阅读说明：本技术 含有经表面处理的芳香族聚醚酮的植入材料及其制造方法 (Implant material containing surface-treated aromatic polyether ketone and method for producing same ) 是由 冲原巧 高田善机 斋藤直人 青木薰 西村直之 羽二生久夫 森山茂章 上田胜也 于 2020-03-27 设计创作，主要内容包括：本发明的目的在于,通过不需要昂贵的制造装置的方法提供具有比含有芳香族聚醚酮的植入材料更优秀的骨传导性的植入材料。本发明涉及上述方法及通过上述方法得到的植入材料,上述方法包括：在不存在钙离子的情况下在强碱溶液中浸渍处理芳香族聚醚酮,在含有含磷化合物的溶液中浸渍处理通过该浸渍处理得到的芳香族聚醚酮的步骤。(The purpose of the present invention is to provide an implant material having excellent bone conductivity compared to an implant material containing an aromatic polyether ketone by a method that does not require an expensive manufacturing apparatus. The present invention relates to the above method and an implant material obtained by the above method, the above method comprising: and a step of immersing the aromatic polyether ketone in a strong alkaline solution in the absence of calcium ions, and immersing the aromatic polyether ketone obtained by the immersion treatment in a solution containing a phosphorus-containing compound.)

1. A method for producing an implant material comprising a surface-treated aromatic polyether ketone, comprising:

the surface treatment is a step of immersing the aromatic polyether ketone in a strong alkaline solution in the absence of calcium ions, and immersing the aromatic polyether ketone obtained by the immersion treatment in a solution containing a phosphorus-containing compound.

2. The method of claim 1, comprising the step of adsorbing calcium ions on the surface treated aromatic polyether ketone in the absence of calcium ions.

3. The method according to claim 1 or 2, wherein the aromatic polyether ketone is polyether ether ketone or polyether ketone.

4. The method according to any one of claims 1 to 3, wherein the strong alkaline solution is selected from the group consisting of lithium hydroxide solution, sodium hydroxide solution, potassium hydroxide solution, rubidium hydroxide solution, cesium hydroxide solution, tetraalkylammonium hydroxide solution, calcium hydroxide solution, strontium hydroxide solution, barium hydroxide solution, europium hydroxide solution, and thallium hydroxide solution.

5. The method according to any one of claims 1 to 4, wherein the strong alkaline solution is a sodium hydroxide solution of 3N or more.

6. The method according to any one of claims 1 to 5, wherein the solution containing the phosphorus-containing compound is selected from the group consisting of phosphorous acid diester chlorides including phosphorus oxychloride solution, phosphorus trichloride solution, phosphorus oxybromide solution, phosphorus tribromide solution, polyphosphoric acid solution, phosphorus pentachloride solution, pyrophosphoric acid solution, dimethyl chlorophosphite, diethyl chlorophosphite, and propyl chlorophosphite.

7. The method of any one of claims 1-6, wherein the surface treatment does not include a step of plasma treating the aromatic polyetherketone.

8. An implant material comprising surface-treated aromatic polyether ketone, characterized by being produced by the method of any one of claims 1 to 7.

9. The implant material of claim 8, wherein the surface-treated aromatic polyether ketone has 0.11 weight percent or more of phosphorus atoms in the surface component.

10. An implant material characterized by having a surface component containing 0.11 weight% or more of phosphorus atoms and containing an aromatic polyether ketone having a phosphate group formed on the surface.

11. The implant material of claim 10, wherein calcium ions are adsorbed on the surface on which the phosphate group is formed.

12. The implant material according to claim 10 or 11, wherein the surface of the aromatic polyether ketone having the phosphate group formed thereon is not subjected to plasma treatment.

Technical Field

The present invention relates to an implant material containing aromatic polyether ketone used for a vertebral body fusion cage for spinal treatment and the like, and a method for producing the same.

Background

Implants for cervical spine treatment, such as vertebral spacers, are typically made of titanium alloys. The titanium alloy has high biocompatibility and high osteoconductivity. However, since metal is used, it not only has high rigidity to bones but also may damage bones. Therefore, care is needed for elderly people with weak bones. Further, magnetic properties cause halation in image diagnosis such as MRI, and are considered to be one of the disturbing factors affecting accurate image diagnosis.

Vertebral spacers using Polyetheretherketone (PEEK), one of the super engineering plastics, have been developed in recent years. It is well known that polyetheretherketone is chemically very stable and has a rigidity that is very close to that of human bone, and the likelihood of bone destruction is lower than for the titanium vertebral body spacers described above.

