Antibacterial medical equipment and method for producing the same

An object of the present invention is to provide an antibacterial medical equipment which has sufficient antibacterial activity in vivo and is excellent in compatibility with living tissues, and also can maintain antibacterial activity over a long period and has high safety.An antibacterial medical equipment characterized in that inositol phosphate is bonded to a Ca compound of a medical equipment whose surface is at least coated with a layer of the Ca compound, or a medical equipment comprising the Ca compound. The antibacterial medical equipment as described above, wherein silver ions are bonded to the inositol phosphate. A method for producing an antibacterial medical equipment, which comprises bringing a medical equipment whose surface is at least coated with a layer of a Ca compound, or a medical equipment comprising a Ca compound into contact with an aqueous solution of inositol phosphate to obtain an antibacterial medical equipment in which inositol phosphate is bonded to the Ca compound. The method for producing an antibacterial medical equipment, wherein inositol phosphate is bonded to the Ca compound and then the Ca compound is brought into contact with an aqueous solution containing silver ions to obtain an antibacterial medical equipment in which silver ions are bonded to the inositol phosphate.

TECHNICAL FIELD

The present invention relates to medical equipments such as an implant and a surgical instrument used in the medical field, and more particularly to an antibacterial medical equipment having antibacterial activity, and a method for producing the same.

BACKGROUND ART

Conventionally, a technology disclosed, for example, in WO 2008/081861 has been proposed as a technology for a titanium-based or non-titanium based member having antibacterial activity in an implant.

WO 2008/081861 discloses an antibacterial member coated with titanate, including a substrate, a layer of a nanosheet, nanotube, nanofiber or nanocrystal made of a crystalline alkali titanate formed on the substrate, and a silver titanate layer in which a portion or all of an alkali component of the alkali titanate is substituted with silver ions.

WO 2008/081861 also discloses, as a method for producing the antibacterial member, the method comprising the steps of (i) subjecting a titanium-based substrate to a hydrothermal treatment in an aqueous alkali solution at a temperature of 110 to 180° C., (ii) heat-treating the product after subjecting to the hydrothermal treatment at 200 to 700° C., and (iii) washing and drying the product after subjecting to the heat treatment, and immersing the product in an aqueous silver ion-containing solution.

Since the antibacterial member disclosed in WO 2008/081861 includes the silver titanate layer, high antibacterial activity is obtained. However, there was a problem in that when this antibacterial member is used in an implant, titanate or silver titanate makes contacted with living tissues, resulting in poor compatibility between the implant and living tissues.

In the silver titanate layer of the antibacterial member, since a portion or all of the alkali component of the alkali titanate is substituted with silver ions, the degree of elution of silver ions varies drastically depending on a usage state of an implantation site, as described in Example 10. Therefore, this antibacterial member may not exhibit antibacterial activity when silver ions are not eluted, or exhibit toxicity when silver ions are excessively eluted. Also, there may arise a problem that antibacterial activity cannot be maintained over a long period because the degree of elution of silver ions is unstable.

DISCLOSURE OF INVENTION

Under these circumstances, the present invention has been made and an object thereof is to provide an antibacterial medical equipment which has sufficient antibacterial activity in vivo and is excellent in compatibility with living tissues, and also can maintain antibacterial activity over a long period and has high safety.

In order to achieve the above object, the present invention provides an antibacterial medical equipment characterized in that inositol phosphate is bonded to a Ca compound of a medical equipment whose surface is at least coated with a layer of the Ca, or a medical equipment comprising the Ca compound.

The antibacterial medical equipment of the present invention may have a constitution such that silver ions are bonded to the inositol phosphate.

The inositol phosphate is preferably phytic acid (inositol hexaphosphate, hereinafter sometimes abbreviated to IP6).

The Ca compound is preferably hydroxyapatite (hereinafter referred to as HAp).

The antibacterial medical equipment is preferably an antibacterial implant.

The present invention provides a method for producing an antibacterial medical equipment, which comprises bringing a medical equipment whose surface is at least coated with a layer of a Ca compound layer, or a medical equipment comprising a Ca compound into contact with an aqueous solution of inositol phosphate to obtain an antibacterial medical equipment in which inositol phosphate is bonded to the Ca compound.

In the method for producing an antibacterial medical equipment of the present invention, it is also possible to obtain an antibacterial medical equipment in which silver ions are bonded to the inositol phosphate by bonding inositol phosphate to the Ca compound and bringing the Ca compound into contact with an aqueous solution containing silver ions.

The inositol phosphate is preferably phytic acid.

The Ca compound is preferably HAp.

The antibacterial medical equipment is preferably an antibacterial implant.

According to the antibacterial medical equipment of the present invention, since inositol phosphate is bonded to a Ca compound of a medical equipment whose surface is at least coated with a layer of the Ca compound, or a medical equipment comprising the Ca compound, it is possible to provide an antibacterial medical equipment which has sufficient antibacterial activity in vivo and is excellent in compatibility with living tissues, and also can maintain antibacterial activity over a long period and has high safety.

When the antibacterial medical equipment of the present invention has a constitution such that silver ions are bonded to the inositol phosphate, it is possible to provide an antibacterial medical equipment having more instantaneous antibacterial activity.

