JP2012205656A - Coating material for living body implant and implant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new coating material for a living body implant which is effective to prevent infection of a living body implant and an implant having the coating material.SOLUTION: The coating material for a living body implant is a coating material used for preventing infection of a living tissue around a burial region of the living body implant and contains polysaccharides. A monosaccharide which is represented by a chemical formula of (CHO) and/or (CHO) and is linked via at least one of a β-1,3 linkage, β-1,6 linkage, β-2,1 linkage, β-2,6 linkage, α-1,4 linkage, and α-1,6 linkage is preferably used for polysaccharides.

Description

  The present invention relates to a coating material for living body implants used for preventing infection of living tissue around a living implant implantation site, and an implant provided with the covering material.

  Biological implants such as artificial tooth roots, artificial joints, osteosynthesis members, and metal artificial bones are widely used in the medical field, but infection around the implants due to pathogenic microorganisms is a problem. Infection by microorganisms occurs when microorganisms invade the affected area and proliferate during or after the living implant. It is said that when an artificial object such as a living body implant is present, infection occurs with about one-tenth of the number of bacteria compared to the case where no artificial object is present. In recent years, there has been a problem of infection caused by biofilm-forming bacteria that adhere to the implant surface and secrete a polysaccharide film called a biofilm that is resistant to antibacterial agents.

  Therefore, for example, Patent Document 1 discloses a method of applying an antibacterial drug to the surface of a substrate in order to prevent infection of a biological implant due to microorganisms. Patent Document 2 discloses a method for applying immunoglobulin to the surface of an implant.

  On the other hand, the applicant of the present application is also able to prevent microbial infection, and is a biological implant having a metal oxide layer having an isoelectric point of less than 7 on at least a part of the substrate as a biological implant having little toxicity to the human body (Patent Document 3). ) And a biological implant having a surface layer containing the metal oxide and an antibacterial inorganic material (Patent Document 4).

JP 2002-128697 A Special table 2000-508620 JP 2008-61897 A JP 2009-189683 A

  However, in general, many antibacterial agents used in Patent Document 1 exhibit selective effects only on specific microorganisms. Moreover, although the antibacterial inorganic material used in Patent Document 4 exhibits efficacy against a wide range of microorganisms, the antibacterial property is low. Further, in any case, there is a concern about generation of resistant bacteria due to long-term use.

  As described above, among microorganisms, biofilm-forming bacteria that secrete a polysaccharide film (biofilm) after attaching to the surface of a base material such as a biological implant are known. Antibacterial action against bacteria is remarkably low. In this regard, Patent Documents 1 and 2 described above do not examine the antibacterial action against biofilm-forming bacteria at all. On the other hand, the methods described in Patent Documents 3 and 4 described above are extremely useful as a technique that exerts an effect on not only a normal microorganism but also a biofilm-forming bacterium. It is desired.

  This invention is made | formed in view of the said situation, The objective is to provide the implant provided with the novel coating material for biological implants useful for the infection prevention of a biological implant, and the said coating material.

  The coating material for living body implants of the present invention that has solved the above problems is a covering material used for preventing infection of living tissue around the living body implant site, and has a gist in that it contains a polysaccharide. is there.

In a preferred embodiment of the present invention, the polysaccharide is a monosaccharide represented by a chemical formula of (C ₆ H ₁₀ O ₅ ) and / or (C ₆ H ₁₂ O ₆ ), wherein β-1,3 bond, β -1,6 bond, β-2,1 bond, β-2,6 bond, α-1,4 bond, and α-1,6 bond.

  In addition, the implant of the present invention that has solved the above problems has a gist where at least a part of the surface of the substrate constituting the biological implant is coated with the coating material for biological implant.

  According to the present invention, it is possible to effectively prevent infection of a biological implant without using an antibacterial drug. By using the coating material of the present invention, it is possible to prevent infection by biofilm-forming bacteria or resistant bacteria, which has been difficult to prevent infection with antibacterial drugs. It is extremely useful in that it can be realized.

FIG. 1 is a graph showing the results of the phagocytosis rate of Staphylococcus aureus in Example 1. FIG. 2 is a graph showing the results of the phagocytosis rate of biofilm-forming S. aureus in Example 1.