On the other hand, however, bone conductivity is lower than that of titanium, which is said to be a disadvantage of a vertebral body spacer made of a titanium alloy.

To date, various attempts have been made to improve osteoconductivity with respect to these.

For example, there have been reported a method of depositing a metal ion plasma on a substrate to attach cells such as osteoblasts (for example, patent document 1), a method of mixing with a bioactive fine-particle ceramic containing hydroxyapatite (for example, patent document 2), a method of forming a metal oxide adhesive layer on a polymer surface (for example, patent document 3), and the like.

Further, as for the spinal fusion cage, there has been reported a spinal fusion cage in which an integrated osteoconductive member is filled in a long axis portion penetrating and extending a bone contact surface on both sides of a structural member made of polyetheretherketone or the like (for example, patent document 4).

Further, there have been reported a method of supplying a soluble calcium ion supply source and a soluble phosphate ion supply source to a base material at a predetermined concentration (patent document 6), and a method of producing a body implant in which surface foam is immersed in both a solution containing calcium ions and a solution containing phosphate ions (patent document 7), but the methods of these patent documents 6 and 7 are methods of depositing and applying calcium phosphate or the like on the surface of a base material, and there is no chemical bond between the base material and a phosphorus atom.

Even by these methods, a substance having the same osteoconductivity as titanium cannot be obtained.

Further, a method of using sodium hydroxide or fluorine gas when a hydrophilic functional group such as a carboxyl group is added to the surface of polyetheretherketone has been proposed (patent document 5), but since this production method uses a dangerous fluorine gas, a large amount of equipment investment is required for the corrosion resistance and safety of the production apparatus.

Further, although a method of introducing a phosphoric group into a surface of polyetheretherketone is provided (non-patent document 1), a special technique using plasma or the like is required, and a large amount of equipment investment is required.

Documents of the prior art

Patent document

Patent document 1: japanese patent application laid-open No. 2009-

Patent document 2: japanese unexamined patent publication No. 2010-246934

Patent document 3: japanese patent publication No. 2011-500216

Patent document 4: japanese Kokai publication No. 2007-512874

Patent document 5: japanese laid-open patent publication No. 2016-209197

Patent document 6: WO2009/095960

Patent document 7: japanese unexamined patent publication No. Hei 4-224747

Non-patent document 1: colloid and surface (Colloids and Surfaces) B: biological interfaces (Biointerfaces), 197, 36-42, 2019; futian zhi, guxiang, Sunarso (sunaso), well-field power of the family, wide zhi, senyuexiu, shi chuanfu; surface plasma treatment and phosphorylation to enhance the biological Performance of polyetheretherketone (ether ketone)

Disclosure of Invention

Technical problem

The present inventors have recognized that: in order to safely and inexpensively provide an implant material having bone conductivity comparable to that of titanium without causing problems of high rigidity, bone destruction, and halo when titanium is used, it is necessary to develop a new surface treatment technique for a material such as polyetheretherketone. Accordingly, an object of the present invention is to provide an implant material that can be used practically by surface-treating a material such as polyetheretherketone safely and inexpensively.

In order to solve the above problems, the present inventors found that: the present inventors have further studied and found that an implant material having sufficient osteoconductivity can be obtained by immersing an aromatic polyether ketone such as polyether ether ketone in a strongly basic solution such as sodium hydroxide and immersing the aromatic polyether ketone obtained by the immersion treatment in a solution containing a phosphorus compound such as a phosphorus oxychloride solution.

Means for solving the problems

Accordingly, the present invention relates to the following.

[1] A method of manufacturing an implant material comprising a surface-treated aromatic polyether ketone, comprising: the surface treatment is a step of immersing the aromatic polyether ketone in a strong alkaline solution in the absence of calcium ions, and immersing the aromatic polyether ketone obtained by the immersion treatment in a solution containing a phosphorus-containing compound.

[2] The method according to the above [1], comprising a step of adsorbing calcium ions on the surface-treated aromatic polyether ketone in the absence of calcium ions.

[3] The method according to the above [1] or [2], wherein the aromatic polyether ketone is poly-ether-ketone or polyether ketone.

[4] The method according to any one of the above [1] to [3], wherein the strong alkaline solution is selected from the group consisting of a lithium hydroxide solution, a sodium hydroxide solution, a potassium hydroxide solution, a rubidium hydroxide solution, a cesium hydroxide solution, a tetraalkylammonium hydroxide solution, a calcium hydroxide solution, a strontium hydroxide solution, a barium hydroxide solution, a europium hydroxide solution, and a thallium hydroxide solution.

