a fluorescence microscope. In the order of placement, the damage of the head area that received the shear force from the beginning to the end of the placement was the highest, and the damage was decreased in the order of the trunk and tail. As a result of the placement, more than 40% of the damaged area was 28% of the total area, 10-40% of the damaged area was 22% and less than 10% of the damaged area was 40%. In calculating this, it is determined that the damaged part of the silane membrane which is damaged at the time of implantation is 25-40% of the total area (FIG. 16).

Example 3 BMOE Fixation

<3-1> BMOE fixing method

We determined the BMOE fixation conditions based on 20 specimens. Therefore, when the amount of specimens to be treated is changed, the concentration, the treatment time, etc. are constant, but the volume of the solution and the amount of the cleaning liquid used are adjusted accordingly.

The reacted BMOE 10 mg was weighed using a microbalance and dissolved in 1 ml of anhydrous DMSO (DMSO anhydrous, aldrich, Cat. No. 276855) to make a stock solution and stored in a refrigerator.

Twenty specimens of APTES or PLL were placed in a petri dish with the reaction side facing up. After dissolving DMSO stock solution in which BMOE was dissolved, 500 µl was diluted in 9.5 ml of pH 7.4 PBS, and then poured into a petri dish containing specimens within 5 minutes for 1 hour. Four 100 ml beakers were prepared for washing, two beakers filled with 50 ml pH 7.4 PBS and the other two with 50 ml tertiary distilled water. After the reaction was completed, washed twice by shaking 8-10 times in pH 7.4 PBS and then washed twice by shaking 3-10 times in distilled water, and the substrate was dried using nitrogen gas or clean air.

<3-2> BMOE Fixed Results

1) Check for reaction

Fixation of BMOE monomolecules on APTES and PLL polymers was performed using contact angle measurement and BPB method. As a result of evaluating APTES, PLL, APTES-BMOE, and PLL-BMOE using the contact angle, it was confirmed that both materials changed to hydrophilic surfaces around 10 ° after BMOE fixation (FIG. 17). In addition, it was confirmed that about 30% of the amine functional group in the APTES monolayer thin film and about 50% of the amine functional group in the PLL polymer thin film were replaced by BMOE after BMOE fixation using the BPB method (FIG. 18).

Example 3 Peptide Fixation

<3-1> Peptide Fixation Method

We prepared peptide fixation based on 20 specimens. Therefore, the volume and amount of each solution vary depending on the number of substrates.

2.3 mg of oligopeptide (PEP111) was dissolved in 1 ml of tertiary distilled water to make a stock solution, and then divided into 100 μl and stored in a freezer. PEP111 is the oligopeptide described in the applicant's prior application 2008-0054730, whose sequence is Cys Lys Ile Pro Lys Pro Ser Ser Ala Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu (CKIPKPSSAPTELSAISMLYL) and the sequence is of known amino acids. It can be synthesized by the synthesis method.

Twenty specimens of BMOE were placed in a Petri dish with the reaction side facing up. After dissolving the stock solution in which the oligopeptide is dissolved, 100 μl was diluted in 900 μl of pH 6.6 phosphate buffer, and 50 μl of peptide solution per substrate was dropped on the surface using a 100 μl pipette and evenly spread on the surface. The reaction was carried out for 12 hours. Four 100 ml beakers were prepared for washing, two beakers were filled with 50 ml pH 6.6 phosphate buffer and the other two were filled with 50 ml tertiary distilled water. After completion of the reaction, the solution was washed twice by shaking 8-10 times in pH 6.6 phosphate buffer, and then washed twice by shaking 8-10 times in distilled water, followed by drying the substrate using nitrogen gas or clean air.

<3-1> Oligopeptide Fixation Results

1) Check for reaction

① Evaluation using SPR

SPR was used to confirm peptide fixation via SPR signal changes following peptide fixation under the same chemical conditions. As a result of the SPR measurement, a fixed amount of 85-110 ng / cm ² was fixed under the 1 hour reaction condition, and a fixed amount increase of 20% was confirmed upon further 1 hour reaction. This confirms that the longer the reaction time can increase the fixed concentration of the peptide (Fig. 19).

