Bone regeneration material, and method and device for producing bone regeneration material

Abstract

Provided are a bone regeneration material, a method for producing a bone regeneration material, and a device for producing a bone regeneration material, whereby the amount of protein adsorbed to octacalcium phosphate can be controlled. A bone regeneration material comprising a complex, the complex comprising: a base material comprising octacalcium phosphate; and a functional molecule adsorbed to the octacalcium phosphate.

Description

Technical Field

[0001] The present invention relates to a bone regeneration material, a method for manufacturing the bone regeneration material and a device for manufacturing the bone regeneration material. Background Art

[0002] Conventionally, there is known a method for increasing the amount of protein adsorbed onto octacalcium phosphate by promoting the precipitation of novel octacalcium phosphate on octacalcium phosphate seed crystals under supersaturated conditions (see, for example, Patent Document 1).

[0003] [Prior art literature]

[0004] [Patent Document]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2021-16724 Summary of the Invention

[0006] [Problems to be solved by the invention]

[0007] However, the method of Patent Document 1 has a problem in that the amount of protein adsorbed to octacalcium phosphate cannot be controlled and the amount of protein adsorbed cannot be adjusted according to the intended use.

[0008] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a bone regeneration material having a controlled amount of protein adsorbed onto octacalcium phosphate, a method for producing the bone regeneration material, and an apparatus for producing the bone regeneration material.

[0009] [Technical means to solve the problem]

[0010] The present invention has the following aspects.

[0011] [1] A bone regeneration material comprises a composite, wherein the composite comprises: a substrate comprising octacalcium phosphate; and a functional molecule adsorbed on the octacalcium phosphate.

[0012] [2] The bone regeneration material according to [1], wherein the functional molecule is at least one selected from growth factors and serum-derived proteins.

[0013] [3] The bone regeneration material according to [1], wherein the base material comprises a lactic acid-glycolic acid copolymer.

[0014] [4] A method for producing a bone regeneration material, comprising: dissolving a functional molecule in phosphate-buffered saline to prepare a phosphate-buffered saline solution;

[0015] a step of bringing the phosphate-buffered saline solution into contact with a substrate containing octacalcium phosphate to allow the functional molecule to be adsorbed on the octacalcium phosphate; and

[0016] A step of freeze-drying the substrate containing the octacalcium phosphate adsorbed with the functional molecule.

[0017] [5] A method for producing a bone regeneration material, comprising: dissolving a functional molecule in a buffer solution that is saturated or supersaturated with respect to octacalcium phosphate to prepare a buffer solution containing the functional molecule;

[0018] a step of immersing a substrate comprising octacalcium phosphate in the buffer solution containing the functional molecule to allow the functional molecule to be adsorbed on the octacalcium phosphate; and

[0019] A step of freeze-drying the substrate containing the octacalcium phosphate adsorbed with the functional molecule.

[0020] [6] A bone regeneration material manufacturing device, comprising: a reaction tank for receiving a slurry, wherein the slurry contains a base material containing octacalcium phosphate;

[0021] an acid / alkali supply unit for supplying acid or alkali to the slurry in the reaction tank;

[0022] a calcium ion supplying unit for supplying calcium ions to the slurry in the reaction tank;

[0023] an inorganic phosphate ion supply unit for supplying inorganic phosphate ions to the slurry in the reaction tank;

[0024] a functional molecule supplying unit, supplying functional molecules to the slurry in the reaction tank;

[0025] a functional molecule concentrating unit, which, in the reaction tank, recovers and concentrates the functional molecules remaining in the supernatant of the slurry after the functional molecules are adsorbed on the substrate, and supplies the recovered functional molecules to the reaction tank again;

[0026] a pH electrode for measuring the pH of the slurry in the reaction tank;

[0027] a calcium ion electrode for measuring the concentration of calcium ions in the slurry in the reaction tank; and

[0028] The control unit calculates a supersaturation of the slurry relative to octacalcium phosphate based on the pH of the slurry in the reaction tank measured by the pH electrode and the calcium ion concentration of the slurry in the reaction tank measured by the calcium ion electrode, and instructs the acid / base supply unit, the calcium ion supply unit, and the inorganic phosphate ion supply unit to supply acid or base, calcium ions, and inorganic phosphate ions to the slurry in the reaction tank based on the supersaturation so that the supersaturation of the slurry in the reaction tank reaches a set value.

[0029] [Effects of the Invention]

[0030] According to the present invention, a bone regeneration material having a controlled amount of protein adsorbed onto octacalcium phosphate, a method for producing the bone regeneration material, and an apparatus for producing the bone regeneration material can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram showing a bone regeneration material production apparatus according to one embodiment of the present invention.

[0032] Figure 2 This is a diagram explaining the mechanism for promoting the adsorption of functional molecules onto a substrate containing OCPs in a buffer solution supersaturated with OCPs.

[0033] Figure 3 This is a diagram explaining the mechanism for promoting the adsorption of functional molecules onto a substrate containing OCPs in a buffer solution supersaturated with OCPs.

[0034] Figure 4 This is a graph showing the ratio of the new bone area to the defect area in Example 1.

[0035] Figure 5 This is a graph showing the ratio of the area of ​​the remaining OCP particles to the area of ​​the defect in Example 1.

[0036] Figure 6 This is a graph showing the DNA concentration of cells on the 14th or 21st day from the start of culture in Example 2.

[0037] Figure 7 This is a graph showing the ALP activity on the 14th or 21st day from the start of culture in Example 2.

[0038] Figure 8 This is a graph showing the relationship between the initial concentration of cytochrome c in a buffer solution saturated with OCP or a buffer solution supersaturated with OCP in Example 4 and the amount of cytochrome c adsorbed to OCP.

[0039] Figure 9 This is a diagram showing the results of X-ray diffraction analysis of the crystal structure of OCP adsorbing cytochrome c in Example 4.

[0040] Figure 10 This is a transmission electron microscope image of the OCP crystal before adsorption of cytochrome c in Example 5.