[5] The process according to any one of the above [1] to [4], wherein the alkali solution is a sodium hydroxide solution of 3N or more.

[6] The method according to any one of the above [1] to [5], wherein the solution containing a phosphorus-containing compound is selected from the group consisting of phosphorus oxychloride solution, phosphorus trichloride solution, phosphorus oxybromide solution, phosphorus tribromide solution, polyphosphoric acid solution, phosphorus pentachloride solution, pyrophosphoric acid solution, dimethyl chlorophosphite, diethyl chlorophosphite, propyl chlorophosphite and other phosphite diester chlorides.

[7] The method according to any one of the above [1] to [6], wherein the surface treatment does not include a step of subjecting the aromatic polyether ketone to plasma treatment.

[8] An implant material comprising surface-treated aromatic polyether ketone produced by the method of any one of the above [1] to [7 ].

[9] The implant material according to the above [8], wherein the surface-treated aromatic polyether ketone has 0.11 weight% or more of phosphorus atoms in the surface component.

[10] An implant material having a surface component containing 0.11 weight% or more of phosphorus atoms and a surface containing aromatic polyether ketone having a phosphate group formed thereon.

[11] The implant material according to the above [10], wherein calcium ions are adsorbed on the surface on which phosphate groups are formed.

[12] The implant material according to the above [10] or [11], wherein the surface of the aromatic polyether ketone having the phosphoric acid group formed thereon is not subjected to plasma treatment.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention can provide an implant material having excellent bone conductivity as compared with aromatic polyether ketone without surface treatment and having bone conductivity comparable to titanium. In addition, in the titanium implant at the time of follow-up observation using post-operation MRI, a problem of halo occurs and observation is difficult, but in the implant material of the present invention, post-operation follow-up observation can be performed together with accurate image diagnosis without causing such a problem. Further, the present invention can provide an implant material for cervical vertebra treatment or an implant material for dental treatment, which has a wide range of applications as a vertebral spacer or the like, because the implant material can be manufactured by a simple method using an inexpensive material without using an expensive manufacturing apparatus or manufacturing equipment such as a fluorine gas apparatus.

Also, since the method of the present invention can manufacture an implant material without going through a step of immersion into calcium ions, the implant material can be provided extremely easily. In addition, since the method of the present invention can manufacture an implant material by a method that does not include plasma treatment, it is superior in terms of cost and ease of manufacture, because plasma treatment equipment is not required and the number of steps is reduced. Also, the method including plasma treatment causes large surface damage, whereas the method of the present invention uses alkali such as sodium hydroxide, etc., which minimizes surface damage compared to plasma treatment. The implant material obtained by the manufacturing method of the present invention shows high antibacterial activity and can be used with less risk of infection. Also, the implant material obtained by the manufacturing method of the present invention can endure more severe movement since it shows higher bone contact rate and higher maximum punching stress from the bone when it is actually used in vivo.

Drawings

Fig. 1 is a graph showing a comparison of relative proliferation rates of osteoblast-like cells in the case of using an untreated polyetheretherketone material and in the case of using an implant material obtained by the manufacturing method of the present invention.

Figure 2 shows product 1 of the invention and a rabbit femoral implant insertion test of polyetheretherketone.

Figure 3 shows the histopathological examination results of product 1 of the invention and of polyetheretherketone after 4 weeks of intercalation.

Figure 4 shows the histopathological examination results of product 1 of the invention and of polyetheretherketone after 4 weeks of intercalation.

Figure 5 shows the histopathological examination results of product 1 of the invention and polyetheretherketone insertion 4 weeks later.

Figure 6 shows the histopathological examination results of product 1 of the invention and polyetheretherketone insertion 4 weeks later.

Figure 7 shows the bone exposure of product 1 of the invention and polyetheretherketone from a rabbit femoral implant insertion test.

Figure 8 shows the punch test through a mechanical tester after 4 weeks of embedding of product 1 of the invention and polyetheretherketone.

Figure 9 shows the punch test through a mechanical tester after 4, 8 and 12 weeks of embedment of product 1 of the invention and polyetheretherketone.

Detailed Description

The method of manufacturing an implant material containing surface-treated aromatic polyether ketone of the present invention comprises: and a step of immersing the aromatic polyether ketone in a strong alkaline solution and immersing the aromatic polyether ketone obtained by the immersion treatment in a solution containing a phosphorus-containing compound.

The aromatic polyether ketone usable in the present invention is not particularly limited as long as the benzene ring has a linear polymer structure in which ether and ketone are bonded. For example, Polyether Ether Ketone (PEK), poly-ether-ketone (PEK), or the like can be used, but polyether ether ketone is preferably used from the viewpoint of practical performance in medical use or the like.