The present inventors have been able to prevent infection of in vivo implants by microorganisms without using antibacterial drugs, and in particular, biofilm-forming bacteria and drug resistance that have been difficult to prevent with antibacterial drugs. In order to provide a novel coating material for living body implants that can also exert an effect on bacteria, studies have been repeated. Specifically, the study was conducted based on the viewpoint that if a substance having an immunostimulatory action is coated on the substrate surface, infection with microorganisms can be prevented without using an antibacterial agent. As a result, a coating material containing a polysaccharide, preferably a monosaccharide represented by the chemical formula of (C ₆ H ₁₀ O ₅ ) and / or (C ₆ H ₁₂ O ₆ ), has a β-1,3 bond, A coating material containing a polysaccharide bound by at least one of β-1,6 bond, β-2,1 bond, β-2,6 bond, α-1,4 bond, and α-1,6 bond As a result, the inventors have found that the intended purpose can be achieved, and that a technique capable of both preventing proliferation of a wide range of microorganisms and preventing adhesion to an implant can be provided, and the present invention has been completed.

  Thus, the coating material for living body implants of the present invention is characterized by containing a polysaccharide. It is known that some polysaccharides have an immunostimulatory action, but no polysaccharide or immunostimulant used for preventing infection of living implants has been known so far. If the present inventors prey on and digest pathogenic microorganisms that have entered the living body (called phagocytic ability) and activate macrophages that activate the immune mechanism, the result is a marked suppression of infection by microorganisms. I found out that I can do it. Such an immunostimulatory mechanism is effectively exhibited regardless of the type of microorganism, and the application range is greatly expanded as compared with the case of using an antibacterial agent having extremely high selectivity for microorganisms. In the present invention, the antibacterial agent is not gradually eluted from the applied site (sustained release action) and exerts the antibacterial effect like the antibacterial agent; it remains stably (fixed) on the applied site. As a result, infection of microorganisms is prevented by exerting an immunostimulatory effect.

  In Patent Document 2 described above, an immunoglobulin having an immunoactive activity is used. However, an immunoglobulin is a substance that is handled with great care, such as possibly causing damage to surrounding tissues depending on the method of use. It is. On the other hand, any of the polysaccharides used in the present invention is a highly safe substance with extremely low toxicity. In addition, the immunoglobulin is gradually eluted from the applied site (sustained release action), and exerts its effect by binding to the invading bacteria, and is stable to the applied site as in the present invention. It remains in (fixed), and the immune structure of the living body recognizes its chemical structure so that the immunostimulatory effect is not exhibited (described later).

  As used herein, “infection prevention” includes prevention (prevention) and / or treatment of microbial infection.

  In the present specification, the microorganisms to be prevented from infection are not particularly limited, and examples include microorganisms that are problematic during and after treatment of biological implants. For example, Staphylococcus aureus, Staphylococcus epidermidis, and coagulase. Negative staphylococci, enterococci, Pseudomonas aeruginosa, Escherichia coli, streptococci; methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, methicillin-resistant coagulase-negative staphylococci, vancomycin-resistant enterococci, multidrug-resistant Pseudomonas aeruginosa Etc. are exemplified. Biofilm forming Staphylococcus aureus, biofilm forming methicillin resistant Staphylococcus aureus, biofilm forming Staphylococcus epidermidis, biofilm forming methicillin resistant Staphylococcus epidermidis, biofilm forming coagulase negative staphylococci, biofilm Examples include methicillin-resistant coagulase-negative staphylococci, biofilm-forming enterococci, biofilm-forming vancomycin-resistant enterococci, and biofilm-forming Escherichia coli.

As the polysaccharide used in the present invention, a monosaccharide represented by the chemical formula of (C ₆ H ₁₀ O ₅ ) and / or (C ₆ H ₁₂ O ₆ ) is preferably a β-1,3 bond, β- It is a polysaccharide linked by 1,6 bond, β-2,1 bond, β-2,6 bond, α-1,4 bond, or α-1,6 bond. This is because many of these polysaccharides have a high immunostimulatory effect. The polysaccharide used for this invention should just contain at least 1 of the coupling | bonding mentioned above, and may contain the 2 or more coupling | bonding. Moreover, in this invention, said polysaccharide may be used independently and may use 2 or more types together. A commercial item can also be used for the said polysaccharide.

Furthermore, in the present invention, it is preferable to select one having extremely low toxicity, particularly considering application to a substrate constituting a biological implant. Examples of preferable polysaccharides including such a viewpoint include, for example, dextran [α-1,6- (C ₆ H ₁₀ O ₅ ) _n ], β-1,3-glucan [β-1,3- ( C ₆ H ₁₀ O ₅ ) _n ], inulin [(β-2,1- (C ₆ H ₁₀ O ₅ ) _n (β-2,1- (β-2,1- (C ₆ H ₁₂ O ₆ )) _n ] and the like, and β-1,3 glucan is more preferable, and these polysaccharides are extremely resistant to not only normal bacteria but also biofilm-forming bacteria, as shown in the examples described later. Commercially available products can be used for these.

