Bone regeneration material and method for producing bone regeneration material

Abstract

To provide a bone regeneration material having both antibacterial properties and high aesthetic properties, and to provide a method for producing the bone regeneration material.SOLUTION: A bone regeneration material in a cotton form comprising a biodegradable fiber, wherein the biodegradable fiber contains calcium phosphate, silver ion-solid solution calcium phosphate, and a biodegradable polymer, and a content ratio of the silver ion-solid solution calcium phosphate to a total amount of the calcium phosphate and the silver ion-solid solution calcium phosphate is 60 mass% or more. The silver ion solid solution calcium phosphate is preferably silver ion solid solution β - tricalcium phosphate.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a bone regeneration material and a method for producing the bone regeneration material. [Background technology]

[0002] As a treatment for bone defects caused by disease or accident, a method has been investigated in which bone regeneration materials are filled into the defect site and bone is regenerated using the filled bone regeneration materials as a scaffold. In surgery where artificial bone regeneration materials are implanted into the affected area, there is a risk of surgical site infection, so attempts have been made to impart antibacterial properties to bone regeneration materials.

[0003] In addition, various shapes of bone regeneration materials are being considered, such as granular, cylindrical, and disc-shaped, but cotton-shaped bone regeneration materials are being considered as they are easy to handle and fill.

[0004] The present inventors have disclosed in Patent Document 1 a bone-forming material comprising biodegradable fibers containing silver-loaded β-phase tricalcium phosphate particles as a fibrous bone regeneration material with antibacterial properties, and a method for producing the same. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-130417 Summary of the Invention [Problem to be solved by the invention]

[0006] In addition to antibacterial properties, bone regeneration materials are required to have high aesthetics, so that the repaired area is indistinguishable from natural bone. From the viewpoint of achieving both antibacterial properties and high aesthetics, there was room for improvement in the bone regeneration material described in Patent Document 1.

[0007] The present invention has been made in view of the above circumstances, and aims to provide a bone regeneration material that is both antibacterial and highly aesthetic, and a method for producing the bone regeneration material. [Means for solving the problem]

[0008] The present invention includes the following aspects. [1] A cotton-shaped bone regeneration material comprising biodegradable fibers, the biodegradable fibers comprising calcium phosphate, silver ion-dissolved calcium phosphate, and a biodegradable polymer, wherein the content of the silver ion-dissolved calcium phosphate relative to the total amount of the calcium phosphate and the silver ion-dissolved calcium phosphate is 60 mass% or more. [2] The bone regeneration material according to [1], wherein the content of the silver ion-dissolved calcium phosphate relative to the total amount of the calcium phosphate and the silver ion-dissolved calcium phosphate is 60% by mass or more and 90% by mass or less. [3] The bone regeneration material according to [1] or [2], wherein the silver ion-doped calcium phosphate is silver ion-doped β-tricalcium phosphate. [4] The bone regeneration material according to any one of [1] to [3], wherein the content of the biodegradable polymer relative to the total amount of the biodegradable fibers is less than 50 mass %. [5] A method for producing a bone regeneration material, comprising the steps of: preparing a spinning solution by mixing powder of silver ion-doped calcium phosphate, calcium phosphate, and a biodegradable polymer; and forming fibers from the spinning solution by an electrospinning method, wherein the silver ion-doped calcium phosphate has a silver ion content of more than 5 mol% and less than 20 mol%. [6] The silver ion solid solution calcium phosphate has a specific surface area of ​​1.0 m 2 ·g -1 The method for producing a bone regenerating material described in [5] above. [7] The method for producing a bone regeneration material according to [5] or [6], wherein the silver ion-doped calcium phosphate has a particle size of 1.0 μm or more and 60 μm or less. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a bone regeneration material that is both antibacterial and highly aesthetic, and a method for producing the bone regeneration material.