According to the method for producing an antibacterial medical equipment of the present invention, since an aqueous solution of inositol phosphate is brought into contact with the Ca compound of a medical equipment to obtain an antibacterial medical equipment in which inositol phosphate is bonded to the Ca compound, it is possible to provide an antibacterial medical equipment which has sufficient antibacterial activity in vivo and is excellent in compatibility with living tissues, and also can maintain antibacterial activity over a long period and has high safety, efficiently at low cost.

According to the method for producing an antibacterial medical equipment of the present invention, since inositol phosphate is bonded to the Ca compound and then the Ca compound is brought into contact with an aqueous solution containing silver ions to obtain an antibacterial medical equipment in which silver ions are bonded to the inositol phosphate, it is possible to produce an antibacterial medical equipment having more instantaneous antibacterial activity, efficiently at low cost.

EXEMPLARY EMBODIMENT FOR CARRYING OUT THE INVENTION

In the present invention, the “medical equipment” includes medical equipments defined in Pharmaceutical Affairs Act revised in 2002. Among such medical equipments, a medical equipment made of metallic materials such as pure titanium, titanium alloy (Ti-6Al-4V alloy, etc.), stainless steel, low carbon steel, copper or copper alloy, silver or silver alloy, gold or gold alloy, platinum group elements such as platinum or alloy thereof, and cobalt-chromium alloy; a medical equipment made of synthetic resins such as polyether ether ketone (PEEK), carbon, carbon fiber-reinforced PEEK, polyethylene, polypropylene, polyethylene terephthalate (PET), fluororesin, silicone resin, and polylactic acid; a medical equipment made of Ca compounds such as HAp; and a medical equipment made of ceramics such as silica, alumina, and zirconia are preferred. The “medical equipment” of the present invention is also applied to tableware and toys which are desirably imparted with antibacterial activity.

Preferred medical equipment in the present invention will be exemplified below.

[Implants which can be Subjected to an Antibacterial Treatment]

FIG. 1is a schematic diagram showing the first embodiment of an antibacterial medical equipment of the present invention. An antibacterial medical equipment1A of the present embodiment has a structure in which a HAp layer3as a Ca compound layer is formed on a surface of a medical equipment2made of a material other than a Ca compound and inositol phosphate4is bonded to the HAp layer3.

The HAp layer3is made of HAp represented by Ca10(PO4)6(OH)2or made of HAp in which a molar ratio of each constituent element slightly varies and a trace amount of carbonate ions is contained, and at least one portion, preferably all of a surface of the medical equipment2is coated with the HAp layer. The thickness of this HAp layer3is preferably 1 μm or more. The Ca compound layer is not limited to this HAp layer3and another insoluble Ca compound, for example, octacalcium phosphate, calcium hydrogen phosphate, calcium sulfate, calcium carbonate, and insoluble salts in which a portion of Ca is substituted with Mg may also be used.

There is no particular limitation in the method for formation of the HAp layer3and the method includes, for example, a dry film formation method such as a plasma spraying method, a vacuum deposition method, or a chemical vapor phase deposition (CVD) method; and a wet method such as a method in which HAp is precipitate/adhered on a surface of a medical equipment2in an aqueous solution containing a Ca compound and a P compound. Among these methods, the wet method is preferred because it can be applied to the medical equipment2made of various materials and also a HAp layer3can be formed in abundance and at low cost. The wet method is particularly preferably a method in which urea and urease are added in a solution containing a Ca compound and a P compound dissolved therein in a ratio corresponding to a molar ratio of the composition of HAp and a medical equipment2is immersed in the mixed solution, and then incubation is conducted at a temperature of about 30 to 60° C., preferably about 50° C. thereby to precipitate HAp and to adhere the HAp to a surface of the medical equipment2.

The inositol phosphate is inositol phosphate in which at least one of six hydroxyl groups of inositol (1,2,3,4,5,6-cyclohexanehexaol) is phosphorylated. In the present invention, the inositol phosphate is preferably inositol phosphate in which three or more of hydroxyl groups are phosphorylated, more preferably inositol phosphate in which four or more of hydroxyl groups are phosphorylated, and most preferably phytic acid (inositol hexaphosphate, IP6in which all hydroxyl groups of inositol are phosphorylated. Phytic acid has a strong chelate effect and exists in the state of being bonded to Ca ions of the HAp layer3in an antibacterial medical equipment1A of the present embodiment.

As described above, the antibacterial medical equipment1A of the present embodiment can be simply produced by forming the HAp layer3on a surface of the medical equipment2and immersing in a solution of inositol phosphate such as IP6thereby bonding inositol phosphate4to the HAp layer3. It is also possible that an inositol phosphate solution is spray-coated on the surface of the HAp layer3instead of immersion in the inositol phosphate solution thereby bonding inositol phosphate to Ca on the surface.

Although it is predicted that inositol phosphate such as the phytic acid has an antitumor effect, regarding the in vivo effect thereof, sufficient elucidation of the detailed effect is not yet known.

On the other hand, in the present invention, we have already found new operational advantages such that inositol phosphate in the state of being bonded to a Ca compound of the HAp layer3, and the like, has antibacterial activity.