② Evaluation using fluorescence

After the peptide was fixed, the reaction and uniformity of the peptide were confirmed using FITC. As a result, as shown in FIG. 20, the fluorescence image uniformly fixed to the 1 mm ² area was confirmed.

2) Quantitative Evaluation

① Quantification using BPB method

In the AET (2-aminoethanthiol) thin film formed by the self-assembly thin film on the Au substrate, the concentration of the peptide fixed by BMOE was quantified using an SPR apparatus, and the concentration of the peptide fixed on the Cellnest substrate was quantified based on this. The fixed concentration of peptide immobilized on Au substrate showed a fixed concentration of 51 pmol / cm ² as shown in FIG. 19. This was quantified by the difference in absorbance of BPB in the BMOE and peptide fixation step using the BPB method.

Figure 21 shows the absorbance per unit area of the peptide by subtracting the absorbance at the peptide and BMOE stage and then divided by the area of the substrate. Based on the following values, fixed concentrations of peptides immobilized on CellNest were found to be 130-150 pmol / cm ^{2 in} APTES thin films and 1.2-1.4 nmol / cm ² in PLL.

Theoretically calculating the fixed peptide concentration in APTES, the molar absorption coefficient of BPB is 60000 and the number of amines present in the oligopeptide (PEP111) is 3, so the theoretical absorbance value is 3 (the number of amines in the peptide) X 130 X 10 ^-12 (fixed peptide concentration) X 60000 (molar extinction coefficient of BBP) = 2.64 X 10 ^-4

This is the theoretical absorbance and the test results are 2.21 X 10 ^-4 , 2.48 X 10 ^-4 . This is an error of about 15% of the theoretical value, and the quantitative value using BPB can be regarded as reliable data.

The same theoretical calculation of the fixed peptide concentration in the PLL shows that the molar absorptivity of BPB is 60000 and the number of amines present in the peptide 111 is 3, so the theoretical absorbance value is 3 (the number of amines in the peptide) X 1.2 X 10 ^-9 (fixed peptide concentration) X 60000 (molar extinction coefficient of BBP) = 2.16 X 10 ^-3

This is the theoretical absorbance and the test result is 2.47 X 10 ^-3 , 3.21 X 10 ^-3 . This is about 15-30% of the theoretical value, which shows a slightly larger error value than in the monomolecular film, but through this, the quantitative value using BPB can be regarded as reliable data.

1 is a high-resolution XPS spectrum analysis data graph of carbon (a) and silicon (b) atoms of an APTES red line and anodized titanium oxide black line before TSBA reaction,

FIG. 2 is a high-resolution XPS spectrum analysis graph of carbon (a) and silicon (b) atoms of an APTES red line and anodized titanium oxide black line after TSBA reaction.

FIG. 3 is a graph of high resolution XPS spectral analysis data under a nitrogen source of TSBA red and PLL green lines after PLL fixation to TSBA.

4 is a fluorescence image of each step after a) anodized titanium surface, b) after APTES treatment, c) after TSBA and FITC treatment, and d) after PLL and TSBA treatment.

5 is a schematic diagram of a BPB experiment.

6 is a schematic diagram of the hydrolysis stability evaluation experiment method of the titanium oxide surface stability evaluation experimental method,

7 is a FITC fluorescence titration graph,

8 is a graph of hydrolysis stability evaluation for 30 days, wherein a) a comparison of before and after anodization of an APTES thin film, and b) a comparison of PLL and APTES on an anodization substrate.