  The coating material of this invention may be comprised only from said polysaccharide, and in the range which does not impair the effect | action of this invention, you may add another component. For example, a preservative or a stabilizer may be added for the purpose of long-term storage. In the present invention, the use of antibacterial drugs is basically excluded, but known antibacterial drugs may be used in combination for the purpose of further enhancing the effect of preventing infection by microorganisms.

  Next, the implant which has the said coating material is demonstrated.

  In the implant of the present invention, at least a part of the surface of the substrate constituting the biological implant is coated with the above-described coating material for biological implants. Since the said coating | coated material can coat | cover the whole base | substrate, a stronger infection prevention effect is exhibited.

  The material of the substrate is not particularly limited, and any material used for implants (for example, metal, ceramics, plastics, etc.) can be used. The kind of the metal material is not particularly limited as long as it is used for an implant, and examples thereof include pure Ti or Ti alloy, stainless steel, cobalt chromium molybdenum alloy, zirconium alloy, tantalum, or an alloy thereof. The kind of Ti alloy is not particularly limited, and examples thereof include Ti alloys containing at least one element such as Al, V, Zr, Mo, Nb, and Ta. Specifically, Ti-6 mass% Al-4 Examples include mass% V alloy, Ti-15 mass% Mo-5 mass% Zr-3 mass% Al alloy, Ti-6 mass% Al-2 mass% Nb-1 mass% Ta-0.8 mass% Mo alloy. Is done.

  The surface state of the implant is not particularly limited, and may be a smooth surface or a rough surface, and the surface properties may be appropriately adjusted according to required characteristics. For example, if it is desired to increase the amount of polysaccharide coated on the substrate surface as much as possible per apparent area of the substrate, it is recommended that the surface of the implant be in a rough state that increases the true surface area. Is done. The surface roughening method is not particularly limited. For example, when a metal is used as the base material, a commonly used surface roughening method such as sandblasting or thermal spraying can be used.

  Next, a method for producing an implant using the coating material will be described.

  First, the polysaccharide mentioned above is prepared. In coating the surface of the substrate, it is preferable to adjust the concentration of the polysaccharide as appropriate so that the desired action and effect are exhibited.

Specifically, according to the type of polysaccharide, an appropriate dispersion medium such as ethanol, DMSO, and water is selected to disperse the polysaccharide, and the amount of the polysaccharide applied at the time of application is approximately 0.00001 mg / cm. it is preferably adjusted to be within the range of ^{2 ~200mg} / cm ^2. Especially it is more preferably adjusted to the range of ^{0.01mg / cm 2 ~5mg / cm 2} .

  Then, the polysaccharide dispersion prepared as described above is coated on at least a part of a substrate made of a biomaterial such as Ti. The coating means is not particularly limited, and examples thereof include coating, spraying, and dipping. These coating means may be combined with a chemical method such as a surface graft polymerization method. For example, in application, the polysaccharide can be uniformly applied by applying the dispersion liquid in which the above-mentioned polysaccharide is dispersed after appropriately shaking with Vortex or the like.

  In the present invention, an implant in which at least a part of the surface of the substrate is coated with the polysaccharide is also included in the scope of the present invention. Examples of such implants include artificial joints such as artificial hip joints, artificial knee joints, and artificial shoulder joints in the orthopedic field; osteosynthesis members such as bone screws, bone plates, spinal fixation members; and other metal artificial bones Typical examples include artificial tooth roots in the dental field. The coating material of the present invention can also be applied to vascular stents, artificial heart valves, artificial blood vessels, and the like.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and can be implemented with modifications within a range that can meet the purpose described above and below. They are all included in the technical scope of the present invention.

Example 1
In this example, the phagocytosis rate of each microorganism was compared when the following four types of test materials were coated on a titanium material.

(Test material)
(A) Dextran (DT) [α-1,6- (C ₆ H ₁₀ O ₅ ) _n ] 2 mg: Use Dextran 60,000 manufactured by Wako (a) β-1,3-glucan (GL) [β −1,3- (C ₆ H ₁₀ O ₅ ) _n ]: β-1,3 Glucan (for biochemistry) manufactured by Wako is used. (C) Inulin (IN) [(β-2,1- (C ₆ H ₁₀ O ₅ ) _n (β-2,1- (β-2,1- (C ₆ H ₁₂ O ₆ ) _n ) 1 mg: Inulin manufactured by Wako is used. (D) Aluminum hydroxide gel (AO) 0. 64 mg: Uses Alu-Gel-S suspense manufactured by SERVA

  Among the above materials, aluminum hydroxide gel (AO) is generally used as an adjuvant at the time of vaccination, and was used for comparison in this example.

  In coating the titanium material, each of the above test materials (a) to (c) was dispersed in 100 μL of ethanol, and a dispersion liquid stirred with Vortex was used. Further, the above test material (d) was used as it was without being dispersed in a dispersion medium.