In the antibacterial medical equipment of the present invention1A, it is preferred to bond the inositol phosphate4to the Ca compound of the HAp layer3as much as possible. The amount of the inositol phosphate4bonded to the Ca compound can be appropriately adjusted by the concentration of or the time of immersion in inositol phosphate in the case of immersing the medical equipment2with the HAp layer3formed on the surface in a solution of the inositol phosphate4.

The antibacterial medical equipment1A of the present embodiment has a structure in which a HAp layer3as a Ca compound layer is formed on a surface of a medical equipment2and inositol phosphate4is bonded to the HAp layer3, and has operational advantages such as antibacterial activity which could have never been predicted by conventional knowledge with respect to inositol phosphate. Thus, it is possible to provide an antibacterial medical equipment which has practically sufficient antibacterial activity and is excellent in compatibility with living tissues, and also can maintain antibacterial activity over a long period and has high safety.

FIG. 2is a schematic diagram showing the second embodiment of the antibacterial medical equipment of the present invention. The antibacterial medical equipment1B of the present embodiment has a structure in which a HAp layer3as a Ca compound layer is formed on a surface of a medical equipment2made of a material other than a Ca compound and inositol phosphate4is bonded to the HAp layer3, and also silver ions5are bonded to the inositol phosphate4.

The inositol phosphate4bonded to the HAp layer3can still be chelate-bonded and silver ions5can be bonded to inositol phosphate4by bringing silver ions into contact with the inositol phosphate. Silver ions5bonded to the inositol phosphate4are gradually released by bringing into contact with body fluids and tissues in vivo and exert strong antibacterial activity.

The antibacterial medical equipment1B of the present embodiment can be easily produced by immersing the above-described antibacterial medical equipment1A of the first embodiment in an Ag ion-containing solution such as an aqueous AgNO3solution, followed by taking out, washing and drying, or spray-coating the solution, followed by washing and drying. There is no particular limitation on the amount of silver ions5bonded, and a proper amount of silver ions5may be bonded according to the kind of the antibacterial medical equipment1B and purposes. The amount of silver ions5bonded can be appropriately adjusted by the concentration of silver ions used for immersion and the immersion time.

Since the antibacterial medical equipment1B of the present embodiment has a structure in which silver ions5are bonded to the inositol phosphate4in the above-described antibacterial medical equipment1A of the first embodiment, it is possible to provide an antibacterial medical equipment having more instantaneous antibacterial activity.

FIG. 3is a schematic diagram showing the third embodiment of an antibacterial medical equipment of the present invention. The antibacterial medical equipment10of the present embodiment has a structure in which inositol phosphate4is bonded directly to a surface of a medical equipment6which is made of a Ca compound or contains a Ca compound.

The antibacterial medical equipment10of the present embodiment can be simply produced by optionally polishing or washing the surface of a medical equipment6and immersing the medical equipment6in an inositol phosphate solution thereby bonding inositol phosphate to Ca on the surface, followed by separation from the solution, washing and drying. It is also possible that an inositol phosphate solution is spray-coated on the surface of the medical equipment6instead of immersion in the inositol phosphate solution thereby bonding inositol phosphate to Ca on the surface.

Similar to the above-described antibacterial medical equipment1A of the first embodiment, the antibacterial medical equipment10of the present embodiment has a structure in which inositol phosphate4is bonded to the surface of a medical equipment6which is made of a Ca compound or contains a Ca compound, and has operational advantages such as antibacterial activity which could have never been predicted by conventional knowledge with respect to inositol phosphate. Thus, it is possible to provide an antibacterial medical equipment which has practically sufficient antibacterial activity and is excellent in compatibility with living tissues, and also can maintain antibacterial activity over a long period and has high safety.

FIG. 4is a schematic diagram showing the fourth embodiment of an antibacterial medical equipment of the present invention. The antibacterial medical equipment1D of the present embodiment has a structure in which inositol phosphate4is bonded directly to a surface of a medical equipment6which is made of a Ca compound or contains a Ca compound, and also silver ions5are bonded to the inositol phosphate4.

The antibacterial medical equipment1D of the present embodiment can be easily produced by immersing the above-described antibacterial medical equipment1C of the third embodiment in an Ag ion-containing solution such as an aqueous AgNO3solution, followed by taking out, washing and drying, or spray-coating the solution, followed by washing and drying. There is no particular limitation on the amount of silver ions5bonded, and a proper amount of silver ions5may be bonded according to the kind of the antibacterial medical equipment1D and purposes. The amount of silver ions5bonded can be appropriately adjusted by the concentration of silver ions used for immersion and the immersion time.

Since the antibacterial medical equipment1D of the present embodiment has a structure in which silver ions5are bonded to the inositol phosphate4in the above-described antibacterial medical equipment1C of the third embodiment, it is possible to provide an antibacterial medical equipment having more instantaneous antibacterial activity.

EXAMPLES

The effects of the present invention will be proved by way of Examples. The following Examples of the present invention are provided merely for illustrative purposes of the present invention and the present invention is not limited by the description of Examples.

