WO2014208824A1 - Method for producing implant having bioactive material immobilized on surface thereof and implant produced thereby - Google Patents

Abstract

The present invention relates to a method for immobilizing bone morphogenetic proteins for promoting osseointegration of an implant that is implanted in the body and a method for producing an implant. The present invention enables growth factors to be coated through a two-step coating method including a primary coating and a secondary coating. According to the present invention, the effect of the loaded growth factors can be maximized by adjusting a release rate of the bone morphogenetic proteins. Therefore, the hydrophilicity of a mineral biomaterial can be improved by the loading of the growth factors and the engraftment and osseointegration of the implant that is implanted in the body is enhanced by improving the adhesion and differentiation of cells.

Description

Method for preparing an implant in which a bioactive substance is immobilized on a surface and an implant prepared according to the method

The present invention relates to a method for producing an implant in which a bioactive substance is immobilized on a surface, and to an implant prepared accordingly.

In order for the dental and orthopedic implants to be inserted into the body to be maintained for a long time, the joint surface of the bone and the biomaterial must be kept stable. Rapid bone fusion and biocompatibility of the implant procedure is associated with this long-term implant use.

Recently, surface treatment methods for stimulating cellular action between bone and implant surface have been increasing. In the medical application of implants, it is important to fix bioactive materials on the surface of biomaterials used in implants, and the research on metal surface treatments for controlling cell and tissue reactions has been widely conducted.

Therefore, the method of transferring the growth factor to the bone-implant junction by implant coating technology has been widely used for effective fixation of the implant and healing of the treatment site.

Over the past few years, it has been shown that the bone fusion of implants can be increased by adsorbing bone forming protein (BMP-2) on the surface of titanium or hydroxyapatite (HAp). Calcium phosphate, mainly composed of hydroxyapatite, is also a ceramic material, which is typical and very good biocompatibility for implant applications.

Hydroxyapatite was first known by DeJong et al in 1926 to be an inorganic component of bone, but it was only 35 years before such hydroxyapatite was approved as a biomaterial for orthopedics and dentistry.

However, while hydroxyapatite has an advantage of increasing bone fusion, it has a disadvantage of being difficult to use as an implant due to its brittleness. In order to overcome this problem, a technique of coating hydroxyapatite on titanium using plasma spray, sputtering, electrolysis sol-gel, and the like has been studied. The method of coating hydroxyapatite on titanium with strong physical properties could compensate for the disadvantages of physically weak hydroxyapatite.

However, because there are no functional groups on the surface of the hydroxyapatite, it is difficult to effectively fix the bone morphogenic protein and control the bone morphogenic protein in vivo. Therefore, a simple and effective growth factor fixing or coating technique is required, excluding such cumbersome methods.

The present invention aims to provide a coating method that improves the fusion and regeneration efficiency of implants equipped with the same growth factor by eliminating chemical bonds, minimizing potential inflammation, and controlling the release rate of the growth factor in a two-step coating method. .

In addition, the present invention is to provide a growth factor immobilization technology or growth factor coating technology that can induce rapid bone fusion by improving the immobilization efficiency of growth factors.

In addition, the present invention is to provide a method for producing a dental and orthopedic bone fusion biomaterial for the bone fusion.

According to one aspect of the invention, the step of first coating a bioactive material on the implant surface; And a second coating of a hydrogel containing a bioactive material on the surface of the first coated implant, and a method of preparing an implant which can control the release rate of the bioactive material by the secondary coating. Can be.

The surface of the implant may be etched or RBM treated.

The bioactive substance may include an osteoinducible protein (BMP-2 or BMP-12), PDGF, IGF-I, or an amino acid (or protein) capable of promoting bone formation.

The bioactive substance may be used by dissolving in physiological saline and tertiary distilled water.

The hydrogel may be prepared using biodegradable synthetic polymer or biodegradable natural polymer.

The biodegradable natural polymer comprises one or more selected from the group consisting of collagen, gelatin, hyaluronic acid, chitosan, and alginate, and the synthetic polymer may comprise one or more selected from the group consisting of PLA, PGA, PLGA, and PCL. It may include.

The hydrogel may further contain one or more components in the form of collagen alone or a mixture of hyaluronic acid and glycosaminoglycan.

The method may further include crosslinking the coated hydrogel.

The crosslinking can be carried out by heat drying under vacuum or UV irradiation.

The crosslinking can be carried out in the presence of a crosslinking agent.