[0041] Figure 11 This is a transmission electron microscope image of OCP crystals adsorbed with cytochrome c using buffer E containing cytochrome c in Example 5.

[0042] Figure 12This is a transmission electron microscope image of OCP crystals adsorbed with cytochrome c using buffer F containing cytochrome c in Example 5.

[0043] Figure 13 This is a graph showing the adsorption isotherms of fetuin to OCP in a fetuin-containing 0.5Ca0.5Pi buffer solution and a fetuin-containing 3.0Ca1.0Pi buffer solution in Example 6.

[0044] Figure 14 Graphs showing the results of CD spectrum measurement of fetuin in 0.5Ca0.5Pi buffer and in 3.0Ca1.0Pi buffer before and after OCP immersion in Example 6.

[0045] Figure 15 This is a diagram showing the secondary structural elements of fetuin in 0.5Ca0.5Pi buffer and 3.0Ca1.0Pi buffer in Example 6.

[0046] [Explanation of Symbols]

[0047] 1: Bone regeneration material manufacturing device

[0048] 2: Reaction tank

[0049] 3: Acid / alkali supply unit

[0050] 4: Calcium ion supply unit

[0051] 5: Inorganic phosphate ion supply unit

[0052] 6: Functional molecule supply department

[0053] 7: Functional molecule concentration section

[0054] 8: pH electrode

[0055] 9: Calcium ion electrode

[0056] 10: Control Department

[0057] 11-16: Pump

[0058] 21~25: Piping

[0059] 31-36: Wiring DETAILED DESCRIPTION

[0060] Embodiments of the bone regeneration material, the method for producing the bone regeneration material, and the apparatus for producing the bone regeneration material of the present invention will be described.

[0061] In addition, this embodiment is specifically described for better understanding of the gist of the invention, and does not limit the present invention unless otherwise specified.

[0062] [Bone regeneration materials]

[0063] A bone regeneration material according to one embodiment of the present invention comprises a composite comprising a base material comprising octacalcium phosphate and functional molecules (eg, biofunctional molecules) adsorbed on the octacalcium phosphate.

[0064] The shape of the complex can be arbitrarily set in consideration of the shape and size of the affected area to be filled with the complex, etc. For example, the complex is preferably a rectangular parallelepiped (block), a cube, a cylinder, a sheet, or a granule.

[0065] "Substrate"

[0066] The substrate includes octacalcium phosphate (Ca8H2(PO4)6·5H2O, hereinafter sometimes referred to as "OCP"). The substrate may include only OCP or may include OCP and other components.

[0067] When the substrate contains OCP and other components, the other component is preferably a poly(lactic-co-glycolic acid) copolymer (hereinafter sometimes also referred to as "PLGA").

[0068] When the substrate comprises OCP and PLGA, the ratio of OCP to PLGA is not particularly limited. For example, based on the total amount of OCP and PLGA, the OCP content is preferably 20% to 65% by mass, more preferably 20% to 40% by mass. Furthermore, the PLGA content is preferably 35% to 80% by mass, more preferably 60% to 80% by mass. This balance between the osteoconductivity of OCP and the bioabsorbability of PLGA allows for a bone regeneration material with excellent osteoconductivity.

[0069] OCP is a known substance and can be produced, for example, by the dropping method of LeGeros (LeGeros RZ, Calcified Tissue International, Calcif Tissue Int. 37: 194-197, 1985) or a method using the synthesis apparatus (three-flow tube) described in Japanese Patent No. 3115642.

[0070] "Functional molecules"

[0071] Functional molecules adsorbed on the substrate promote bone regeneration through OCP enlargement and also promote cell migration and proliferation. For example, as reported in Tohoku Journal of Experimental Medicine (Tohoku J Exp Med) 164: 37-50, 1991 and Bone and Mineral Research (Bone Miner) 20: 151-166, 1993, it is believed that functional molecules accumulate in the gaps between OCP crystals and exert their functions.

[0072] As the functional molecule, growth factors and serum-derived proteins are preferred.

[0073] Examples of growth factors include stromal cell-derived factor 1 (SDF-1), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF). For example, by adsorbing SDF-1 onto a substrate, it is expected to promote the migration of mesenchymal stem cells (MSCs), which can differentiate into osteoblasts.

[0074] Examples of serum-derived proteins include fetuin (α2-HS glycoprotein), albumin, and fibronectin. For example, by adsorbing fetuin onto a substrate, it can promote bone formation by binding to surrounding calcium and phosphate ions. By adsorbing fibronectin onto a substrate, it can enhance cell adhesion.

[0075] The functional molecules can be used alone or in combination of two or more. By using two or more functional molecules in combination, two or more effects can be exerted on organisms.

[0076] The amount of functional molecules adsorbed on the substrate is preferably per unit area (cm 2) in an amount of 50 ng or more and 70 μg or less, more preferably 100 ng or more and 60 μg or less, and further preferably 200 ng or more and 20 μg or less. If the adsorption amount of the functional molecule is above the lower limit, bone regeneration can be expected to be promoted. If the adsorption amount of the functional molecule is less than the lower limit, the effect of promoting bone regeneration cannot be obtained. If the adsorption amount of the functional molecule exceeds the upper limit, cytotoxicity or inflammation may be induced, and sufficient bone regeneration effect cannot be obtained.

[0077] Without prejudice to the effects of the present invention, the bone regeneration material of this embodiment may also contain components generally contained in bone regeneration materials. Examples of such components include: collagen, gelatin, alginic acid, hyaluronic acid, chitosan, bioabsorbable polymers (polylactic acid, polylactic acid-polyethylene glycol copolymer, etc.), bioabsorbable calcium phosphate (β-tricalcium phosphate (β-TCP), α-tricalcium phosphate (α-TCP), tetracalcium phosphate (Ca4(PO4)2O; TTCP (tetracalcium phosphate)), calcium hydrogen phosphate (CaHPO4; dicalcium phosphate (DCP)), calcium hydrogen phosphate dihydrate (CaHPO4·2H2O; DCPD (dicalcium phosphate dihydrous)), low-crystalline hydroxyapatite (Ca 10 (PO4)6(OH)2); HA), nano HA, HA containing carbonate, etc.), bio-non-absorbable materials (HA ceramics, etc.).