(1) Coating of Titanium Substrate with HAp

(1-1) Preparation of Reagent

Preparation of Simulated Body Fluid (Hereinafter Referred to as SBF)

SBF is a solution in which the concentration of inorganic ions remaining after removing organic matter such as human cells and proteins from blood plasma in human blood is made nearly identical to that of blood plasma. The ion concentration of SBF and blood plasma is shown in Table 1.

In Table 1, SBF (1.5) means that the amount of all solutes of a standard concentration of SBF (SBF (1.0)) was increased by 1.5 times.

Regarding SBF, using reagents described in Table 2, each reagent was added in the amount described in the table to make the entire amount to 1 dm3(36.5° C., pH 7.40). In the following Examples, SBF (1.5) was used as SBF.

A surface treating solution was prepared by adding urea to SEE.

Urea was added to SBF so as to adjust the concentration of urea to 2.0 mol/dm3. When titanium substrate was immersed in the surface treating solution, a 1.0 mass % urease solution prepared by dissolving urease as a hydrolase of urea in pure water was added.

These two kinds of solutions were prepared.

(1-2) Preparation of Titanium Substrate

Using a polishing machine, a surface of titanium substrate was polished with a #240 abrasive paper. Then, titanium substrate was ultrasonic-washed in turn with pure water, ethanol and acetone twice for 5 minutes each, followed by air drying.FIG. 5(a) is an enlarged image of a surface of titanium substrate thus prepared,FIG. 5(b) is a graph showing measurement results of elements contained, andFIG. 5(c) is an appearance of titanium substrate subjected to a polishing treatment (left) and an appearance of untreated titanium substrate (right).

(1-3) Experiment Procedure

An experiment procedure is shown inFIG. 6. Titanium substrate subjected to surface polishing, washing and drying as described above was heated to 200° C. in air, immersed in SBF containing urea and urease, and then allowed to stand in an incubator at 50° C. for several days thereby precipitating HAp.

Solutions used in HAp coating are shown in Table 3.

New SBF was replaced on the 2nd day, 4th day and 6th day.

On the seventh day, the solution was completely drained off, followed by washing with pure water and further air drying.

A HAp-coated titanium substrate was obtained by the above procedure.FIG. 7(a) is a SEM (scanning electron microscope) image of a surface of HAp-coated titanium substrate (magnified 500 times),FIG. 7(b) is an image magnified 5,000 times,FIG. 7(c) is an image magnified 10,000 times,FIG. 7(d) is an image magnified 20,000 times, andFIG. 7(e) is a graph showing measurement results by EDX (energy-dispersive X-ray microanalysis) of elements on the surface of HAp-coated titanium substrate. As is apparent from these results, a coating of HAp was formed on a surface of the resultant HAp-coated titanium substrate.

(2) Immobilization of Silver Ions to HAp-Coated Titanium Substrate

(2-1) Preparation of Reagent

Inositol Phosphate

An inositol phosphate aqueous solution was used for immobilization of silver ions. In the present Example, IP6was used as inositol phosphate. IP6is a compound in which all six hydroxyl groups of inositol are esterified with phosphoric acid and is a biogenic related compound having a high cheleting effect. It has been found that inositol phosphate has higher effect of metal cheletes than that of ethylenediaminetetraacetic acid (EDTA) and also has a metal corrosion prevention effect, metal removal effect and antioxidative effect.

It is considered that, by bringing an aqueous solution of inositol phosphate into contact with HAp layer, calcium ions in HAp are bonded to inositol phosphate and also the bonded inositol phosphate chelates silver ions, and thus silver ions are immobilized to a HAp layer via inositol phosphate.

In the present Example, an aqueous 1,000 ppm solution of IP6was prepared by diluting 50% IP6solution. Using the resultant solution as an undiluted solution, four kinds of aqueous solutions were prepared by further diluting the undiluted solution.

Silver Ions

In the present Example, an aqueous AgNO3solution was used as a silver ion source.

A commercially available AgNO3(1.71 g) was dissolved in pure water to make 100 mL of a solution. Using the resultant solution as an undiluted solution, six kinds of aqueous solutions (0.00005 mol/dm3, 0.0001 mol/dm3, 0.0005 mol/dm3, 0.001 mol/dm3, 0.005 mol/dm3and 0.01 mol/dm3) were prepared by further diluting the undiluted solution. AgNO3has higher solubility in pure water than that of the other silver compound. In pure water, silver ions exist as Ag+.

(2-2) Experiment Method

FIG. 8is a flow chart showing a procedure for immobilization of inositol phosphate and silver ions to the surface of HAp-coated titanium.

In the present experiment, first, the HAp-coated titanium was set in a 6-well plate and 5.0 cm3of each of IP6solutions (concentration: 250 ppm, 500 ppm, 750 ppm and 1,000 ppm) was injected into the plate and then the plate was allowed to stand at 50° C. for 1 day, and thus IP6was bonded to the surface of HAp-coated titanium.

Thereafter, the IP6solution was removed from the plate and the inside of the plate was washed several times with pure water.