[0010] Hereinafter, "silver ion solid-solubilized β-tricalcium phosphate" may be referred to as "Ag-TCP." The number in parentheses following Ag-TCP is the silver ion content (unit: mol%). X-ray diffraction is sometimes abbreviated as "XRD." [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows XRD profiles of Ag-TCP(10) heat-treated at various heating temperatures. [Figure 2] This is a diagram obtained by measuring the lattice constant by XRD for Ag-TCP(10) heat-treated at 800°C. [Figure 3] FIG. 1 shows the results of the viable cell count (growth value) and silver ion concentration for Ag-TCP(10) heat-treated at each heating temperature. [Figure 4] This is a photograph obtained as a result of evaluating inhibition zone formation using the inhibition circle method. [Figure 5] FIG. 1 shows the XRD profiles of Ag-TCP(0), Ag-TCP(5), Ag-TCP(10) and Ag-TCP(20). [Figure 6] This figure shows the results of evaluating bacterial growth using the turbidity method for Ag-TCP(0), Ag-TCP(5), Ag-TCP(10), and Ag-TCP(20). [Figure 7] FIG. 1 is a photograph showing the appearance of a bone regeneration material. [Figure 8] 1 is a graph showing the results of investigating the elution behavior of silver ions. [Figure 9] FIG. 1 is a graph showing the results of evaluating bacterial growth in bone regenerative materials using the turbidity method. [Figure 10] 1 is a graph showing the relationship between the number of days of culture and the number of cells obtained as a result of cytotoxicity evaluation. [Figure 11]1 is a graph showing the silver ion concentration in the extraction medium. DETAILED DESCRIPTION OF THE INVENTION

[0012] <Bone regeneration material> The present invention is a cotton-shaped bone regeneration material comprising biodegradable fibers. The bone regeneration material of this embodiment is in the form of a cotton ball, and multiple biodegradable fibers are intertwined with each other, forming interfiber spaces. When the bone regeneration material of this embodiment is implanted into a bone defect site, cells invade the interfiber spaces using the biodegradable fibers as a scaffold, promoting bone regeneration.

[0013] The biodegradable fiber contains calcium phosphate and silver ion-doped calcium phosphate. Each material will be described below.

[0014] [Calcium phosphate] Calcium phosphate is a component that is absorbed in the body and promotes bone regeneration. The calcium phosphate is preferably one or more selected from α-tricalcium phosphate, β-tricalcium phosphate, and tetracalcium phosphate. In this embodiment, the calcium phosphate is more preferably β-tricalcium phosphate. Hereinafter, "β-tricalcium phosphate" may be referred to as "β-TCP."

[0015] [Silver ion solid solution calcium phosphate] Silver ion solid solution calcium phosphate is a substitutional solid solution in which some of the calcium ions or vacancies in the crystal lattice constituting calcium phosphate are substituted with silver ions. Silver ion solid-solution calcium phosphate exhibits higher antibacterial properties than materials in which silver is fixed as metallic silver. If silver ions do not dissolve but precipitate to form metallic silver, the bone regeneration material will become discolored and its aesthetics will be impaired. In this embodiment, the silver ions are present within the crystal lattice of calcium phosphate and do not precipitate, thereby imparting high antibacterial properties and aesthetics.

[0016] In this embodiment, the silver ion-doped calcium phosphate is preferably silver ion-doped β-tricalcium phosphate, which has a structure in which some of the calcium ions or vacancies in the crystal lattice constituting β-TCP are substituted with silver ions.

[0017] The content of silver ion-doped calcium phosphate relative to the total amount of calcium phosphate and silver ion-doped calcium phosphate is 60% by mass or more, preferably 61% by mass or more, and more preferably 62% by mass or more, from the viewpoint of exerting antibacterial properties. From the viewpoint of achieving aesthetic appeal, the content of silver ion-doped calcium phosphate relative to the total amount of calcium phosphate and silver ion-doped calcium phosphate is preferably 90 mass % or less, more preferably 80 mass % or less, and even more preferably 72 mass % or less. The upper and lower limits of the content of silver ion-doped calcium phosphate relative to the total amount of calcium phosphate and silver ion-doped calcium phosphate can be arbitrarily combined, such as 60% by mass or more and 90% by mass or less, 61% by mass or more and 80% by mass or less, and 62% by mass or more and 72% by mass or less.