[0078] The bone regeneration material of this embodiment comprises a composite comprising a substrate containing an OCP and a functional molecule adsorbed onto the OCP. Therefore, when used in a biological setting, the composite not only enhances bone regeneration through the OCP but also promotes the effects of the functional molecule. Furthermore, the bone regeneration material of this embodiment can be configured to control the amount of functional molecule adsorbed through the bone regeneration material manufacturing method described below.

[0079] [Method for producing bone regeneration material]

[0080] (First embodiment)

[0081] A method for producing a bone regeneration material according to one embodiment of the present invention comprises: dissolving a functional molecule in phosphate-buffered saline to prepare a phosphate-buffered saline solution (hereinafter referred to as the "first step"); contacting the phosphate-buffered saline solution with a substrate containing octacalcium phosphate to allow the functional molecule to adsorb onto the octacalcium phosphate (hereinafter referred to as the "second step"); and freeze-drying the substrate containing the octacalcium phosphate adsorbed with the functional molecule (hereinafter referred to as the "third step").

[0082] "First process"

[0083] The concentration of phosphate-buffered saline is preferably 1.0 mmol / L or more and 30.0 mmol / L or less, more preferably 5.0 mmol / L or more and 15.0 mmol / L or less. If the concentration of phosphate-buffered saline is less than the lower limit, the pH buffering capacity decreases. If the concentration of phosphate-buffered saline exceeds the upper limit, cytotoxicity due to precipitated phosphate may occur.

[0084] The content (concentration) of the functional molecule in the phosphate buffered saline solution is preferably more than 3.0 μmol / L and less than 30.0 μmol / L, more preferably more than 4.0 μmol / L and less than 10.0 μmol / L, and then preferably more than 4.5 μmol / L and less than 7.5 μmol / L. If the content of functional molecule is less than the lower limit, the carrying amount of functional molecule is few, and it is possible not to obtain sufficient bone regeneration effect. If the content of functional molecule exceeds the upper limit, cytotoxicity or inflammation etc. can be induced, and sufficient bone regeneration effect cannot be obtained.

[0085] "Second Process"

[0086] In the second step, bringing the phosphate-buffered saline solution into contact with the substrate containing the OCP includes, for example, dropping the phosphate-buffered saline solution onto the substrate containing the OCP or immersing the substrate containing the OCP in the phosphate-buffered saline solution.

[0087] When the substrate containing the OCP is immersed in a phosphate-buffered saline solution, the immersion time is not particularly limited, but is preferably, for example, 1 second or longer and 10 minutes or shorter.

[0088] When a phosphate-buffered saline solution is added dropwise to a substrate containing an OCP, the amount of the phosphate-buffered saline solution added per unit mass (mg) of the substrate is preferably 2.0 μL or more and 8.0 μL or less, more preferably 3.0 μL or more and 7.0 μL or less, and even more preferably 4.0 μL or more and 6.0 μL or less. If the amount of the phosphate-buffered saline solution added is at least the lower limit, the functional molecules can be uniformly supported on the surface of the OCP. If the amount of the phosphate-buffered saline solution added is at most the upper limit, the dissolution of the OCP crystals can be suppressed.

[0089] When the substrate containing the OCP is immersed in a phosphate-buffered saline solution, the mass (mg) of the substrate containing the OCP added per unit volume (μL) of the phosphate-buffered saline solution is preferably 0.10 mg to 0.50 mg, more preferably 0.15 mg to 0.30 mg, and even more preferably 0.15 mg to 0.25 mg, in terms of OCP. If the mass of the substrate containing the OCP is above the lower limit, dissolution of the OCP crystals can be suppressed. If the mass of the substrate containing the OCP is below the upper limit, the functional molecules can be uniformly supported on the OCP.

[0090] "Third Process"

[0091] In the third step, the substrate containing the OCP having the functional molecule adsorbed thereon is preferably pre-frozen before freeze-drying the substrate containing the OCP having the functional molecule adsorbed thereon. The pre-freezing temperature is preferably -196°C or higher and -10°C or lower.

[0092] The freeze-drying time is not particularly limited, but is preferably, for example, 12 hours to 120 hours.

[0093] According to the bone regeneration material of this embodiment, the functional molecule is adsorbed to the OCP using the phosphate-buffered saline solution in which the content of the functional molecule is adjusted. Therefore, the amount of the functional molecule adsorbed to the OCP can be controlled.

[0094] (Second embodiment)

[0095] A method for producing a bone regeneration material according to one embodiment of the present invention comprises: a step of dissolving a functional molecule in a buffer solution that is saturated or supersaturated with respect to octacalcium phosphate to prepare a buffer solution containing the functional molecule (hereinafter referred to as the "first step"); a step of immersing a substrate containing octacalcium phosphate in the buffer solution containing the functional molecule to allow the functional molecule to be adsorbed on the octacalcium phosphate (hereinafter referred to as the "second step"); and a step of freeze-drying the substrate containing the octacalcium phosphate adsorbed with the functional molecule (hereinafter referred to as the "third step").

[0096] "First process"

[0097] Examples of buffer solutions saturated with OCP include those containing calcium ions (Ca 2+ ), inorganic phosphate ions, and tris-HCl buffer, and a buffer with a pH of 5.0 or higher and 8.0 or lower.

[0098] The calcium ion concentration in the buffer solution saturated with OCP is preferably 0.1 mmol / L to 1.0 mmol / L, more preferably 0.4 mmol / L to 0.6 mmol / L. If the calcium ion concentration is below the lower limit, the OCP serving as the substrate dissolves. If the calcium ion concentration exceeds the upper limit, hydroxyapatite precipitates.