Subsequently, 5.0 cm3of each of aqueous AgNO3solutions with the concentration adjusted within a range from 0.00005 to 0.01 mol/dm3was injected into the plate. After immersion for 15 minutes, 30 minutes and 60 minutes (immersion time), the titanium was taken out, sufficiently washed with pure water and then air-dried to obtain samples in which silver ions are immobilized to the HAp-coated titanium.

In the present Example, the following tests (1) to (3) were conducted with respect to immobilization of silver ions.

(1) Influence of Concentration of Inositol Phosphate

FIG. 9is a graph showing X-ray diffraction results of a surface of each sample obtained by varying the concentration of inositol phosphate (IP6). As is apparent fromFIG. 9, in samples using inositol phosphate, existence of silver (Ag3PO4) on the surface can be confirmed and silver was immobilized onto the surface of each sample via inositol phosphate. In contrast, in samples made of only HAp, silver was not observed.

FIG. 10is a SEM image of a surface of each sample produced by varying the concentration of inositol phosphate (IP6).

FIG. 11is a graph showing measurement results by EDX of elements contained on a surface coated with HAp of each sample. In the samples, a peak assigned to Ag was not observed.

FIG. 12is a graph showing measurement results by EDX of elements contained in a surface of each sample obtained by varying the concentration of inositol phosphate (IP6).

FIG. 13is a graph showing a relationship between the concentration of inositol phosphate (IP6) and the content of silver on a surface of each sample.

FIG. 14is a graph showing a relationship between the concentration of AgNO3and the ratio of Ag/(Ca+Ag) of a surface of each sample.

Summarizing the results of the test (1), it could be confirmed that silver was contained in any case where the concentration of inositol phosphate is varied within a range from 250 ppm to 1,000 ppm (four kinds). Even when the concentration of inositol phosphate varied, % by mass of silver was from 20% to 30% and thus a large variation was not recognized. As is apparent from the results shown inFIG. 14, when the concentration of AgNO3becomes less than 0.005 ppm, dependency on the concentration of inositol phosphate becomes unstable.

The following tests were conducted at the concentration of inositol phosphate of 1,000 ppm.

(2) Influence of Variation of the Concentration of AgNO3in Aqueous Solution

FIG. 15is a graph showing X-ray diffraction results of a surface of each sample made by varying the concentration of AgNO3.

FIG. 16is SEM image of a surface of each sample made by varying the concentration of AgNO3.

FIG. 17is a graph showing measurement results by EDX of elements contained in a surface of each sample made under the conditions of the IP6concentration of 1,000 ppm, the AgNO3concentration of 0.001 mol/dm3and the immersion time of 15 minutes.

FIG. 18is a graph showing measurement results by EDX of elements contained in a surface of each sample made under the conditions of the IP6concentration of 1,000 ppm, the AgNO3concentration of 0.005 mol/dm3and the immersion time of 15 minutes.

FIG. 19is a graph showing measurement results by EDX of elements contained in a surface of each sample made under the conditions of the IP6concentration of 1,000 ppm, the AgNO3concentration of 0.01 mol/dm3and the immersion time of 15 minutes.

FIG. 20is a graph showing a relationship between the concentration of AgNO3and the content of silver on a surface of each sample.

FIG. 21is a graph showing a relationship between the concentration of IP6and the ratio of Ag/(Ca+Ag) of the surface of each sample.

Summarizing the results of the test (2), it was found that when the time of immersion in an aqueous AgNO3solution and the concentration of inositol phosphate were made to be constant, the content of silver increases as the concentration of AgNO3becomes higher. Thus, it is considered that it is possible to adjust the amount of silver ions to be added to the surface in the production of an antibacterial medical equipment by controlling the concentration of AgNO3.

However, it is known that the resultant antibacterial medical equipment exhibits cytotoxicity when the amount of silver is too large. It is important to control so that a proper amount of silver can be added to the antibacterial medical equipment.

(3) Influence of Variation of Time of Immersion in AgNO3Solution

FIG. 22is a graph showing X-ray diffraction results of a surface of each sample made by varying the immersion time under the conditions of the IP6concentration of 1,000 ppm and the AgNO3concentration of 0.01 mol/dm3.

FIG. 23is a SEM image of a surface of each sample made by varying the immersion time.

FIG. 24is a graph showing measurement results by EDX of elements contained in a surface of each sample made under the conditions of the IP6concentration of 1,000 ppm, the AgNO3concentration of 0.01 mol/dm3and the immersion time of 15 minutes.

FIG. 25is a graph showing examination results of elements contained in a surface of each sample made under the conditions of the IP6concentration of 1,000 ppm, the AgNO3concentration of 0.01 mol/dm3and the immersion time of 30 minutes.

FIG. 26is a graph showing examination results by EDX of elements contained in a surface of each sample made under the conditions of the IP6concentration of 1,000 ppm, the AgNO3concentration of 0.01 mol/dm3and the immersion time of 60 minutes.

FIG. 27is a graph showing a relationship between the immersion time in an aqueous AgNO3solution and the content of silver of a surface of each sample.

FIG. 28is a graph showing a relationship between immersion time in an aqueous AgNO3solution and the ratio of Ag/(Ca+Ag) of the surface of each sample.