The crosslinking agent may include one or more selected from the group consisting of diphenylphosphoryl azide, glutaraldehyde, hexamethylene isocyanate, succinimide and carbodiimide.

The concentration of the bioactive substance may be 0.3 to 3.0 μg.

The concentration of the bioactive substance may be 1.2 to 2.5 μg.

According to another aspect of the invention, the first bioactive material layer formed on the surface of the implant; And a second bioactive material layer formed on the first bioactive material layer using a hydrogel.

According to the bone-forming protein immobilization technology according to the present invention, it is possible to reduce the loss of bone-forming protein and maintain an effective release effect.

Therefore, implantation can be prepared to minimize the immune response after transplantation, improve the hydrophilicity of the implant by introducing cell compatibility, biocompatibility, and growth factor, thereby improving the adhesion and differentiation of cells and inducing fast bone fusion with high engraftment rate. have.

1 is a photograph showing a contact angle measurement result (a: comparative example, b: Example).

2 is a graph showing the change in BMP elution amount over time.

Figure 3 is an X-ray picture of the mandible bone implanted.

4 is a graph showing a change in unwinding torque according to a change in BMP concentration.

5 is a photograph of the results of Masson's Trichrome (MT) staining according to the change in BMP concentration.

Figure 6 is a photograph showing the process of implanting a two-stage coated implant.

FIG. 7 is an x-ray photograph of three uncoated and three coated implants (A: left mandible, B: right mandible).

8 is an implant photograph at 4 weeks after implantation.

9 is a graph showing the unwinding torque of uncoated, primary coated and secondary coated implants.

Hereinafter, preferred aspects of the present invention will be described with reference to the accompanying drawings. However, aspects of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Accordingly, the shape and size of elements in the drawings may be exaggerated for clarity, and the elements denoted by the same reference numerals in the drawings are the same elements.

One aspect of the present invention includes the step of first coating the bioactive material on the surface of the implant, and the second step of coating the hydrogel containing the bioactive material on the first coated implant surface, by a secondary coating It may be a method for producing an implant that can control the release rate of the bioactive material.

First, the bioactive material may be first coated on the implant surface.

The implant may use titanium and metal materials such as titanium alloys such as Ti-6Al-4V and Ti-8Ta-3Nb, cobalt alloys, stainless steel, nitinol, platinum, iridium, niobium, tantalum, gold, silver, and the like. In addition, Ultra High Molecular Weight Poly Ethylene (UHMWPE), Poly Ether Ether Ketone (PEEK), Polyurethane (polyurethane), Silicone Elastomers (silicone elastomers), and Bioabsorbable Polymers (PLA (poly lactic acid), PGA (poly glycolic) acid), poly capro lactone, poly methyl metha acrylate).

Ceramic materials such as aluminum oxide, zirconium, physiologically active glass fibers, silicon nitrogen compounds, calcium phosphate and carbon may also be used. However, it is not limited thereto.

The implant can be modified using a variety of surface treatments, such as titanium powder treatment, etching, restorable blasted media (RBM) treatment, and subtractive methods, depending on the material.

By modifying the implant surface, a coating layer can be more easily formed on the implant surface.

The bioactive substance may be a bone morphogenetic protein (BMP-2 (bone morphogenetic protein-2) or BMP-12), platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-I), or bone formation It may include an amino acid (or protein) that can promote.

Growth factors are used to promote bone fusion, and may include proteins such as BMP-2, BMP-4, BMP-12, PDGF, and IGF. Growth factors may be used, such as bone morphogenetic protein to promote bone fusion, it can be diluted in distilled water or saline phosphate buffer (PBS) at a concentration of 0.3 ~ 3 μg / 20 ul. However, the dilution concentration is not limited thereto.

The concentration of the bioactive substance may be 0.3-3.0 μg / 20 ul, more preferably 1.2-2.5 μg / 20 ul. The initial bone adhesion rate is excellent when the concentration of the bioactive substance is within the above range.

The bioactive substance may be dissolved in physiological saline or tertiary distilled water.

Primary coating can be carried out by immersion in BMP solution under sonication.

BMP can be dissolved in tertiary distilled water and the solution can be used to form a BMP thin film on the implant surface. The BMP solution used for manufacturing the thin film may be dissolved in pH 3 to 8, such as distilled water and physiological saline.

After the primary coating it can be dried. Drying can be carried out in vacuo and even at room temperature.