[0099] The concentration of inorganic phosphate ions in the buffer solution saturated with OCP is preferably 0.1 mmol / L or higher and 1.0 mmol / L or lower, and more preferably 0.4 mmol / L or higher and 0.6 mmol / L or lower. If the concentration of inorganic phosphate ions is below the lower limit, the OCP serving as the substrate dissolves. If the concentration of inorganic phosphate ions exceeds the upper limit, hydroxyapatite precipitates.

[0100] The concentration of Tris in the Tris-HCl buffer solution in the OCP-saturated buffer solution is preferably 50 mmol / L or more and 300 mmol / L or less, more preferably 100 mmol / L or more and 200 mmol / L or less. If the concentration of the Tris-HCl buffer solution is less than the lower limit, the pH buffering capacity is lost. If the concentration of the Tris-HCl buffer solution exceeds the upper limit, cytotoxicity may occur due to residual Tris.

[0101] The pH of the buffer solution saturated with OCP is preferably 5.0 to 8.0, more preferably 6.8 to 8.0, and even more preferably 7.2 to 7.6. If the pH is below the lower limit, OCP dissolves. If the pH exceeds the upper limit, hydroxyapatite precipitates.

[0102] Examples of buffer solutions supersaturated with respect to OCP include those containing calcium ions (Ca 2+ ), inorganic phosphate ions, and tris-hydrochloric acid buffer, and a buffer with a pH of 5.0 or higher and 8.0 or lower.

[0103] The calcium ion concentration in the buffer solution supersaturated with OCP is preferably 0.5 mmol / L or higher and 5.0 mmol / L or lower, more preferably 1.0 mmol / L or higher and 4.0 mmol / L or lower, and even more preferably 2.5 mmol / L or higher and 3.5 mmol / L or lower. When the calcium ion concentration is within this range, novel OCP crystals precipitate on the OCP crystals. When the calcium ion concentration is below the lower limit, the OCP crystals dissolve. When the calcium ion concentration exceeds the upper limit, hydroxyapatite precipitates.

[0104] The concentration of inorganic phosphate ions in the buffer solution supersaturated with OCP is preferably 0.5 mmol / L to 5.0 mmol / L, more preferably 0.75 mmol / L to 4.0 mmol / L, and even more preferably 1.0 mmol / L to 3.0 mmol / L. When the concentration of inorganic phosphate ions is within this range, novel OCP crystals precipitate on the OCP crystals. When the concentration of inorganic phosphate ions is below this lower limit, the OCP crystals dissolve. When the concentration of inorganic phosphate ions exceeds this upper limit, hydroxyapatite precipitates.

[0105] The concentration of Tris in the Tris-HCl buffer solution in the buffer supersaturated with OCP is preferably 50 mmol / L or more and 300 mmol / L or less, more preferably 100 mmol / L or more and 200 mmol / L or less. If the concentration of the Tris-HCl buffer solution is less than the lower limit, the pH buffering capacity is lost. If the concentration of the Tris-HCl buffer solution exceeds the upper limit, cytotoxicity may occur due to residual Tris.

[0106] The pH of the buffer solution supersaturated with OCP is preferably 5.0 to 8.0, more preferably 6.8 to 8.0, and even more preferably 7.2 to 7.6. If the pH is below the lower limit, OCP dissolves. If the pH exceeds the upper limit, hydroxyapatite precipitates.

[0107] "Second Process"

[0108] When immersing the OCP-containing substrate in a saturated buffer solution, the mass (mg) of the OCP-containing substrate added per unit volume (mL) of the saturated buffer solution is preferably 0.5 mg to 10 mg, more preferably 1.0 mg to 7.5 mg, and even more preferably 3.0 mg to 6.0 mg, in terms of OCP. If the mass of the OCP-containing substrate is less than the lower limit, the surface area of ​​the substrate decreases, and the amount of functional molecules adsorbed decreases. If the mass of the OCP-containing substrate exceeds the upper limit, the amount of adsorption per unit area of ​​the substrate decreases.

[0109] When immersing the OCP-containing substrate in a supersaturated buffer solution, the mass (mg) of the OCP-containing substrate added per unit volume (mL) of the supersaturated buffer solution is preferably 0.5 mg to 10 mg, more preferably 1.0 mg to 7.5 mg, and even more preferably 3.0 mg to 6.0 mg, in terms of OCP. If the mass of the OCP-containing substrate is less than the lower limit, the surface area of ​​the substrate decreases, reducing the amount of functional molecules adsorbed. If the mass of the OCP-containing substrate exceeds the upper limit, the amount of novel OCP crystals precipitated on the OCP crystals decreases.

[0110] When the substrate including the OCP is immersed in a saturated buffer solution, the immersion time is not particularly limited, but is preferably, for example, 10 hours to 120 hours.

[0111] When the substrate including the OCP is immersed in the supersaturated buffer solution, the immersion time is not particularly limited, but is preferably, for example, 10 hours to 120 hours.

[0112] "Third Process"

[0113] In the third step, it is preferred that the substrate containing the OCP having the functional molecule adsorbed thereon be washed with pure water before freeze-drying the substrate containing the OCP having the functional molecule adsorbed thereon.

[0114] After washing the substrate containing the OCP adsorbed with the functional molecule, it is preferably pre-frozen before freeze-drying. The pre-freezing temperature is preferably -196°C or higher and -10°C or lower.

[0115] The freeze-drying time is not particularly limited, but is preferably, for example, 12 hours to 120 hours.

[0116] According to the bone regeneration material of this embodiment, the functional molecule is adsorbed to OCP using a buffer solution containing the functional molecule and being saturated or supersaturated with respect to OCP with an adjusted content of the functional molecule. Therefore, the amount of the functional molecule adsorbed to OCP can be controlled.

[0117] [Bone regeneration material manufacturing device]

[0118] Figure 1 This is a schematic diagram showing a bone regeneration material production apparatus according to one embodiment of the present invention.