[0018] [Biodegradable polymers] The biodegradable polymer can be, for example, polylactic acid-co-glycolic acid (PLGA) or polylactic acid (PLLA). These biodegradable polymers can contain calcium phosphate and calcium phosphate with silver ions dissolved therein. PLGA is preferred because it is more easily absorbed by the body than PLLA.

[0019] When the bone forming material is implanted in the body, the biodegradable polymer decomposes, exposing the silver ion-doped calcium phosphate and calcium phosphate from the biodegradable fiber. The exposed silver ion-doped calcium phosphate gradually releases silver ions, exhibiting antibacterial properties. Calcium ions and phosphate ions are eluted from the exposed calcium phosphate, promoting bone formation.

[0020] Furthermore, areas of the body where bacterial infection occurs tend to have a localized acidic pH. In an acidic environment, silver ions are more likely to be eluted from the silver ion-doped calcium phosphate. This allows localized antibacterial activity to be exerted in areas where bacterial infection has occurred.

[0021] In this embodiment, the content of the biodegradable polymer relative to the total amount of the biodegradable fiber is preferably less than 50% by mass, more preferably 45% by mass or less, and even more preferably 40% by mass or less. The content of the biodegradable polymer relative to the total amount of the biodegradable fiber is, for example, 5% by mass or more, 10% by mass or more, or 20% by mass or more. The upper and lower limits can be combined in any desired manner, such as 5% by mass or more but less than 50% by mass, 10% by mass or more but less than 45% by mass, or 20% by mass or more but less than 40% by mass.

[0022] <Method of manufacturing bone regenerative material> The method for producing a bone regenerating material of the present invention includes the steps of mixing powder of silver ion-doped calcium phosphate, calcium phosphate, and a biodegradable polymer to prepare a spinning solution, and then forming fibers from the spinning solution by electrospinning. Furthermore, a step of obtaining powder of silver ion-doped calcium phosphate may be included before the fiberizing step. Each step will be described below.

[0023] [Step of obtaining silver ion solid-solution calcium phosphate powder] First, a suspension containing a calcium raw material is prepared, to which a mixed solution of a phosphorus raw material and a silver raw material is added dropwise, and the mixture is reacted to produce silver ion-doped calcium phosphate by a wet method.

[0024] The type of calcium raw material is not particularly limited, and examples thereof include inorganic salts and organic acid salts. Specific examples of inorganic salts include calcium chloride, calcium nitrate, calcium carbonate, calcium oxide, and calcium hydroxide. Specific examples of organic acid salts include calcium formate, calcium acetate, calcium lactate, calcium gluconate, and calcium citrate.

[0025] The type of phosphorus raw material is not particularly limited, and examples thereof include alkali metal salts of phosphoric acid, ammonium salts, etc. Examples of alkali metal salts of phosphoric acid include sodium salts and potassium salts, and more specific examples include disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, tripotassium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, triammonium phosphate, etc. Phosphoric acid may also be used as a raw material.

[0026] The silver raw material is preferably a silver compound that does not exhibit cytotoxicity, and examples thereof include silver oxide, silver thiocyanate, silver cyanide, silver cyanate, silver carbonate, silver nitrate, silver nitrite, silver sulfate, silver phosphate, silver perchlorate, silver tetrafluoroborate, silver acetylacetonate, silver acetate, silver lactate, silver oxalate, etc. Among these, silver nitrate, silver nitrite, silver acetate, and silver oxalate are preferred, and silver nitrate is particularly preferred.

[0027] In this embodiment, the blending amount is preferably set appropriately so that the molar ratio of Ca / P at the end of the dropwise addition is 1.0 or more and 2.0 or less, more preferably 1.2 or more and 1.8 or less, and even more preferably 1.3 or more and 1.6 or less.