Next, the hydrogel containing the bioactive material may be secondarily coated on the surface of the first coated implant. Secondary coating can control the release rate of the bioactive material.

Hydrogels can be prepared using biodegradable synthetic polymers or biodegradable natural polymers. Biodegradable natural polymers include one or more selected from the group consisting of collagen, gelatin, hyaluronic acid, chitosan, and alginate, and synthetic polymers include PLA (polylactic acid), PGA (polyglycol acid), and PLGA (poly (lactic-co) -glycol acid)), and PCL (polycaprolactone) may include one or more selected from the group consisting of.

The hydrogel may further contain one or more components in the form of collagen alone or a mixture of hyaluronic acid and glycosaminoglycan.

Secondary coating can be carried out using a method of immersion under sonication in a hydrogel solution containing BMP. It is desirable to block the light during hydrogel coating and sonication in refrigerated state.

After the secondary coating it can be dried. Drying can be carried out in vacuo and even at room temperature.

Cross-linking the surface hydrogel of the BMP-coated implant may be further included. By controlling the crosslinking properties of the BMP release rate of the implant can be improved.

Crosslinking can be carried out by heat drying under vacuum or UV irradiation, and crosslinking can be carried out in the presence of a crosslinking agent.

The crosslinking agent may comprise one or more selected from the group consisting of diphenylphosphoryl azide, glutaraldehyde, hexamethylene isocyanate, succinimide and carbodiimide.

Another aspect of the invention may be an implant comprising a primary bioactive material layer formed on the implant surface, and a secondary bioactive material layer formed on the primary bioactive material layer through a hydrogel.

The release rate of the bioactive material may be controlled by forming the second bioactive material layer using a hydrogel.

Other implants, bioactive substances, hydrogels, etc. are the same as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples.

In the present invention, the growth factor was first coated and dried, and the hydrogel containing the growth factor was coated and dried secondly, and then placed in a cell culture and a beagle dog to confirm the effect by evaluating initial fixation and bone fusion.

Hydrophilicity evaluation

In order to fix the BMP on the disk-shaped titanium (grade 4) surface having a diameter of 14mm, two-stage coating was performed as follows.

First, a solution of 0.3 μg / 20 μl concentration was prepared by dissolving BMP in tertiary distilled water, and the solution was coated on a titanium surface, and then dried under vacuum for 15 minutes. Next, a solution of 0.3 μg / 20 μl was prepared by dissolving BMP in a 0.5% atelocollagen solution, and using this solution, a secondary coating was applied to the dried titanium surface and dried under vacuum for 30 minutes. In this way, an implant prepared according to the two-stage coating was prepared.

The contact angle was measured to determine the hydrophilic properties of the hydrogel and bone forming protein-coated titanium surface. The contact angle was measured by a contact angle measuring device (KRUSS, Germany) using the third distilled water, the results are shown in b of FIG. It was shown in comparison with the case where the hydrogel coating was not performed after the etching treatment (a of FIG. 1).

Referring to FIG. 1, when the hydrogel is coated, the contact angle is about 41.4 °, and when the hydrogel is coated, the contact angle is about 70.5 °, and when the hydrogel is coated, the contact angle is significantly reduced as compared with the etching. You can check it. From this, it can be seen that the hydrophilicity of the titanium surface is greatly increased when the bone forming protein is immobilized using the hydrogel.

In vitro Elution Characteristics of Growth Factors

In order to evaluate the release rate of the growth factor (BMP) coated in two stages using a hydrogel, the coating was applied to the implant under the following conditions and soaked in physiological saline 500 at 1, 2, 3, 5, Each eluted BMP was quantitatively analyzed on day 7.

Ⓐ uncoated group

Ⓑ Dissolve 300 ng of BMP in 20 μl of tertiary distilled water and coat it on the implant surface

Ⓒ After fixing 100 ng / 20 μl of BMP directly to the implant surface, secondary coating with BMP 200 ng / 20 μl atelo collagen solution

Ⓓ Directly dry 200 ng / 20 μl of BMP onto the implant surface and apply secondary coating with BMP 100 ng / 20 μl atelo collagen solution

Quantitative analysis of bone morphogenetic protein was carried out using the 'Human BMP-2 Super X-ELISA' kit (Antigenix, USA) as follows.