[0119] like Figure 1 As shown, the bone regeneration material manufacturing device 1 of this embodiment includes: a reaction tank 2, an acid / base supply part 3, a calcium ion supply part 4, an inorganic phosphate ion supply part 5, a functional molecule supply part 6, a functional molecule concentration part 7, a pH electrode 8, a calcium ion electrode 9 and a control part 10.

[0120] Reaction tank 2 is a tank for reacting the substrate containing OCP with the functional molecule, thereby adsorbing the functional molecule onto the OCP. Specifically, in reaction tank 2, a slurry containing the substrate containing OCP and dissolved functional molecules (hereinafter referred to as "slurry") is reacted with a buffer solution supersaturated with OCP.

[0121] A stirring unit may be provided in the reaction tank 2 , and the stirring unit is used to stir the slurry containing the functional molecules and the buffer solution that is supersaturated with respect to the OCP.

[0122] Examples of the stirring means include a stirring blade and a magnetic stirrer.

[0123] The acid / base supply unit 3 is used to supply acid or base to the reaction tank 2. The acid or base is supplied from the acid / base supply unit 3 to the reaction tank 2 to adjust the pH of the slurry in the reaction tank 2. A pump 11 is provided in the pipe 21. The pump 11 is connected to the control unit 10 via a line 31.

[0124] Examples of the acid include hydrochloric acid, nitric acid, acetic acid, and citric acid.

[0125] Examples of the base include sodium hydroxide, potassium hydroxide, and ammonia.

[0126] The calcium ion supply unit 4 is used to supply calcium ions to the reaction tank 2. Calcium ions are supplied from the calcium ion supply unit 4 to the reaction tank 2 to adjust the calcium ion concentration of the slurry in the reaction tank 2. The calcium ion supply unit 4 is connected to the reaction tank 2 via a pipe 22. A pump 12 is provided in the pipe 22. The pump 12 is connected to the control unit 10 via a line 32.

[0127] The inorganic phosphate ion supply unit 5 is used to supply inorganic phosphate ions to the reaction tank 2. Inorganic phosphate ions are supplied from the inorganic phosphate ion supply unit 5 to the reaction tank 2 to adjust the inorganic phosphate ion concentration of the slurry in the reaction tank 2. The inorganic phosphate ion supply unit 5 is connected to the reaction tank 2 via a pipe 23. A pump 13 is provided in the pipe 23. The pump 13 is connected to the control unit 10 via a line 33.

[0128] The functional molecule may be pre-incorporated into the reaction tank 2. The functional molecule supply unit 6 is connected to the reaction tank 2 via a pipe 24. A pump 14 is provided in the pipe 24. The pump 14 is connected to the control unit 10 via a line 34. The functional molecule can be supplied from the functional molecule supply unit 6 to the reaction tank 2 via the pump 14, and the functional molecule can be dissolved in the slurry in the reaction tank 2.

[0129] After the functional molecules are adsorbed on the substrate, the functional molecule concentrator 7 recovers and concentrates the functional molecules remaining in the supernatant of the slurry and supplies them back to the reaction tank 2 (for reuse). The functional molecule concentrator 7 is connected to the reaction tank 2 via a pipe 25. The pipe 25 is provided with a pump 15 and a pump 16.

[0130] The pH electrode 8 is provided in the reaction tank 2 and is used to measure the pH of the slurry in the reaction tank 2. The pH electrode 8 is connected to the control unit 10 via a wiring 35.

[0131] The calcium ion electrode 9 is provided in the reaction tank 2 and is used to measure the concentration of calcium ions in the slurry in the reaction tank 2. The calcium ion electrode 9 is connected to the control unit 10 via a wiring 36.

[0132] In the bone regeneration material manufacturing apparatus 1, the pH of the slurry in the reaction tank 2 is measured using a pH electrode 8, and the calcium ion concentration of the slurry in the reaction tank 2 is measured using a calcium ion electrode 9. Based on these measurement results (concentrations), the control unit 10 calculates the supersaturation of the slurry relative to the OCP. Based on the calculated supersaturation, the control unit 10 operates pumps 11, 12, and 13 as needed, so that the supersaturation of the slurry in the reaction tank 2 reaches a set value, thereby supplying acid or alkali, calcium ions, and inorganic phosphate ions to the slurry in the reaction tank 2 as needed.

[0133] When the pH and calcium ion concentration of the slurry being stirred in the reaction tank 2 are set to predetermined values, the functional molecules are adsorbed on the OCP.

[0134] Thus, a bone regeneration material can be obtained.

[0135] According to the bone regeneration material manufacturing device of this embodiment, the control unit 10 calculates the supersaturation relative to OCP based on the pH of the slurry in the reaction tank 2 measured by the pH electrode 8 and the calcium ion concentration of the slurry in the reaction tank 2 measured by the calcium ion electrode 9. Based on the supersaturation, the control unit 10 supplies acid or base, calcium ions and inorganic phosphate ions to the slurry in the reaction tank 2 as needed in such a manner that the supersaturation of the slurry in the reaction tank 2 becomes a set value, thereby controlling the adsorption amount of the functional molecule relative to the OCP.

[0136] Here, refer to Figure 2 and Figure 3 The mechanism of promoting the adsorption of functional molecules to a substrate containing OCP in a buffer solution supersaturated with OCP in this embodiment will be described.

[0137] In a buffer solution saturated with OCP, the functional molecules are adsorbed onto the substrate containing the OCP through the interaction between the surface of the substrate and the functional molecules.

[0138] On the other hand, in Figure 2 As shown in the buffer supersaturated with respect to OCP, Figure 3 As shown, a novel OCP 110 is selectively precipitated on the surface of the substrate 100 , and the functional molecules are adsorbed on the novel OCP 110 , thereby increasing the adsorption amount of the functional molecules 200 .

[0139] [Example]

[0140] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0141] [Example 1]

[0142] SDF-1 was dissolved in 10 mmol / L phosphate-buffered saline to prepare a phosphate-buffered saline solution. The SDF-1 content in the phosphate-buffered saline solution was set to 0.5 μg per 5 mg of OCP particles (OCP 0.5 μg SDF-1), 1.0 μg per 5 mg of OCP particles (OCP 1.0 μg SDF-1), and 5.0 μg per 5 mg of OCP particles (OCP 5.0 μg SDF-1).