[0028] In this embodiment, it is preferable to appropriately set the blending amount so that the molar ratio of (Ca+Ag) / P at the end of the dropwise addition is 1.0 or more and 2.0 or less, more preferably 1.2 or more and 1.8 or less, and even more preferably 1.3 or more and 1.6 or less.

[0029] After the dropwise addition, it is preferable to age the mixture at room temperature for about 24 hours. Ageing means leaving the mixture to stand for a certain period of time either still or under stirring. After aging, a precursor is obtained in which the calcium raw material, the phosphorus raw material, and the silver raw material have reacted with each other.

[0030] The precursor is appropriately pulverized and then heat-treated to obtain a powder of silver ion-doped calcium phosphate. Silver ion solid solution calcium phosphate has a specific surface area of ​​1.0m 2 ·g -1 It is preferable that it is 3m or more. 2 ·g -1 More than 5m is preferable. 2 ·g -1 The above is even more preferable. By using a silver ion-doped calcium phosphate having a specific surface area equal to or greater than the above lower limit, a bone regeneration material from which silver ions are easily eluted can be produced.

[0031] The upper limit of the specific surface area of ​​the silver ion solid-solution calcium phosphate is not particularly limited, but it is, for example, 60 m 2 ·g -1 Below, 50m 2 ·g -1 Below, 40m 2 ·g -1 The following is the result.

[0032] The particle size of the silver ion solid-doped calcium phosphate is preferably 1.0 μm or more and 60 μm or less, more preferably 5 μm or more and 50 μm or less, and even more preferably 15 μm or more and 30 μm or less. By using silver ion-doped calcium phosphate having a particle size within the above range, a bone regeneration material from which silver ions are easily eluted can be produced.

[0033] In order to control the specific surface area and particle size of the silver ion solid-doped calcium phosphate within the above ranges, the heating temperature during heat treatment is preferably 500°C or higher and lower than 1000°C, more preferably 600°C or higher and 900°C or lower, and even more preferably 750°C or higher and 850°C or lower.

[0034] The silver ion-doped calcium phosphate produced in this process preferably has a silver ion content exceeding 5 mol% and less than 20 mol%, more preferably 6 mol% or more and 15 mol% or less, and even more preferably 8 mol% or more and 12 mol% or less. When silver ion-doped calcium phosphate with a silver ion content within the above range is used, biodegradable fibers with uniformly dispersed silver ions are easily obtained in the subsequent fibrillation process described below.

[0035] [Fibrillation process] The powder of silver ion-doped calcium phosphate obtained by the above method, calcium phosphate, and a biodegradable polymer are mixed to prepare a spinning solution, and the spinning solution is fibrillated by the electrospinning method. By this process, a cotton-shaped bone regeneration material is obtained. The fibrillation process can be carried out by the methods described in International Publication No. 2017 / 188435 and International Publication No. 2016 / 159240.

Examples

[0036] Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the examples shown below.

[0037] [Process for obtaining Ag-TCP powder] 1 of 0.600 mol·dm -3 Ca(OH)2 suspension was prepared at 250 cm 3 . 1 of 0.400 mol·dm -3 H3PO4 and a mixed solution 1 containing 0.020 mol·dm -3 AgNO3 were prepared at 250 cm 3 . The mixed solution 1 was dropped into the suspension 1 at 6 cm 3 / min. After dropping, the pH was adjusted to 6 - 7 using 25% aqueous ammonia, and the mixture was stirred at 700 rpm for 3 hours. After stirring, it was left standing at room temperature for 24 hours. After standing, the precursor obtained by filtration was dried at 110 °C for 24 hours, the obtained precursor was pulverized, and heat-treated at a predetermined temperature.

[0038] For the heat treatment, a box-type electric furnace was used, and the samples were heated for 3 hours at each heat treatment temperature (600°C, 700°C, 800°C, 900°C, 1000°C, 1100°C, 1200°C). The temperature rise rate was 10°C / min. Through the above steps, Ag-TCP powder was obtained.