(Test bacteria)
Staphylococcus aureus: NBRC 12732 (S. aureus)
Here, NBRS is an abbreviation for the biological genetic resource section of the National Institute of Technology and Technology.
Biofilm-forming S. aureus: NBRC 14462 (BF-MSSA)

  As a metal material, a pure titanium test piece (size: φ14 mm × 1 mm) blasted with commercially available alumina powder is prepared, and the dispersion of the test material described above is applied to the entire surface of the test piece at 100 μL per test piece. Applied. Next, it dried at room temperature and produced each test piece for a poor food test. As a control group, a sample not coated with the above-mentioned test material (pure titanium with blast treatment only) was prepared (control group).

(experimental method)
As bacterial phagocytic cells, RAW 264 cell line (European Collection of Cell Culture, standard cell list catalog number EC85062803) was used.

First, each test piece (size φ14 mm × 1 mm) is immersed in a medium in a commercially available 24-well plate [RPMI1640 made by SIGMA-Aldrich plus 10% GIBCO fetal calf serum (FCS)]. RAW 264 cell line was inoculated with 5 × 10 ⁵ cells and cultured in a CO ₂ incubator at 37 ° C. for 24 hours. Subsequently, after inoculating the said test microbe pre-cultured in the BHI culture medium (Eiken Chemical Co., Ltd.) of 2 * 10 < ⁶ > CFU (colony formation unit) in the said culture medium, it is 10 at 1800 * g at 4 degreeC. Centrifugation was performed for minutes to allow airborne bacteria to adhere to each test piece. Next, the cells were cultured in a CO ₂ incubator at 37 ° C. for 20 minutes, and the RAW 264 cell line was phagocytosed by the test bacteria. Next, each test piece was washed three times with 1 mL of PBS (−) to remove the test bacteria that were not phagocytosed (not taken up) by the RAW 264 cell line. Next, in order to recover the above-mentioned test bacteria phagocytosed in the RAW 264 cell line, 0.2% saponin (Merck saponin was dissolved to 0.2% with milli-Q water, filtered and filtered. The RAW 264 cell line was destroyed by adding 500 μL of the sterilized product, and the above-mentioned test bacteria that had been taken up by the RAW 264 cell line were recovered. The solution of the test bacteria collected in this manner is transferred to a test tube, and 0.5 mL of sterilized water is added to the well for washing, and the well is transferred to the test tube and mixed with the solution of the test bacteria. did. 1 mL of these mixed solutions were washed with ultrasonic waves for 5 minutes, stirred with vortex, serially diluted, spread on a 110 medium plate (manufactured by Eiken Chemical Co., Ltd.), and cultured at 37 ° C. for 24 hours. The number of colonies grown on the medium plate was measured as the number of viable bacteria phagocytosed by the RAW 264 cell line.

  The same operation was repeated 3 times, and the average value of the phagocytosis rate ± standard error was calculated for each test bacterium. These results are shown in FIG. 1 (phagocytosis against S. aureus) and FIG. 2 (biofilm-forming S. aureus). In these figures, the greater the number of phagocytosis (that is, the number of viable bacteria phagocytosed by the RAW 264 cell line), the higher the immunostimulatory action and the better the antibacterial action.

  From FIG. 1 and FIG. 2, it can be seen that when the polysaccharide (DT, GL, IN) used in the present invention is used as a coating material, the phagocytic rate is improved compared to the control group for any fungus. . In particular, when GL was used as a polysaccharide, the phagocytosis rate for Staphylococcus aureus was remarkably high and the phagocytosis rate for biofilm-forming Staphylococcus aureus was very high as compared with the case where other polysaccharides were used. . In contrast, when aluminum hydroxide gel (AO) was used, only an effect lower than that of the control group was obtained.

  From the above experimental results, it was demonstrated that the use of the coating material of the present invention effectively prevents the growth of microorganisms that enter the periphery of the living body implant without using an antibacterial drug. Moreover, it was demonstrated that the bacterial growth inhibitory effect by this invention is effectively exhibited also with respect to a biofilm formation microbe.

Claims (3)

  A coating material for living body implants, which is used for preventing infection of living tissue around a living body implant site, and contains a polysaccharide. The polysaccharide is a monosaccharide represented by a chemical formula of (C ₆ H ₁₀ O ₅ ) and / or (C ₆ H ₁₂ O ₆ ), which is β-1,3 bond, β-1,6 bond, β- The coating material for living body implants according to claim 1, which is bonded by at least one of 2,1 bond, β-2,6 bond, α-1,4 bond, and α-1,6 bond.   An implant in which at least a part of the surface of a substrate constituting the biological implant is coated with the coating material for a biological implant according to claim 1 or 2.