First, put the titanium disks coated with bone-forming protein-fixed hydroxyapatite having a diameter of 8 mm and the titanium disks of the comparative group in a 48-well plate, and completely pour 0.1 ml / ml bovine serum albumin (BSA) in 1 ml. Incubation was performed at room temperature for 2 hours while shielding from light.

It was then washed four times with the wash solution inherent in the kit. After washing, 0.5 μg / ml of the biotin-Labeled tracer inherent in the kit was titrated 20 μl on each titanium surface, and then incubated at room temperature while shielded from light for 2 hours.

It was then washed four more times with the wash solution inherent in the kit.

After washing, the conjugate solution (streptavidin-HRP) contained in the kit was diluted 1: 500 with a solution of 0.05% Tween-20 (Uniqema, USA) diluted in 0.1% bovine serum albumin. After titration of 20 μl on the surface, the cells were incubated at room temperature while blocked from light for 30 minutes.

It was then washed four more times with the wash solution inherent in the kit and transferred to another 48 well plate with the titanium coated surface facing the bottom. The TMB substrate solution A and the TMB substrate solution ratio inherent in the kit were mixed in a ratio of 100 μl per well and incubated at room temperature while being blocked from light for 30 minutes.

After the incubation, the stop solution (2N sulfuric acid) was added 100μl per well to stop the color development and the absorbance was measured at 450nm.

As shown in FIG. 2, when BMP was coated with 1 step (lime green ⓑ), the initial release amount was maintained at about 25 ng for 2 days and the elution amount was reduced to about 10 ng from day 3.

When BMP was coated with 2 step (ⓒ, ⓓ), the initial release amount was about 35 ng for 1 day. Faster BMP elution was observed in the experimental group (blue ⓒ) fixed with collagen 200 ng over time.

In the experimental group coated with only 100 ng of collagen (red ⓓ), it was determined that 200 ng of BMP directly immobilized on the surface was eluted after 3 days, thereby increasing the amount of BMP.

Therefore, BMP elution was a 1 step high information and 2 step fixation was found to be an efficient way to control the initial elution amount.

Animal efficacy evaluation for optimization of bone forming protein concentration

The optimal concentration of BMP was confirmed by evaluating the degree of bone fusion according to the BMP-2 concentration in the beagle dog oral model. Implant size was used 3.5mm × 6.0m, according to the implant surface treatment was classified into four groups as follows.

G1: SLA (Sandblasted Large grit, Acid etched) Treatment

G2: 0.3 μg / 20 ul BMP-2 / collagen coating after SLA treatment

G3: 0.7 μg / 20 ul BMP-2 / collagen coating after SLA treatment

G4: 1.5 μg / 20 ul BMP-2 / collagen coating after SLA treatment

Four 15 kg healthy beagle dogs were used, and three implants were placed in each of the left and right mandibles (Fig. 3). The concrete method is as follows.

First, zoletil (0.03 mL / kg) and lumpoon (0.03 mL / kg) were mixed and administered intravenously, and respiratory anesthesia was maintained with isofuluran.

Next, bilateral anesthesia with lidocaine was applied to both mandibles and the mandibles were exposed.

Next, the mandible placement point was marked as Lance Drill and implant implantation holes were formed at 800 rpm in the order of 2.0 mm diameter and 2.8 mm twist drill.

Next, all implants were implanted with a mounting torque of 15 NCm and the cover screw was covered.

Next, the magnetic resonance dimensions and x-rays of all implants were measured immediately after implantation, and the skin was closed.

Next, four weeks after the procedure, four beagle dogs were euthanized with CO ₂ gas, and then the loosening torque was measured.

As shown in FIG. 4, higher releasing torque was observed in the 0.3 μg / 20 ul and 0.7 μg / 20 ul BMP-2 treatment groups than in the SLA comparison group, and in the 1.5 μg / 20ul BMP-2 treatment group, the lowering torque was lower than the comparison group. Was observed.

Therefore, it was confirmed that when the BMP-2 concentration is high, it inhibits bone fusion. In particular, the 0.3μg / 20ul BMP-2 treatment group showed a good initial bone adhesion rate with 96.3 NCm of loosening torque.

In the oral model of 15 kg beagle dog, it was confirmed that the BMP-2 concentration was 0.3 ~ 0.7 ㎍ / 20 ul for the initial bone adhesion rate.

5A-D shows a photograph of Masson's Trichrome (MT) staining result, it can be observed that the new bone was formed as a whole (middle photo).