Summarizing the results of the test (3), when the concentration of inositol phosphate and that of AgNO3were made to be constant and the immersion time was varied, the content of silver increased as the immersion times became longer. However, as is apparent from the results by EDX, when the immersion time was 60 minutes, the content of “calcium” was very low and that of silver was considerably high.

During the immersion time in the aqueous AgNO3solution, the content of silver increased, while that of calcium decreased.

The following facts have been found from a series of the above tests.

Regarding Form of Silver:

It could be confirmed that silver was contained when inositol phosphate was used. As is apparent from a graph of XRD, silver exists in the form of “silver phosphate”. It could also be confirmed from the results of EDX that silver and calcium coexist. Since particles having a characteristic squamous form were observed in HAp which was precipitated from SBF in SEM observation, it is considered that an original HAp layer was converted into a mixed layer of silver phosphate and HAp in the present process.

Regarding Content of Silver:

It has been found that the content of silver can be controlled by the concentration of AgNO3and the immersion time. When immersed in the aqueous AgNO3solution for a long time, or when immersed in the solution having a high concentration, the amount of calcium decreased. It has been found that since HAp is dissolved under the acidic condition, it must be immersed in the aqueous AgNO3solution in as short a time as possible.

In accordance with preferred production conditions obtained from the above-described results, samples for carrying out an antibacterial activity test were produced. These samples were produced using a pure titanium implant (0.5 mm in diameter and 8 mm in length) as a material so as to transplant to the thighbone of a mouse.

A pure titanium implant (0.5 mm in diameter and 8 mm in length) was used without being treated (hereinafter sometimes referred to as “Ti”).

The surface of the Ti was polished with a #240 abrasive paper, ultrasonic-washed in turn with pure water, ethanol and acetone twice for 5 minutes each, followed by air drying to obtain samples (hereinafter sometimes referred to as “Ti (acetone treatment)”).

A pure titanium implant was subjected to polishing, washing and drying in the same manner as in the sample No. 2, heated to 200° C. in air, followed by standing to cool to obtain samples (hereinafter sometimes referred to as “Ti (heat treatment)”).

A pure titanium implant was heated to 200° C. in air in the same manner as in the sample No. 3, immersed in a SBF solution of urea+urease as shown inFIG. 6, allowed to stand for 7 days while replacing with the fresh solution, thereby precipitating and coating HAp on a surface, washed with pure water and then air-dried to obtain samples (hereinafter sometimes referred to as “HAp-Ti”).

After coating HAp on a surface of a pure titanium implant in the same manner as in the sample No. 4, the coated pure titanium implant was immersed in an aqueous IP6solution (temperature 50° C.) having a concentration of 1,000 ppm for 1 day, taken out, washed with pure water and then air-dried to obtain samples (hereinafter sometimes referred to as “HAp-IP6-Ti”).

A pure titanium implant was immersed in an aqueous IP6solution in the same manner as in the case of the implant sample No. 5, immersed in an aqueous AgNO3solution having a concentration of 0.001 mol/dm3for 15 minutes thereby immobilizing silver ions, washed with pure water and then air-dried to obtain samples (hereinafter sometimes referred to as “HAp-IP6-Ag—Ti Ag 0.001M”).

A pure titanium implant was immersed in an aqueous IP6solution in the same manner as in the case of the implant sample No. 5, immersed in an aqueous AgNO3solution having a concentration of 0.005 mol/dm3for 15 minutes thereby immobilizing silver ions, washed with pure water and then air-dried to obtain samples (hereinafter sometimes referred to as “HAp-IP6-Ag—Ti Ag 0.005M”).

A pure titanium implant was immersed in an aqueous IP6solution in the same manner as in the case of the implant sample No. 5, immersed in an aqueous AgNO3solution having a concentration of 0.01 mol/dm3for 15 minutes thereby immobilizing silver ions, washed with pure water and then air-dried to obtain samples (hereinafter sometimes referred to as “HAp-IP6-Ag—Ti Ag 0.01M”).

(1) In Vitro Petri Dish Test

Samples Nos. 1 to 8 were radially placed on luciferase-expressingStaphylococcus aureusin a LB agar medium in a Petri dish at 37° C. for 24 hours, and then a bacterial growth state around each implant was examined.

FIG. 29is a phase-contrast image of the entire Petri dish with bacteria after placement of sample Nos. 1 to 8.FIG. 31shows phase-contrast images of bacterial growth around each of implants Nos. 1 to 8 in the Petri dish. As is apparent from these drawings, the non-growing sites (black) of bacteria were observed around implants only in samples Nos. 6 to 8 with immobilized silver ions, suggesting the strong antibacterial activity. It has also been detected that the sample No. 5, which has immobilized inositol phosphate and does not have immobilized Ag, has slight antibacterial activity.

FIG. 30is a bluc image obtained when high-sensitivity observation of trapping of light emitted from luciferase-expressingStaphylococcus aureusof the same entire Petri dish as inFIG. 29using an imaging system IVIS® (manufactured by Xenogen Co.).FIG. 32is bluc images around each of samples Nos. 1 to 8 of the Petri dish after culture. As is apparent from these drawings, the non-growing site (black) of bacteria is observed around the samples Nos. 6 to 8 with immobilized silver ions among samples Nos. 1 to 8 and the implants have strong antibacterial activity. It has also been recognized that the sample No. 5, which has immobilized inositol phosphate and does not have immobilized Ag, also has slight antibacterial activity.