[0039] [Quantitative analysis of elements] Table 1 shows the quantitative analysis results of the elements contained in Ag-TCP heat-treated at each heating temperature, measured by ICP atomic emission spectroscopy (ICP-AES) using high-frequency inductively coupled plasma (ICP) as the light source.

[0040] [Table 1]

[0041] After the heat treatment, Ag-TCP containing 10 mol% silver ions was obtained, as shown in Table 1. Hereinafter, Ag-TCP containing 10 mol% silver ions will be referred to as Ag-TCP(10).

[0042] [XRD measurement] Figure 1 shows the XRD profiles of Ag-TCP(10) heat-treated at each heating temperature. (XRD profile measurement conditions) Measuring equipment: Rigaku MiniFlex ·Radiation source:CuKα Measurement range (2θ): 4°~50° Scan speed: 4° / min Sampling: 0.04° Voltage 30kV, current 15mA

[0043] As shown in Figure 1, the crystalline structure of β-TCP was confirmed when heated between 600°C and 1200°C. In Figure 1, "Non-heat" is a reference example in which the precursor was not heat-treated. When no heat treatment was performed, hydroxyapatite was the dominant crystalline structure. As shown in Figure 1, when heat treatment was performed at 1000 to 1200°C, silver precipitation was confirmed.

[0044] [Lattice constant measurement by XRD] The lattice constant of Ag-TCP(10) heat-treated at 800°C was measured by XRD. The results are shown in Figure 2. As shown in Figure 2, when comparing the amount of silver added at 0 mol% and 10 mol%, there was no change in the a-axis length, but when 10 mol% silver was added, a degeneration of the c-axis length was confirmed. The degeneration of the c-axis length confirmed that it was a substitutional solid solution, in which some of the calcium ions or vacancies in the crystal lattice that makes up β-TCP were substituted with silver ions.

[0045] [Particle size distribution measurement] Table 2 shows the particle size distribution of the Ag-TCP(10) heat-treated at each heating temperature.

[0046] [Table 2]

[0047] In Table 2, the particle size distribution of β-TCP alone is shown as a reference example. As shown in Table 2, it was confirmed that heat treatment at temperatures exceeding 1000°C caused sintering to proceed and the grain size to increase.

[0048] [Measurement of specific surface area] The specific surface area of ​​the Ag-TCP(10) heat-treated at each heating temperature was measured and the results are shown in Table 3.

[0049] [Table 3]

[0050] In Table 3, the particle size distribution of β-TCP alone is shown as a reference example. As shown in Table 3, it was confirmed that the specific surface area was significantly reduced when the material was heat-treated at a temperature exceeding 1000°C.

[0051] [Evaluation of bacterial growth using turbidity method] The bacterial growth of Ag-TCP(10) heat-treated at each heating temperature was evaluated by the turbidity method. The samples used were Ag-TCP(10) (non-heat, 600°C, 700°C, 800°C, 900°C, 1000°C, 1100°C, 1200°C) sterilized by dry heat at 160°C for 90 minutes, and a control (β-TCP only). 10cm 3 0.5 g of each sample was added to the LB liquid medium (diluted 10 times with sterilized ultrapure water) and shaken at 37°C for 24 hours. After that, the mixture was centrifuged at 9000 rpm for 10 minutes, and the supernatant was extracted to obtain an extract. The silver ion concentration of the extract was measured by ICP-AES.

[0052] 4.9cm 3 0.1cm 3 The bacterial solution 1 was inoculated and cultured at 37°C for 24 hours with shaking. After that, the absorbance at a wavelength of 600 nm was measured using a spectrophotometer. The absorbance value is proportional to the turbidity of the culture solution, so the viable cell count (growth value) was measured using this value.

[0053] Bacterial solution 1 contains Staphylococcus aureus, a gram-positive bacterium (bacterial count: 5 × 10 6 CFU·cm -3 )

[0054] Figure 3 shows the results of the viable cell count (growth value) and silver ion concentration. In FIG. 3, "ND" is an abbreviation for "Not detected," meaning that it was not detected. As shown in Figure 3, it was confirmed that Ag-TCP (10) produced by heating at 600 to 900 °C had high antibacterial activity. The Ag-TCP(10) produced by heating above 1000°C had low antibacterial activity, which is presumably due to the fact that silver ions were less likely to be eluted.