In the present invention, the shape of the aromatic polyether ketone is not particularly limited as long as it can be used as an implant material. For example, machining may be performed by a compound lathe, a machining center, or the like.

In the production method of the present invention, the surface treatment may be performed in the absence of calcium ions.

In the manufacturing method of the present invention, the surface treatment may not include the step of plasma treatment.

The strong alkaline solution used in the present invention is not particularly limited as long as it is a solution having a basicity capable of introducing a functional group capable of substituting a substituent having a phosphorus atom such as a phosphoric group into the impregnated aromatic polyether ketone. Such functional groups include, for example, hydroxyl, alkoxy, siloxy, and the like. Also, in one aspect, the strong alkaline solution used in the present invention does not contain calcium ions.

Specific strong alkaline solutions include, for example, aqueous sodium hydroxide solution, aqueous lithium hydroxide solution, aqueous potassium hydroxide solution, aqueous rubidium hydroxide solution, aqueous cesium hydroxide solution, aqueous tetraalkylammonium hydroxide solution, aqueous calcium hydroxide solution, aqueous strontium hydroxide solution, aqueous barium hydroxide solution, aqueous europium hydroxide solution, aqueous thallium hydroxide solution, and the like. Hydroxyl groups can be introduced into the aromatic polyether by using these aqueous solutions. The concentration of the strong alkali solution is not particularly limited as long as the functional group can be introduced, and is preferably 3N or more, and more preferably 5N or more. The strong alkaline solution is a sodium hydroxide solution having a concentration of preferably 3N or more, more preferably 5N or more.

Also, the alkali solution may be a solution of sodium alkoxide such as sodium methoxide, sodium ethoxide, sodium butoxide and the like, lithium alkoxide such as lithium methoxide, lithium ethoxide, lithium butoxide and the like, potassium alkoxide such as potassium methoxide, potassium ethoxide, potassium butoxide and the like, or a solution of sodium alkylsilanol such as sodium trimethylsilanolate, sodium triethylsilanolate, sodium tripropylsilanolate and the like, lithium alkylsilanol such as lithium trimethylsilanolate, lithium triethylsilanolate, lithium tripropylsilanolate and the like. Solvents that can be used for the above solution of alkoxide or alkylsilane alkoxide include alcohols such as methanol, ethanol, propanol, butanol, THF, dichloromethane, chloroform, DMF, DMSO, acetonitrile, water, any combination thereof, and the like. In the case of using these strong alkali solutions, an alkoxy group or a siloxy group may be introduced into the aromatic polyether.

The functional group introduced as described above, such as a hydroxyl group, an alkoxy group, a siloxy group, or the like, may be used as the functional group which may be substituted with a substituent having a phosphorus atom. For example, a substituent having a phosphorus atom such as a phosphate group can be substituted by directly acting on a solution containing a phosphorus-containing compound after introduction of a hydroxyl group. Further, the alkoxy group or the siloxy group may be substituted with a substituent having a phosphorus atom such as a phosphoric group by acting on the solution containing a phosphorus-containing compound after being converted into a hydroxyl group. The alkoxy group or siloxy group can be converted into a hydroxyl group by treatment with a solution of a fluoride reagent such as aqueous sodium hydroxide solution, aqueous lithium hydroxide solution, aqueous potassium hydroxide solution, aqueous rubidium hydroxide solution, aqueous cesium hydroxide solution, aqueous tetraalkylammonium hydroxide solution, aqueous calcium hydroxide solution, aqueous strontium hydroxide solution, aqueous barium hydroxide solution, aqueous europium hydroxide solution, aqueous thallium hydroxide solution, hydrochloric acid, sulfuric acid, nitric acid, etc., tetrabutylammonium fluoride, sodium fluoride, potassium fluoride, rubidium fluoride, cesium fluoride, etc., in water, alcohol, THF, etc.

The strong alkali solution may be used alone or in combination of two or more kinds as long as a functional group capable of substituting a substituent having a phosphorus atom such as a phosphoric group can be introduced into the aromatic polyether ether ketone. Preferably, an aqueous sodium hydroxide solution, an aqueous lithium hydroxide solution, an aqueous potassium hydroxide solution are used alone, and more preferably, an aqueous sodium hydroxide solution is used alone.

The solution containing a phosphorus-containing compound used in the method for producing an implant material of the present invention is not particularly limited as long as it can introduce a phosphorus atom into aromatic polyether ketone. Also, in one aspect, the solution containing a phosphorus-containing compound used in the present invention does not contain calcium ions.