9 is a schematic diagram of the evaluation method of shear strength (ASTM F1044) for the coating thin film (in the drawings, the characters are Metal Substrate, Epoxy Resin, Silane coating)

10 is a graph showing the results of evaluation of shear strength,

11 is a graph showing a result of measuring the shear force at the time of implantation in the fixture (X axis: rotational speed of the fixture at implantation (n = 5)),

12 is a SEM image of the fixture surface before and after implantation (wherein before (a, c), after implantation (b, d), a), b): an image viewed at an angle of 30 degrees, c), d): Image from above)

FIG. 13 is a fluorescence image photograph of the titanium anodic oxide fixture after implantation of the head (A), d) less damage (within 10%), b), e) moderate damage (10-40%), c), f) high damage areas (40% or more); a), b), c) side view, d), e), f) front view),

14 is a fluorescence image after implantation of the trunk portion of the titanium anodization fixture (a), d) less damage (within 10%), b), e) moderate damage (10-40%), c), f) high damage areas (40% or more); a), b), c) side view, d), e), f) front view),

15 is a fluorescence image after the tail portion of the titanium anodization fixture (at this time a), d) less damage (within 10%), b), e) moderate damage (10-40%), c), f) high damage areas (40% or more); a), b), c) side view, d), e), f) front view),

16 is a comprehensive data graph and table for each part of the silane film that is damaged at the time of implantation;

17 is a graph showing contact angle results before and after BMOE modification for APTES and PLL,

18 is a graph showing BPB results for APTES, APTES-BMOE, PLL, and PLL-BMOE.

19 is a graph of SPR signal change data according to peptide fixation;

20 is a fluorescence image using FITC after Peptide fixation,

FIG. 21 is a graph showing absorbance data according to the unit area of peptides in a monomolecular amine thin film (APTES) and a PLL layer.

22 is a titanium anodized surface treatment method,

23 is a graph of hydrolyzate confirmation with time at the anodization surface and the natural oxidation surface.

Claims (11)

Iii) forming an electrochemical oxide film on the surface of the implant for implantation using an anodic oxidation method; Ii) treating the implant surface using one selected from the group consisting of piranha, O ₃ and H ₂ O-Plasma to activate an anodic oxide film; Iii) forming an intermediate reactor layer with an intermediate reactor forming material having a silane group on the surface of the anodization film; Iii) selected from the group consisting of BMOE (bis-maleimidoethane) and SMCC (succinimidyl 4- (N-maleimidoethyl) -cyclohexane-1-carboxylate) connecting the intermediate reactor layer with a bioactive material Forming a linker layer of linker material; And Iii) immobilizing the bioactive material on the surface of the bio-implant implant in which the linker layer is formed. The method of claim 1, wherein the implant for implantation is a biologically active material on the surface of the implant, characterized in that the main component is titanium. The method of claim 1, wherein the electrochemical oxide film of step iii) is formed using an anodization method in a phosphoric acid / sulfuric acid mixed solution. The method of claim 1, wherein the intermediate reactor forming material is of the structure of R _2- (CH ₂ ) _n -Si- (R ₁ ) ₃ , wherein R ₁ is -Cl,- OCH ₃ , and -OCH ₂ CH ₃ , wherein R ₂ is -NH ₂ (amine), -COOH (carboxyl), -CHO (aldehyde), and -CH = CH ₂ (alkyne) It is selected from the group, n is 3 to 18, characterized in that the immobilization of the bioactive material on the implant surface. The method of claim 4, wherein the intermediate reactor forming material is a 3-aminopropyltriethoxysilane (APTES) compound or triethoxysilylbutanaldehyde (TSBA). The method of claim 1, wherein the bioactive material is a synthetic peptide selected from oligopeptides of a new amino acid sequence having a function of Bone Morphogenetic Protein. The method of claim 1, wherein when the bioactive material is immobilized in the step iii), in order to control the surface concentration of the bioactive material and give an active space, the matrix material is additionally introduced to the implant surface, characterized in that Method of immobilization of material. The method according to claim 7, wherein the matrix material is in the form of a single molecule or polymer similar in size to the bioactive material, and at one end of the functional group selected from the group consisting of -NH ₂ , -COOH, -COH, and -SH, Immobilization method of a bioactive material on the surface of the implant, characterized in that the end of the -OH, and -CH ₃ has a functional group that is not reactive with the bioactive material selected from the group consisting of. 10. The method of claim 8, wherein the matrix material is PLL (Poly-L-Lysine hydrochloride). delete delete

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