[0055] [Evaluation of zone of inhibition formation using the inhibition circle method] The formation of the inhibition zone was evaluated using the inhibition circle method for Ag-TCP(10) heat-treated at each heating temperature. The samples used were Ag-TCP(10) (800℃, 900℃) sterilized by dry heat at 160℃ for 90 minutes and a control (β-TCP only). Each powder sample was uniaxially pressed at 200 MPa using a die molding machine with a diameter of 15 mm to produce a green compact with a diameter of 15 mm and a thickness of 3 mm.

[0056] Each compact was placed in the center of the LB agar medium. 3 Add 1 of the bacterial solution to 6.0 cm 3 The bacterial solution was inoculated onto the top agar medium and incubated at 37°C for 24 hours. The results are shown in Figure 4.

[0057] As shown in Figure 4, it was confirmed that no bacteria grew around the compact surrounded by the dashed line in the Ag-TCP (10) produced by heating at 800°C or 900°C. It was confirmed that the area where bacteria did not grow was wider when heated at 800°C than when heated at 900°C, and that heating at 800°C had higher antibacterial activity.

[0058] <Production of Ag-TCP with different silver ion concentrations> 0.600 mol dm -3 of Ca(OH)2 suspension to 250 cm 3 Prepared. 0.400 mol dm -3 of H3PO4 and AgNO3 (0 mol dm -3 , 0.01 mol dm -3 , 0.02 mol dm -3 , 0.10 mol dm -3 ) are mixed with 250cm 3 Prepared. Add 6cm of each mixed solution to suspension 1. 3 After the dropwise addition, the pH was adjusted to 6 to 7 using 25% aqueous ammonia, and the mixture was stirred at 700 rpm for 3 hours. After stirring, the mixture was allowed to stand at room temperature for 24 hours. After standing, the precursor obtained by filtration was dried at 110°C for 24 hours, and the obtained precursor was pulverized and heat-treated at a predetermined temperature.

[0059] The heat treatment was carried out in a box-type electric furnace at a heat treatment temperature of 800°C for 3 hours at a temperature increase rate of 10°C / min. By the above steps, Ag-TCP(0), Ag-TCP(5), Ag-TCP(10) and Ag-TCP(20) powders were obtained, respectively.

[0060] Table 4 shows the synthesis conditions for Ag-TCP with varying silver content (Ag-TCP(0), Ag-TCP(5), Ag-TCP(10), and Ag-TCP(20)).

[0061] [Table 4]

[0062] [XRD measurement] The XRD profiles of Ag-TCP(0), Ag-TCP(5), Ag-TCP(10), and Ag-TCP(20) are shown in Figure 5. The measurement conditions for the XRD profiles were the same as those described above (measurement conditions for XRD profiles).

[0063] As shown in Figure 5, the crystal structure of β-TCP was confirmed in Ag-TCP(0), Ag-TCP(5), Ag-TCP(10), and Ag-TCP(20). As shown in Figure 5, silver deposition was confirmed in Ag-TCP(20).

[0064] [Evaluation of bacterial growth using turbidity method] The bacterial growth of Ag-TCP(0), Ag-TCP(5), Ag-TCP(10) and Ag-TCP(20) was evaluated by turbidity method. The samples used were Ag-TCP(0), Ag-TCP(5), Ag-TCP(10), and Ag-TCP(20), which had been dry-heat sterilized at 160°C for 90 minutes. 10cm 30.5 g of each sample was added to the LB liquid medium (diluted 5 times with sterilized ultrapure water) and shaken at 37°C for 24 hours. After that, the mixture was centrifuged at 9000 rpm for 10 minutes, and the supernatant was extracted to obtain an extract.