The solution containing a phosphorus-containing compound used in the present invention may also contain a phosphorus-containing compound selected from the group consisting of phosphorous acid diester chlorides such as a phosphorus oxychloride solution, a phosphorus trichloride solution, a phosphorus oxybromide solution, a phosphorus tribromide solution, a polyphosphoric acid solution, a phosphorus pentachloride solution, a pyrophosphoric acid solution, dimethyl chlorophosphite, diethyl chlorophosphite, propyl chlorophosphite, and the like. When the phosphorus-containing compound is a liquid under the conditions of the dipping treatment, the solution containing the phosphorus-containing compound of the present invention may be a stock solution of the phosphorus-containing compound.

The substituent having a phosphorus atom to be introduced into the aromatic polyether ketone may be, for example, phosphoric acid, phosphorous acid, phosphonic acid, phosphonous acid, phosphate, phosphite, phosphonate, phosphinate, or the like. The introduced phosphorous acid, phosphonic acid, phosphonous acid, phosphate, phosphite, phosphonate, phosphinate, etc. can also be optionally converted into phosphoric acid, for example, in air or in aqueous solution.

The method of manufacturing an implant material of the present invention may also include a post-treatment to remove impurities in a solution containing a phosphorus-containing compound such as phosphorus oxychloride attached to the implant material after the impregnation treatment of the phosphorus oxychloride solution. Also, the method of manufacturing an implant material of the present invention may also include a post-treatment for removing impurities from the supply agent, simultaneously with or after the post-treatment, for adsorbing calcium ions.

The solution used in the above post-treatment is not particularly limited as long as impurities can be removed. For example, the reaction is carried out by a sodium hydroxide solution, a lithium hydroxide solution, a potassium hydroxide solution, or a calcium hydroxide solution. From the viewpoint of adsorbing calcium ions while performing the post-treatment, a solution containing calcium ions can be selected as the solution used in the post-treatment.

The temperature in the above-mentioned post-treatment is not particularly limited as long as impurities can be removed. At 20 ℃ to 80 ℃, preferably 40 ℃ to 80 ℃, more preferably 60 ℃ to 80 ℃.

The post-treatment time is not particularly limited. For example, the reaction is carried out for 1 to 24 hours, preferably 6 to 24 hours, and more preferably 12 to 24 hours.

The method of the present invention for manufacturing an implant material may also include a cleaning step. The solution for such washing is not particularly limited as long as it can be used for medical treatment. For example, by pure water.

The cleaning temperature is not particularly limited as long as the implant material is sufficiently cleaned. At 20 ℃ to 80 ℃, preferably 40 ℃ to 80 ℃, more preferably 60 ℃ to 80 ℃. The above washing may be performed a plurality of times.

The temperature of the dipping treatment in the strong alkali solution of the present invention is not particularly limited as long as a functional group such as a hydroxyl group can be introduced. For example, at 20 ℃ to 80 ℃, preferably, 40 ℃ to 80 ℃, more preferably, 40 ℃ to 60 ℃.

The temperature of the dipping treatment in the solution containing the phosphorus-containing compound of the present invention is not particularly limited as long as the substituent having a phosphorus atom can be introduced. For example, at 0 ℃ to 40 ℃, preferably 10 ℃ to 30 ℃, more preferably 10 ℃ to 20 ℃.

In the method for producing an implant material of the present invention, the time of the immersion treatment in the strong alkali solution is not particularly limited as long as a functional group such as a hydroxyl group can be introduced. For example, the reaction is carried out for 1 to 24 hours, preferably 6 to 24 hours, and more preferably 12 to 24 hours.

In the method for producing an implant material of the present invention, the time of the immersion treatment in the solution containing the phosphorus-containing compound is not particularly limited as long as the substituent having a phosphorus atom can be introduced. For example, the reaction is carried out for 1 to 6 hours, preferably 1 to 4 hours, and more preferably 1 to 2 hours.

In the method for producing an implant material of the present invention, optionally, before the immersion treatment in the solution containing the phosphorus-containing compound, the moisture existing on the surface of the aromatic polyether ether ketone after the immersion treatment in the strong alkali solution may also be removed. The method of removing moisture is not particularly limited, but includes, for example, wiping, air drying, drying under reduced pressure, drying by heating, and any combination thereof.

The implant material obtained by the method of manufacturing an implant material of the present invention has phosphorus atoms, preferably, 0.11 weight percent or more, more preferably, 0.41 weight percent or more in the surface component.