In particular, referring to D of FIG. 5, when the BMP has a high concentration of 1.5㎍, new bone is formed in the periphery but the collagen content is low (new bone is grey). This may lead to higher results in later BIC or new bone area measurements, but substantially weak bone regeneration was observed.

In addition, bone regeneration within the helix was observed in the 0. / 20 ul 3 and 0.7 μg / 20 ul BMP-2 coated groups (left photo), and the SLA and 1.5 μg / It can be seen that new bone regeneration is better than the 20 ul BMP-2 coating group.

Animal efficacy evaluation of two-stage coated implants

In order to evaluate the bone fusion efficacy of the two-stage coated BMP in the beagle dog oral model, animal efficacy was evaluated by classifying into three groups according to the implant surface treatment. Implant size was 3.5 x 6.0 mm.

G1: Sandblasted Large grit, Acid etched

G2: 0.4 μg / 20 ul BMP-2 / collagen coating after SLA treatment

G3: 0.2 µg / 20 ul BMP-2 after SLA treatment was first fixed on the implant surface, followed by secondary coating with 0.2 µg of BMP-2 collagen

Animal experiments were conducted in the same manner as the previous animal efficacy evaluation using four 15 kg beagle dogs. Specific processes are shown in FIGS. 6 to 8. Figure 6 is a photograph showing the process of implanting a two-stage coated implant. FIG. 7 is an x-ray photograph of three uncoated and three coated implants (A: left mandible, B: right mandible). 8 is an implant photograph at 4 weeks after implantation.

9 shows the evaluation results. Referring to FIG. 9, it was observed that the initial bone adhesion rate of the 0.4 μg / 20 ul BMP-2 coated experimental group was more effective than the SLA control group, and the G3 group in which the release rate was adjusted in two stages. Initial bone adhesion was found to be the best at.

The present invention is not limited by the above-described embodiment and the accompanying drawings, but is intended to be limited by the appended claims. Accordingly, various forms of substitution, modification, and alteration may be made by those skilled in the art without departing from the technical spirit of the present invention described in the claims, which are also within the scope of the present invention. something to do.

The present invention can improve the fusion and regeneration efficiency of implants equipped with the same growth factor by eliminating chemical bonds, minimizing potential inflammation, and controlling the release rate of the growth factor by a two-stage coating method.

Claims (14)

Primary coating the bioactive material on the implant surface; And Secondarily coating a hydrogel containing a bioactive material on the surface of the primary coated implant, Method for producing an implant that can control the release rate of the bioactive material by the secondary coating. The method of claim 1, The surface of the implant is a method of producing an etching treatment or RBM treatment. The method of claim 1, The bioactive substance, bone formation inducing protein (BMP-2 or BMP-12), PDGF, IGF-I, or a method for producing an implant containing an amino acid (or protein) that can promote bone formation. The method of claim 1, The bioactive material is a method of producing an implant used by dissolving in physiological saline and tertiary distilled water. The method of claim 1, The hydrogel is a method for producing an implant prepared using biodegradable synthetic polymer or biodegradable natural polymer. The method of claim 5, The biodegradable natural polymer comprises one or more selected from the group consisting of collagen, gelatin, hyaluronic acid, chitosan, and alginate, and the synthetic polymer may comprise one or more selected from the group consisting of PLA, PGA, PLGA, and PCL. Method for producing an implant comprising. The method of claim 1, The hydrogel is a method for producing an implant further containing one or more components in the form of collagen alone or a mixture of hyaluronic acid and glycosaminoglycans. The method of claim 1, The method of manufacturing an implant further comprising the step of crosslinking the coated hydrogel. The method of claim 8, The crosslinking is a method for producing an implant is carried out by heat drying under vacuum or UV irradiation. The method of claim 8, The crosslinking is carried out in the presence of a crosslinking agent. The method of claim 10, The crosslinking agent is a method for producing an implant comprising at least one selected from the group consisting of diphenylphosphoryl azide, glutaraldehyde, hexamethylene isocyanate, succinimide and carbodiimide. The method of claim 1, The method for producing an implant having a concentration of the bioactive substance is 0.3 to 3.0 μg. The method of claim 1, The concentration of the physiologically active substance is 1.2 ~ 2.5 μg method for producing an implant. A first layer of bioactive material formed on the surface of the implant; And Implant comprising a layer of a secondary bioactive material formed on the primary bioactive material layer using a hydrogel.

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