With respect to the sample No. 7, assuming actual clinical application, the presence or absence of antibacterial activity was confirmed under various conditions. In actual clinical application, since the implant may be contacted with blood or abraded when inserted into the bone, some loss of Ag ion is anticipated.

Here, also with respect to the implant sample No. 7, the presence or absence of antibacterial activity was confirmed by conducting the Petri dish test with respect the implant subjected to washing and rubbing. As the sample, four kinds of samples, such as an implant sample No. 7 (IP6-Ag coating), a washed implant sample No. 7 (IP6-Ag coating washed), a rubbed implant sample No. 7 (IP6-Ag coating rubbed), and an implant sample No. 4 (HAp coating) for comparison.

FIG. 33shows results of a Petri dish test using the above four kinds of implants of interest, in which phase-contrast images around four kinds of implants are shown at the upper side, while bluc images are shown at the lower side.

As shown inFIG. 33, it is apparent that even when the implant sample No. 7 having strong antibacterial activity recognized is washed or rubbed, the non-growing site (black) of bacteria is observed around the implant and the antibacterial activity is sufficiently maintained. Therefore, it was confirmed that the implant sample No. 7 has durability and stability enough to withstand actual clinical application.

Using BALB/c male adult mice, an osteomyelitis model was made.

As the implant, an implant sample No. 4 (HAp-Ti; hereinafter abbreviated to HAp) and a sample No. 7 (HAp-IP6-Ag—Ti Ag 0.005M; hereinafter abbreviated to Ag+) among samples Nos. 1 to 8 were used, and these implants were implanted to the thighbone of a mouse.

Luciferase-expressingStaphylococcus aureuswas injected around the implanted implants. Using the above imaging system, high-sensitivity observation of trapping of light emitted from luciferase-expressingStaphylococcus aureusof this mouse was conducted for 28 days after implantation. The results are shown inFIG. 34. In addition, an inflammatory marker (Interleukin 6: IL-6 and C-reactive protein: CRP) in serum was measured with the lapse of time. The results are shown inFIG. 35. Furthermore, a histological assessment was also performed at 4 weeks after surgery. The results are shown inFIG. 36.

The graph at the upper side ofFIG. 34is a graph showing an average and a variation range of an emission intensity around the implanted site of a mouse implanted with either an implant HAp or Ag+ and also injected with the above bacteria (N=4 in both of HAp group and Ag+ group) measured on the 1st day, 3rd day, 7th day, 14th day, 21st day and 28th day after implantation using the above imaging system. The numerical value of the ordinate of the graph indicates the emission intensity (arbitrary intensity unit), while the numerical value of the abscissa indicates the number of days after implantation.

The images at the lower side ofFIG. 34are images showing a change with the lapse of time in the emission state of the bacteria implanted site of typical mice of both groups photographed on the 1st day, 3rd day, 7th day, 14th day, 21st day and 28th day using the above imaging system.

As is apparent from the results shown inFIG. 34, on the 1st day after implantation, in the HAp group, the emission intensity drastically increased and bacteria (luciferase-expressingStaphylococcus aureus) grew around the implant. In contrast, in the case of the Ag+ group, although the mission intensity increased, the emission intensity was statistically significantly lower than that of the HAp group.

On the 3rd day after implantation, in the case of mice of HAp group, a state of high emission intensity was maintained. In the case of mice of the Ag+ group, the emission intensity increased as compared with that measured on the 1st day, but was lower than that in the mouse of the HAp group.

On the 7th day after implantation, in the case of mice of the HAp group, the emission intensity increased as compared with that measured on the 3rd day. In contrast, in the case of the mouse of the Ag+ group, the emission intensity decreased as compared with that measured on the 3rd day. The emission intensity of the Ag+ group was statistically significantly low as compared with that in the HAp group.

On the 14th day after implantation, in both of the HAp group and the Ag+ group, the emission intensity decreased as compared with that measured on the 7th day due to natural immunity. The fact that bacteria die out by natural immunity is a phenomenon shown in a lot of past bacterial infection tests. In a comparison between both groups, the emission intensity measured on the 14th, 21st or 28th day in Ag+ group was lower than that in HAp group and decreased with the lapse of time. It is noteworthy that the emission intensity measured on the 28th day in the case of the Ag+ group was nearly identical to the background as shown in the image, in other words, bacteria died out almost completely.

As is apparent from the results shown inFIG. 34, the implant Ag+ of the present invention enabled inhibition of bacterial growth around the implant after implantation as compared with the implant HAp. The results proved that the implant according to the present invention exerts high antibacterial activity in vivo, and also exhibits strong antibacterial activity in the short term and can maintain antibacterial activity over a long period.

The graph ofFIG. 35is a graph showing an average and a variation range of Interleukin 6 (IL-6: left drawing) and C-reactive protein (CRP: right drawing) of a mouse implanted with either an implant HAp or Ag+ and also injected with the above bacteria (N=3 in both of HAp group and Ag+ group). Enzyme-linked immunosorbent assay (ELISA) method was used for measurement of the level of two inflammatory markers.