The phosphorus atom contained in the material obtained by the production method of the present invention may be derived from phosphoric acid or a phosphoric acid salt.

The material obtained by the production method of the present invention may contain, for example, a chlorine atom or a sodium atom in addition to the phosphorus atom.

The material obtained by the production method of the present invention is not particularly limited as long as it can be used for medical treatment, and an alkali metal can be adsorbed on the surface. For example, among lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), and francium (Fr), sodium or calcium is preferably adsorbed, and calcium is more preferably adsorbed.

The adsorption of these alkali metals may be performed before the surface treatment, simultaneously with the surface treatment, or after the surface treatment. For example, after the surface treatment, an alkali metal may be adsorbed on the surface on which the phosphate group is formed. Therefore, in the implant material of the present invention, on the one hand, calcium ions are adsorbed on the surface on which the phosphate group is formed.

The alkali metal can be adsorbed by, for example, immersing the substrate in a solution containing the adsorbed alkali metal. For example, when sodium is adsorbed as an alkali metal, the base material may be immersed in a solution of a sodium hydroxide aqueous solution or the like having a predetermined concentration for a predetermined time. For example, when calcium is adsorbed as an alkali metal, the base material may be immersed in a solution such as an aqueous calcium hydroxide solution having a predetermined concentration for a predetermined time.

In the implant material of the present invention, the surface of the aromatic polyether ketone having a phosphate group formed thereon does not need to be treated with plasma.

The implant material of the present invention can also be used in cervical or dental applications. Also, osteoblasts may be adhered prior to application in vivo.

Examples

1. Raw material

Polyether-ether-ketone material

As the polyetheretherketone material used, an implant grade i2P manufactured by celluloid-winning (daicel-evonik) was used. The material was machined into test pieces having a diameter of 14mm and a thickness of 2 mm.

Surface treatment material

Sodium hydroxide: special grade reagent manufactured by Nacalai tesque

Phosphorus oxychloride: manufactured by Nakalettsk corporation

2. Examples of the respective tests

Test example 1

Step 1: adjustment of aqueous sodium hydroxide solution: the aqueous sodium hydroxide solution was adjusted to 5N. The test piece was immersed in the solution for 24 hours.

Step 2: the test piece obtained in step 1 was removed, the surface was wiped with water, and the test piece was immersed in a phosphorus oxychloride solution (stock solution) for 24 hours.

And step 3: adjustment of aqueous sodium hydroxide solution: the aqueous sodium hydroxide solution was adjusted to 1N. The test piece was immersed in the solution for 24 hours. Thereafter, the substrate was washed with pure water.

The steps 1 to 3 are all carried out at room temperature (20 ℃).

And (3) carrying out the treatment of the steps 1-3 to obtain the polyether-ether-ketone material (product 1) subjected to surface phosphorylation treatment.

Test example 2

The surface-phosphorylated polyetheretherketone material (product 2) was obtained in the same manner as in the test example except that the aqueous sodium hydroxide solution was adjusted to 3N in step 1.

Test example 3

The surface-phosphorylated polyetheretherketone material (product 3) was obtained in the same manner as in test example 1 except that the aqueous sodium hydroxide solution was adjusted to 1N in step 1.

3. Engineering and biological assessment

3-1 engineering evaluation

The following analysis was performed to confirm the presence of phosphorus atoms on the surface.

The presence of phosphate groups on the surface was confirmed by electron microscopy (VE-9800 EDX manufactured by KEYENCE: EDAX-GENESIS manufactured by AMETEK). The analysis conditions were as follows: acceleration voltage of 8 kV/amplification of 100 times

The results are shown in Table 1.

TABLE 1 quantification of phosphate groups

In products 1 and 2, a sufficient amount of phosphorus component was confirmed. On the other hand, in product 3, although it was a small amount of 0.03 weight percent, it was confirmed that a phosphorus component was present. It was confirmed that phosphorus atoms were introduced into polyetheretherketone in all the products, and it was presumed that a phosphate group was introduced. In table 1, it is considered that the reason why the total of the atoms is less than 100% is the presence of impurities.

3-2 biological evaluation

3-2-1. relative assessment of cell proliferation Rate

Relative assessment of cell proliferation rates was performed using the assessment method shown below (MC3T3-E1 cell proliferation promotion assay).