[0143] The phosphate-buffered saline solution was added dropwise to 5 mg of OCP particles (particle size 300 μm to 500 μm) to allow SDF-1 to be adsorbed on the OCP particles.

[0144] The OCP particles adsorbed with SDF-1 were pre-frozen at -20°C.

[0145] The pre-frozen OCP particles were freeze-dried to obtain a bone regeneration material in which SDF-1 was adsorbed on the OCP particles. Three types of bone regeneration materials with different SDF-1 contents were prepared.

[0146] A standardized defect of 3 mm in diameter and 3 mm in depth was created in the femur of Sprague-Dawley rats (12 weeks old, male).

[0147] 5 mg of OCP 0.5 μg SDF-1 (n=4), 1.0 μg SDF-1 (n=4), and 5.0 μg SDF-1 (n=4) were implanted in the standardized defect. 5 mg of OCP without SDF-1 adsorption (n=2) was also implanted in the standardized defect as a control group.

[0148] Two weeks after embedding, the femurs were removed and decalcified tissue sections were prepared.

[0149] The decalcified tissue sections were stained with hematoxylin-eosin (HE) and the obtained tissue specimens were observed using an optical microscope. Based on the HE tissue images, the new bone area and the area of ​​residual OCP particles were measured, and the ratios of the new bone area and the area of ​​residual OCP particles to the defect area before implantation were calculated. The results are shown in Figure 4 and Figure 5 middle.

[0150] According to the optical microscopic images of tissue specimens, new bone tissue and residual OCP particles were observed in the defect area regardless of the presence or absence of SDF-1 adsorption. Figure 4 The ratio of the new bone area to the defect area is shown in Figure 5 The ratio of the area of ​​the remaining OCP particles to the area of ​​the defects is shown in FIG.

[0151] according to Figure 4 The results shown in Figure 2 show that the ratio of the new bone area to the defect area was the highest in OCP 1.0 μg SDF-1. Figure 5 The results show that the ratio of the remaining OCP particle area to the defect area was lowest for OCP 1.0 μg SDF-1 and highest for OCP 5.0 μg SDF-1. These results indicate that OCP particles adsorbing SDF-1 have the optimal amount (content) of SDF-1 adsorbed for absorption and replacement with new bone.

[0152] [Example 2]

[0153] 200 ng / mL of SDF-1 was dissolved in a buffer saturated with OCP (Ca concentration 0.5 mmol / L, inorganic phosphate concentration 0.5 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4, hereinafter referred to as "0.5Ca0.5Pi buffer") to prepare SDF-1-containing buffer A. Separately, 200 ng / mL of SDF-1 was dissolved in a buffer supersaturated with OCP (Ca concentration 3.0 mmol / L, inorganic phosphate concentration 1.0 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4, hereinafter referred to as "3.0Ca1.0Pi buffer") to prepare SDF-1-containing buffer B.

[0154] Furthermore, bovine serum albumin as a carrier protein was dissolved at a ratio of 1.0 mg / mL in buffer A containing SDF-1 and buffer B containing SDF-1.

[0155] 5.0 mg of OCP particles (particle size less than 53 μm) were immersed in each of buffer A containing SDF-1 and buffer B containing SDF-1 in which bovine serum albumin was dissolved, so that SDF-1 was adsorbed on the OCP particles.

[0156] The OCP particles adsorbed with SDF-1 were washed with pure water.

[0157] The SDF-1-adsorbed OCP particles were then freeze-dried for 24 hours to obtain bone regeneration materials containing SDF-1 adsorbed on the OCP particles. Two bone regeneration materials were prepared using a 0.5Ca0.5Pi buffer solution (0.5Ca0.5Pi group) and a 3.0Ca1.0Pi buffer solution (3.0Ca1.0Pi group).

[0158] 5 mg of 0.5Ca0.5Pi group or 5 mg of 3.0Ca1.0Pi group were mixed with D1 cell line, which is undifferentiated mesenchymal stem cells (MSC) derived from mouse bone marrow, at a rate of 4×10 4 Cells / well were seeded into a 48-well plate and cultured in osteogenic differentiation induction medium. In addition, 5 mg of OCP particles without SDF-1 adsorption were mixed with D1 cells, which are undifferentiated mesenchymal stem cells (MSCs) derived from mouse bone marrow, at a rate of 4 × 10 4 Cells / well were seeded into a 48-well plate and cultured in an osteodifferentiation induction medium. The resultant was set as a control group.

[0159] The deoxyribonucleic acid (DNA) concentration and alkaline phosphatase (ALP) activity of the cells were measured on the 14th or 21st day from the start of culture. ALP is one of the initial differentiation markers of osteoblasts. The measured ALP activity was normalized to the DNA concentration. The data were presented as mean ± standard deviation, and statistically significant differences were analyzed by Tukey-Kramer test. When p was less than 0.05, it was considered to be a significant difference. The results are shown in Figure 6 and Figure 7 middle.

[0160] according to Figure 6 The results shown show that the DNA concentration on day 14 from the start of culture was significantly higher in the 0.5Ca0.5Pi group and the 3.0Ca1.0Pi group than in the control group. In all groups, DNA concentration increased from day 14 to day 21 from the start of culture, but no significant differences were confirmed between the groups.

[0161] according to Figure 7 The results shown show that ALP activity on day 14 from the start of culture was significantly higher in the 3.0Ca1.0Pi group than in the 0.5Ca0.5Pi group. ALP activity also tended to be higher in the 0.5Ca0.5Pi group compared to the control group, but no significant difference was observed. ALP activity increased in all groups from day 14 to day 21 from the start of culture, but no significant differences were observed between the groups.