The numerical value of the ordinate of the graph indicates the concentration of each substance in serum, while the numerical value of the abscissa indicates the number of days after implantation.

As is apparent from the results shown inFIG. 35, in the acute stage on the 2nd day after implantation, the Ag+ group significantly exhibits a low increase in IL-6, compared with the HAp group, suggesting that the inflammation is remarkably suppressed in the Ag+ group. Also, the Ag+ group significantly exhibits a low increase in IL-6 even at each time point after implantation and approximately exhibits 0 pg/ml on the 7th day after implantation, which shows that inflammation is subsided. Although CRP levels increases in both groups until the 7th day after surgery since an influence of operation invasion is exerted, the Ag+ group significantly exhibits a low value on the 14th day after implantation, which shows that inflammation is subsided. These results are findings reflecting an image (FIG. 34) showing a change with the lapse of time, which is obtained by photographing the emission state of the bacteria implanted site of a mouse at each time after implantation using an imaging system. As is apparent from the above description, the Ag+ implant exhibits strong antibacterial activity not only in the acute stage after implantation, but also in the range from the sub-acute stage on the 14th day to the chronic stage.

FIG. 36shows Hematoxylin and eosin (HE) staining of histological sections from thighbones collected at 4 weeks after implantation in both groups (HAp and Ag+ groups). In the HAp group, subperiosteal reactive bone peculiar to infection, so-called sequestration is remarkably observed and a normal bone structure is fractured, and inside the medullary cavity is filled with inflammatory cell (left drawing: HAp). In contrast, in the Ag+ group, surprisingly, an almost normal bone structure is exhibited and also inside the medullary cavity is filled with a lot of normal myeloid cells. As is apparent from the above results, the Ag+ implant exhibits strong antibacterial activity after implantation thereby to kill bacteria and to subside infection, thus remarkably suppressing an osteoclastic change due to infection.

Similar to the [In vivo antibacterial activity test-1], an osteomyelitis model was made using BALE/c male adult mice.

As the implant, the sample No. 4 (HAp) and the sample No. 5 (HAp-IP6-Ti; hereinafter abbreviated to IP6) among the above samples Nos. 1 to 8 were used, and these implants were implanted to the thighbone of mice.

Luciferase-expressingStaphylococcus aureuswas injected around the implants. Using the above imaging system, high-sensitivity observation of trapping of light emitted from luciferase-expressingStaphylococcus aureusof this mouse was conducted until the 1st to 28th day after implantation. The results are shown inFIG. 37.

The graph at the upper side ofFIG. 37is a graph showing an average and a variation range of an emission intensity around the implanted site of the mouse implanted with either an implant HAp or IP6and also injected with the above bacteria (N=6 in both of HAp group and IP6group) measured on the 1st day, 3rd day, 7th day, 14th day, 21st day and 28th day after implantation using the above imaging system. The numerical value of the ordinate of the graph indicates the emission intensity (arbitrary intensity unit), while the numerical value of the abscissa indicates the number of days after implantation.

The images at the lower side ofFIG. 37show a change with the lapse of time in the emission state of the bacteria-implanted site of typical mice of both groups photographed on the 1st day, 3rd day, 7th day, 14th day, 21st day and 28th day using the above imaging system.

As is apparent from the results shown inFIG. 37, on the 1st day after implantation, in the HAp group, the emission intensity drastically increased and bacteria (luciferase-expressingStaphylococcus aureus) grew around the implant. In IP6group, although the emission intensity increased, the emission intensity was slightly lower than that of the HAp group.

On the 3rd day after implantation, in the case of mice of HAp group, the emission intensity continuously increased. In the IP6group, the emission intensity increased but was lower than that in the HAp group.

At 7th day after implantation, in the HAp group, the emission intensity decreased as compared with that observed on the 3rd day but was still in a high level. In contrast, in the IP6group, the emission intensity drastically decreased as compared with that observed on the 3rd day. The emission intensity of the IP6group was statistically significantly low as compared with that in the HAp group.

On the 14th day to 21st day after implantation, although the emission intensity of both groups tended to decrease, the emission intensity of the mouse of the IP6group was still lower than that in the case of the mouse of the HAp group. At the times of 21st and 28th day after implantation, there was no significant change in the emission intensity of both groups, and the emission intensity of the IP6group was still lower than that in the case of the HAp group. It is noteworthy that the emission intensity measured on the 21st day or the subsequent day in the IP6group was nearly identical to the background as shown in the image, in other words, bacteria died out almost completely.

As is apparent from the results shown inFIG. 37, the implant IP6of the present invention had antibacterial activity similar to the implant Ag+ and enabled inhibition of bacterial growth around the implant after implantation as compared with the implant HAp. The results proved that the implant according to the present invention exerts high antibacterial activity in vivo, and also exhibits strong antibacterial activity over a long period.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to provide an antibacterial medical equipment which has sufficient antibacterial activity in vivo and is excellent in compatibility with living tissues, and also can maintain antibacterial activity over a long period and has high safety.