[0065] 4.9cm 3 0.1cm 3 The bacterial solution 1 was inoculated and cultured at 37°C for 24 hours with shaking. After that, the absorbance at a wavelength of 600 nm was measured using a spectrophotometer. The absorbance value is proportional to the turbidity of the culture solution, so the viable cell count (growth value) was measured using this value.

[0066] Bacterial solution 1 contains Staphylococcus aureus, a gram-positive bacterium (bacterial count: 5 × 10 6 CFU·cm -3 )

[0067] FIG. 6 shows the results of the viable cell count (growth value). As shown in Figure 6, it was confirmed that Ag-TCP(10) and Ag-TCP(20) had high antibacterial activity.

[0068] <Production of bone-forming materials> Ag-TCP (10) was produced by the same method as above, but at a heat treatment temperature of 800°C. Ag-TCP(10), β-TCP, and lactic acid-co-glycolic acid poly(PLGA) that had been melted or softened by heating in a kneader were mixed and kneaded in the proportions shown in Table 5 to obtain a composite containing Ag-TCP(10), β-TCP, and PLGA.

[0069] The obtained complex was dissolved in chloroform to prepare a spinning solution with a complex concentration of 8% by mass. The obtained spinning solution was electrospun using a NANON electrospinning device (MECC Co., Ltd.) at an applied voltage of 28 kV and an extrusion speed of 15 cm 3 The spinning was carried out under the conditions of 1 / hour, needle thickness of 18G, spinneret movement speed of 40mm / sec, and flying distance to the collector of 28cm. The collector container was filled with ethanol, and the spun fibers were deposited, and the cotton-like fibers were collected. By the above method, a cotton-like bone regeneration material containing biodegradable fibers was obtained.

[0070] [Table 5]

[0071] In Table 5, Ag-TCP35 means that the total amount of Ag-TCP in the composite is 35% by mass. The value in parentheses for Ag-TCP (unit: mass%) indicates the proportion of Ag-TCP relative to the total amount of Ag-TCP and β-TCP. "ReBOSSIS-J" in Table 4 is a registered trademark owned by one of the applicants of the present invention, and is a bone regeneration material made of biodegradable fibers containing β-TCP.

[0072] Figure 7 shows the appearance of each sample listed in Table 5. "ReBOSSIS-J (registered trademark)," which does not contain silver ions, was the whitest and most aesthetically pleasing. In contrast, Ag-TCP70, which contains 100% Ag-TCP by mass, was brownish. Ag-TCP35 to 52.5 were aesthetically pleasing, with the bone repair area being indistinguishable from natural bone and white enough. Among them, Ag-TCP35 to Ag-TCP45 were as white as "ReBOSSIS-J (registered trademark)" and had superior aesthetics.

[0073] [Investigation of silver ion elution behavior] The elution behavior of silver ions was investigated for Ag-TCP35, Ag-TCP52.5, and Ag-TCP70 shown in Table 4.

[0074] 10cm, a neutral buffer solution that mimics the in vivo environment 3 0.1 g each of Ag-TCP35, Ag-TCP52.5, and Ag-TCP70 was added to HEPES buffer (20 mM, pH 7.3) and the mixture was shaken in a water bath at 37°C and 100 rpm. Thereafter, the mixture was centrifuged at 1000 rpm for 3 minutes, and the silver ion concentration of the supernatant was measured using ICP-AES, and the amount of elution was calculated using the following formula. Amount of silver ions dissolved (μmol g -1 ) = [amount of detected element (μmol)] / [sample mass (g)]. FIG. 8 is a graph showing the relationship between the water bath shaking time and the amount of eluted silver ions.

[0075] As shown in Figure 8, it was confirmed that Ag-TCP35 and Ag-TCP52.5 slowly released silver ions up to 30 days after immersion. Ag-TCP70 showed a faster sustained release time compared to Ag-TCP35 and Ag-TCP52.5.