[0162] The above results indicate that SDF-1 adsorption to OCP particles promotes early MSC proliferation on OCP. Furthermore, SDF-1 adsorbed to OCP particles in a Tris-HCl buffer supersaturated with OCP promotes osteoblast differentiation of MSCs at an earlier stage than SDF-1 adsorbed to OCP particles in a Tris-HCl buffer saturated with OCP.

[0163] [Example 3]

[0164] use Figure 1 The bone regeneration material manufacturing device shown manufactures bone regeneration material.

[0165] The reaction solution was removed from the precipitate of OCP synthesized by the wet method and washed, and pure water was added to the obtained precipitate of OCP to prepare a slurry.

[0166] A lysozyme aqueous solution was supplied to the slurry in advance to adjust the lysozyme concentration in the slurry to 0.1 mg / mL, and the total amount of the slurry in the reaction tank 2 was adjusted to 1000 mL.

[0167] Pump 12 was operated to supply 40 mmol / L of calcium ions from the calcium ion supply unit 4 to the slurry containing lysozyme. Pump 13 was operated to supply 80 mmol / L of inorganic phosphate ions from the inorganic phosphate ion supply unit 5 to the slurry containing lysozyme. The slurry was stirred at 25°C to allow lysozyme to adsorb to the OCP. During this time, the pH electrode 8 and the calcium ion electrode 9 continued to measure the pH of the slurry in the reaction tank 2 and the calcium ion concentration of the slurry in the reaction tank 2. The total amount of slurry after the supply of calcium ions and inorganic phosphate ions was 2000 mL.

[0168] After lysozyme was adsorbed on the OCP, the OCP and the supernatant were recovered from the slurry.

[0169] The concentration of lysozyme in the supernatant was measured to estimate the amount of lysozyme adsorbed on the OCP. Furthermore, the crystal structure of the lysozyme-adsorbed OCP was analyzed by X-ray diffraction.

[0170] During the production of bone regeneration materials, the calcium ion concentration in the slurry increased for approximately 500 seconds after the start of calcium ion supply, and then decreased. The calcium ion concentration was 4.84 mmol / L when calcium ion supply to the slurry was stopped. Furthermore, the calcium ion concentration was 2.16 mmol / L one hour after the cessation of calcium ion supply to the slurry.

[0171] The pH of the slurry when the supply of calcium ions to the slurry was started was about 7. From about 700 seconds after the start of the supply of calcium ions to the slurry, the pH of the slurry decreased to 5.5 and was maintained at this value.

[0172] 24% of the added lysozyme was adsorbed on the surface of the OCP, and 76% remained in the supernatant.

[0173] Furthermore, it was confirmed that the OCP after adsorbing lysozyme maintained its crystal structure as OCP.

[0174] According to the above results, it is confirmed that the Figure 1 The bone regeneration material production apparatus shown measures the pH and calcium ion concentration of the slurry, controls the supersaturation of the slurry, and adsorbs lysozyme onto the OCP.

[0175] [Example 4]

[0176] Cytochrome c (cytochrome c) 0 mg / mL to 10 mg / mL was dissolved in a buffer saturated with OCP (Ca concentration 0.5 mmol / L, inorganic phosphate concentration 0.5 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4) to prepare cytochrome c-containing buffer C. Separately, cytochrome c 0 mg / mL to 10 mg / mL was dissolved in a buffer supersaturated with OCP (Ca concentration 3.0 mmol / L, inorganic phosphate concentration 1.0 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4) to prepare cytochrome c-containing buffer D.

[0177] 5.0 mg of OCP particles (particle size less than 53 μm) were immersed in each of buffer C containing cytochrome c and buffer D containing cytochrome c to allow cytochrome c to be adsorbed on the OCP particles.

[0178] After cytochrome c was adsorbed to OCP, OCP and the supernatant were recovered from buffer C containing cytochrome c and buffer D containing cytochrome c.

[0179] The concentration of cytochrome c in the supernatant was measured to estimate the amount of cytochrome c adsorbed to OCP. Figure 8 In addition, the crystal structure of OCP adsorbed with cytochrome c was analyzed by X-ray diffraction. The results are shown in Figure 9 middle.

[0180] according to Figure 8 The results shown show that, within the range of the initial cytochrome c concentration of 0 mg / mL to 3.0 mg / mL, the amount of cytochrome c adsorbed in the buffer supersaturated with OCP is greater than that in the buffer saturated with OCP.

[0181] according to Figure 9 The results shown confirmed that the crystal structure of cytochrome c as OCP was maintained after adsorption regardless of the initial cytochrome c concentration.

[0182] [Example 5]

[0183] Cytochrome c 0.1 mg / mL was dissolved in a buffer supersaturated with respect to OCP (Ca concentration 2.5 mmol / L, inorganic phosphate concentration 1.0 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4) to prepare cytochrome c-containing buffer E. Separately, cytochrome c-containing buffer F was prepared by further adding 1.0 mg / mL of bovine serum albumin to cytochrome c-containing buffer E.

[0184] 5.0 mg of OCP particles (particle size less than 53 μm) were immersed in Buffer E and Buffer F containing cytochrome c to allow adsorption of cytochrome c onto the OCP particles. The OCP particles were immersed in each buffer for three days. Furthermore, the buffer was replaced daily to allow adsorption of cytochrome c onto the OCP particles.

[0185] Using a transmission electron microscope (JEOL JEM-2100F, manufactured by JEOL Ltd.), OCP particles before cytochrome c adsorption, OCP crystals adsorbed with cytochrome c using buffer E containing cytochrome c, and OCP crystals adsorbed with cytochrome c using buffer F containing cytochrome c were observed. The results are shown in Figures 10 to 12 middle. Figure 10 This is a transmission electron microscope image of an OCP crystal before cytochrome c adsorption. Figure 11 This is a transmission electron microscopy image of OCP crystals adsorbed with cytochrome c using buffer E containing cytochrome c. Figure 12 This is a transmission electron microscopy image of OCP crystals adsorbed with cytochrome c using buffer F containing cytochrome c.

[0186] exist Figure 11 and Figure 12 As shown by the arrows, it can be seen that novel OCP crystals are formed at the ends of the OCP crystals.