Cell: MC3T3-E1 (model: RCB1126, batch No. 64, institute of physico-chemical research)

Culture medium: alpha-minimum essential medium (. alpha. -minimum essential medium, type (Lot): 21444-05, Lot: L8H5662, Nacalatesk) 10 fetal bovine serum (type: S1820-500, Lot: S1820-500, Biowest) was prepared, and additionally a Penicillin-Streptomycin-Amphotericin B Suspension (penicilin-Streptomycin-Amphotericin B Suspension) (. times.100) (type: 161-23181, Lot: APG7006, and light (Wako)) was added at 1/100.

Reagent: dulbecco's phosphate buffered saline ((model: 045) -29795, lot: ECR7015, and light), alamarBlue (registered trademark) cell viability reagent (model BUF012B, lot: 146809, berle)

< evaluation step >

1) Each gamma sterilized product was brought into close contact with the bottom of a well of a 24-well plate (well assay plate). And, the wells of the product were used as a control group.

2) A cell suspension of MC3T3-E1 cells prepared at 4.0X 104 cells/well in culture medium was added to the wells and cultured in a 5% CO2 incubator at 37 ℃ for 1 day.

3) After 1 day of culture, the medium was aspirated, and alamarBlue (registered trademark) cell viability reagent (cell viability reagent) diluted to 10% with Dulbecco's phosphate buffered saline (-) was added to each well for 1 hour of reaction. In addition, a reagent blank near the addition of the reaction solution was provided in the wells to which no cells were seeded.

4) After 1 hour, 100. mu.l of the reaction solution in each well was transferred to a 96-well assay Plate, and the resultant was analyzed by a microplate Reader (Plate Reader) AF2200 (SEQ ID NO: 1307005705, Eppendorf) at 535/590 nm. Cell proliferation was calculated using cell survival (%) of 100 × (sample value-reagent blank value)/(control value-reagent blank value).

5) The reaction solution in the 24 wells was aspirated, replaced with a fresh medium, and cultured in a 5% CO2 incubator at 37 ℃ for 3 days again for evaluation in the same manner as in 3) to 4) above.

Fig. 1 shows a comparison of relative increment rates.

As can be seen from the figure, the increment rate of MC3T3-E1 was greatly increased compared to that of untreated.

Therefore, it was found that the product of the present invention has a sufficient osteoblast proliferation promoting effect as an implant material and has sufficient osteocyte conductivity.

3-2-2. antibacterial test

The antibacterial activity value of staphylococcus aureus NBRC (s. aureus NBRC) between product 1 of the present invention and polyetheretherketone was measured according to JIS Z2801 as follows.

After the product of the invention is put into a sterile culture dish, a test bacterium solution containing a strain staphylococcus aureus NBRC is dripped, and the culture dish is covered. The dishes were then incubated for 24 hours. Next, agar medium was washed out from the product of the present invention using the SCDLP medium, and after incubating the SCDLP medium on the agar medium for 48 hours, the bacteria were recovered and the number of viable cells was measured.

After the polyetheretherketone was put into a sterile petri dish, a test bacteria solution containing the strain staphylococcus aureus NBRC was added dropwise, and the petri dish was covered. The dishes were then incubated for 24 hours. Next, agar medium was washed out from the product of the present invention using the SCDLP medium, and after incubating the SCDLP medium on the agar medium for 48 hours, the bacteria were recovered and the number of viable cells was measured.

As a control, in placing the raw product in a sterile petri dish, a test bacteria solution containing the strain Staphylococcus aureus NBRC was added dropwise and the petri dish was covered. The dishes were then incubated for 24 hours. Next, agar medium was washed out from the product of the present invention using the SCDLP medium, and after culturing the SCDLP medium on the agar medium for 48 hours, the bacteria were recovered and the number of viable cells was measured.

Similarly, the antibacterial or properties of e.coli NBRC 3972 between product 1 of the present invention and polyetheretherketone were measured as follows.

The antibacterial activity value of staphylococcus aureus NBRC between the product of the invention and polyetheretherketone was measured as follows.

After placing the product of the invention in a sterile petri dish, a test bacteria solution containing the strain e. The dishes were then incubated for 24 hours. Next, agar medium was washed out from the product of the present invention using the SCDLP medium, and after culturing the SCDLP medium on the agar medium for 48 hours, the bacteria were recovered and the number of viable cells was measured.

After the polyetheretherketone was placed in a sterile petri dish, the test bacteria solution containing strain e.coli NBRC 3972 was added dropwise and the dish was covered. Then, the petri dish was cultured for 24 hours, then agar medium was washed out from the product of the present invention using the SCDLP medium, and after culturing the SCDLP medium on the agar medium for 48 hours, the bacteria were recovered, and the number of viable cells was measured.