[0076] [Evaluation of bacterial growth using turbidity method] The bacterial growth of each sample shown in Table 4 was evaluated using the turbidity method. 10cm 3 0.1 g of each gamma-ray sterilized sample was added to the LB liquid medium (diluted 5 times with sterilized ultrapure water) and shaken at 37°C for 24 hours. The supernatant was then extracted to obtain an extract. The silver ion concentration of the extract was measured by ICP-AES.

[0077] 4.9cm 3 0.1cm 3 The bacterial solution 1 was inoculated and cultured at 37°C for 24 hours with shaking. After that, the absorbance at a wavelength of 600 nm was measured using a spectrophotometer. The absorbance value is proportional to the turbidity of the culture solution, so the viable cell count (growth value) was measured using this value.

[0078] Bacterial solution 1 contains Staphylococcus aureus, a gram-positive bacterium (bacterial count: 5 × 10 6 CFU·cm -3 )

[0079] FIG. 9 shows the results of the viable cell count (growth value) and silver ion concentration. As shown in Figure 9, it was confirmed that Ag-TCP45, Ag-TCP50, Ag-TCP52.5 and Ag-TCP70 had high antibacterial activity.

[0080] [Cytotoxicity evaluation] For each sample shown in Table 4, cytotoxicity was evaluated using an extraction medium. 0.2 g of each gamma-ray sterilized sample was placed in a 20 cm 3 The cells were added to a liquid medium (hereinafter referred to as αMEM(+)) prepared by adding 10% FBS medium to α-minimum essential medium (α-MEM medium), and the mixture was shaken at 37°C for 24 hours. Thereafter, the mixture was centrifuged at 1000 rpm for 3 minutes, and the silver ion concentration of the supernatant was measured using ICP-AES.

[0081] 5 × 10 4 Mouse calvarial osteoblast-like cells (MC3T3-E1 cells, P=3) were seeded at 1000 cells / well and cultured in an incubator at 37°C with 5% CO2 for 1 to 5 days. A 24-well polystyrene plate was used as a control. The medium was replaced with extract medium after 1 and 3 days. FIG. 10 is a graph showing the relationship between the number of days in culture and the number of cells. FIG. 11 is a graph showing the silver ion concentration in the extraction medium.

[0082] From the results of Figures 10 and 11, an increase in cell number was confirmed in the medium containing silver ions eluted from the bone regeneration material of the present invention, and no toxic effect was confirmed for the bone regeneration material of the present invention.

Claims

1. A cotton-shaped bone regeneration material comprising biodegradable fibers, the biodegradable fiber includes calcium phosphate, silver ion-doped calcium phosphate, and a biodegradable polymer; A bone regeneration material, wherein the content of the silver ion-dissolved calcium phosphate is 60 mass% or more relative to the total amount of the calcium phosphate and the silver ion-dissolved calcium phosphate.

2. 2. The bone regeneration material according to claim 1, wherein the content of said silver ion-dissolved calcium phosphate is 60% by mass or more and 90% by mass or less relative to the total amount of said calcium phosphate and said silver ion-dissolved calcium phosphate.

3. 3. The bone regeneration material according to claim 1, wherein the silver ion-doped calcium phosphate is silver ion-doped β-tricalcium phosphate.

4. The bone regeneration material according to claim 1 or 2, wherein the content of the biodegradable polymer relative to the total amount of the biodegradable fibers is less than 50 mass %.

5. The method includes a step of preparing a spinning solution by mixing a powder of silver ion-doped calcium phosphate, calcium phosphate, and a biodegradable polymer, and forming the spinning solution into fibers by an electrospinning method, The method for producing a bone regeneration material, wherein the silver ion solid-solubilized calcium phosphate has a silver ion content of more than 5 mol % and less than 20 mol %.

6. The silver ion solid solution calcium phosphate has a specific surface area of ​​1.0 m ２ ・g －１ The method for producing a bone regenerating material according to claim 5, wherein the method is as described above.

7. The method for producing a bone regeneration material according to claim 5 or 6, wherein the silver ion-doped calcium phosphate has a particle size of 1.0 μm or more and 60 μm or less.

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