As a control, after placing the raw product in a sterile petri dish, a test bacteria solution containing strain e.coli NBRC 3972 was added dropwise and the petri dish was covered. The dishes were then incubated for 24 hours. Next, agar medium was washed out from the product of the present invention with the SCDLP medium, and after culturing the SCDLP medium on the agar medium for 48 hours, the bacteria were recovered and the number of viable cells was measured. The antibacterial activity values of e.coli NBRC 3972 and staphylococcus aureus NBRC between the product of the present invention and polyetheretherketone were calculated by the following formula and are shown in table 2.

TABLE 2

	Product 1	Polyether ether ketone
			Staphylococcus aureus NBRC	1.9	0
E.coli	1.2	-1.4

As can be seen from the values in table 2, the product of the present invention showed higher antibacterial activity value than polyetheretherketone against both e.coli NBRC 3972 and staphylococcus aureus NBRC.

3-2-3. Rabbit femoral implant insertion test

Rabbit femoral implant insertion test was performed using product 1 of the present invention. The thigh to the calf of a Japanese white rabbit (Japanese SLC Co., Ltd., male, body weight 2.5-3.5 kg) anesthetized by isoflurane inhalation was shaved with a hair clipper (bariquant). A longitudinal incision was made with a scalpel on the skin from the knee to the lateral thigh, with the lateral vitreous muscle spacing to the lateral femoral part. Next, a bone hole was prepared from the outside of the condyle of the femur using a drill having a diameter of 5 mm. The inside of the bone hole was washed with physiological saline to form a groove in the product of the present invention, and a columnar form of 5mm in diameter × 15mm in height was inserted as an implant. The operation is completed by suturing the femoral fascia and the skin.

Subsequently, 4, 8, 12 weeks after intercalation, rabbits were euthanized by 1% lidocaine injection by otic intravenous injection. The skin, muscle and ligament from the thigh to the shank are exposed out of the bone, the proximal bone cadre of the femur is cut by a saw, and the femur is collected. The collected femurs were immersed in a 10% formalin solution and fixed.

In formalin-fixed rabbit femurs, only the condyles containing the implant are cut from the diaphysis with a saw. The collected condyles of large bone were resin-embedded using a bone resin embedding kit (fuji film and Wako pure chemical industries, Ltd.). The resin-embedded femoral condylar specimens were sectioned with a Zegtome (SP1600, laica Microsystems). Specimens were subjected to hematoxylin-eosin staining with mayer's hematoxylin (fuji film and Wako pure chemical industries, Ltd.) and eosin (70% alcohol solution) (fuji film and Wako pure chemical industries, Ltd.), and subjected to histopathological evaluation with an optical microscope.

Experimental implants (product of the invention and untreated polyetheretherketone) for the rabbit femoral implant insertion test, photographs of femoral implant insertion, photographs 4 weeks after insertion and CT are shown in figure 2. The results of the histopathological examination are shown in fig. 3 to 6.

The bone contact rates after 4 weeks of embedding were measured for the inventive product, which showed a higher bone contact rate than polyetheretherketone, and for polyetheretherketone, 68.7% and 63.8%, respectively (fig. 7).

After 4, 8, and 12 weeks of insertion, the product 1 of the present invention and untreated polyetheretherketone were subjected to a press test using a mechanical tester, and the maximum stress at the time of pressing was measured, as shown in fig. 8. The results are shown in FIG. 9.

Compared with 4 weeks, the maximum punching stress of the product and the polyetheretherketone is stronger at 12 weeks, and the product obviously has stronger maximum punching stress than the polyetheretherketone at 12 weeks. Also, since the maximum punching stress increases with time, it can be predicted from the pathological tissue structure to make the bonding force stronger by long-term embedding such as 8 weeks or 12 weeks.

Further, it was confirmed that a phosphorus atom was introduced into polyetheretherketone, but there are cases where it is impossible or difficult to directly specify an implant material due to the structure or characteristics of an object. That is, although it is predicted that the introduced phosphorus atom is introduced as a phosphate group in the structure of polyetheretherketone at the ortho-position or meta-position to the ether oxygen atom of polyetheretherketone, or at both positions, in order to confirm such prediction, the outermost surface structure of the polymer is analyzed by at least XPS (photoelectron spectroscopy), auger spectroscopy, or the like, and it is necessary to specify the molecular structure thereof. However, these spectroscopic methods alone are not completely specific. In addition, even if the above analysis can be achieved, it is extremely difficult or impossible to directly specify the structure or characteristics of the implant material of the present invention because of the need to further analyze the correlation between the outermost surface structure resulting from such analysis and the biological characteristics.