[0187] [Example 6]

[0188] Fetuin was dissolved in a buffer saturated with OCP (Ca concentration of 0.5 mmol / L, inorganic phosphate concentration of 0.5 mmol / L, Tris-HCl buffer concentration of 150 mmol / L, pH 7.4, hereinafter referred to as "0.5Ca0.5Pi buffer") so that the fetuin concentration became 0 mg / mL, 0.10 mg / mL, 0.25 mg / mL, 0.50 mg / mL, 0.75 mg / mL, 1.0 mg / mL, or 1.5 mg / mL to prepare a 0.5Ca0.5Pi buffer containing fetuin. Separately, fetuin was dissolved in a buffer supersaturated with respect to OCP (Ca concentration 3.0 mmol / L, inorganic phosphate concentration 1.0 mmol / L, Tris-HCl buffer concentration 150 mmol / L, pH 7.4, hereinafter referred to as "3.0Ca1.0Pi buffer") so that the fetuin concentration became 0 mg / mL, 0.10 mg / mL, 0.25 mg / mL, 0.50 mg / mL, 0.75 mg / mL, 1.0 mg / mL, or 1.5 mg / mL to prepare a 3.0Ca1.0Pi buffer containing fetuin.

[0189] 5.0 mg of OCP particles (particle size less than 53 μm) were immersed in 0.5Ca0.5Pi buffer containing fetuin and 3.0Ca1.0Pi buffer containing fetuin dissolved at various concentrations to allow fetuin to adsorb to the OCP particles. Figure 13 middle. Figure 13 This is a graph showing the adsorption isotherms of fetuin to OCP in a 0.5Ca0.5Pi buffer solution containing fetuin and a 3.0Ca1.0Pi buffer solution containing fetuin.

[0190] like Figure 13 As shown, the amount of fetuin adsorbed increased in the equilibrium concentration range from 0 mg / mL to about 0.3 mg / mL in the 0.5Ca0.5Pi buffer containing fetuin and the 3.0Ca1.0Pi buffer containing fetuin.

[0191] In the case of any adsorption isotherm, the adsorption of fetuin tended to be saturated when the equilibrium concentration exceeded 0.3 mg / mL.

[0192] Regardless of the equilibrium concentration of fetuin, the amount of fetuin adsorbed to OCPs immersed in 3.0Ca1.0Pi buffer was greater than that in 0.5Ca0.5Pi buffer.

[0193] The adsorption parameters of fetuin to OCP were calculated by approximating it in the Langmuir model.

[0194] Under any adsorption isotherm, the correlation coefficient is above 0.99.

[0195] The adsorption equilibrium constant of 0.5Ca0.5Pi buffer solution is 33.5mL / mg. The adsorption equilibrium constant of 3.0Ca1.0Pi buffer solution is 69.4mL / mg.

[0196] The saturated adsorption capacity of 0.5Ca0.5Pi buffer solution is 2.9 mg / m 2 The saturated adsorption capacity of 3.0Ca1.0Pi buffer solution is 4.0mg / m 2 .

[0197] Thus, the adsorption equilibrium constant and the saturated adsorption amount of the 3.0Ca1.0Pi buffer solution were higher than those of the 0.5Ca0.5Pi buffer solution.

[0198] Circular dichroism (CD) spectra of fetuin in 0.5Ca0.5Pi buffer and in 3.0Ca1.0Pi buffer containing 0.25 mg / mL fetuin were measured before and after OCP immersion.

[0199] A circular dichroism spectrometer (J-805, manufactured by JASCO Corporation) was used for the measurement of the CD spectrum.

[0200] The CD spectrum measurement results are shown in Figure 14 middle.

[0201] like Figure 14 As shown, the average residual ellipticity decreases with decreasing fetuin concentration due to adsorption to OCP.

[0202] In addition, if Figure 15 As shown, the secondary structural elements of fetuin in 0.5Ca0.5Pi buffer and 3.0Ca1.0Pi buffer were similar before OCP immersion.

[0203] like Figure 14 and Figure 15 As shown, the secondary structural elements of fetuin were maintained even after immersion in OCP in 0.5Ca0.5Pi buffer and 3.0Ca1.0Pi buffer.

Claims

1. A bone regeneration material, comprising a composite, wherein the composite comprises: a substrate comprising octacalcium phosphate; and a functional molecule, which is adsorbed on the octacalcium phosphate and is at least one selected from growth factors and serum-derived proteins.

2. The bone regeneration material according to claim 1, wherein The substrate comprises a lactic acid-glycolic acid copolymer.

3. The bone regeneration material according to claim 1, wherein The growth factor is at least one selected from stromal cell-derived factor 1, bone morphogenetic protein and vascular endothelial cell proliferation factor.

4. The bone regeneration material according to claim 1, wherein The serum-derived protein is at least one selected from fetuin, albumin and fibronectin.

5. A method for producing a bone regeneration material, comprising: A step of dissolving at least one functional molecule selected from growth factors and serum-derived proteins in phosphate-buffered saline to prepare a phosphate-buffered saline solution; a step of bringing the phosphate-buffered saline solution into contact with a substrate containing octacalcium phosphate to allow the functional molecule to be adsorbed on the octacalcium phosphate; and A step of freeze-drying the substrate including the octacalcium phosphate adsorbed with the functional molecule.

6. The method for producing a bone regeneration material according to claim 5, wherein: The growth factor is at least one selected from stromal cell-derived factor 1, bone morphogenetic protein and vascular endothelial cell proliferation factor.

7. The method for producing a bone regeneration material according to claim 5, wherein: The serum-derived protein is at least one selected from fetuin, albumin and fibronectin.

8. A method for producing a bone regeneration material, comprising: dissolving at least one functional molecule selected from a growth factor and a serum-derived protein in a buffer solution that is saturated or supersaturated with respect to octacalcium phosphate to prepare a buffer solution containing the functional molecule; A step of immersing a substrate including octacalcium phosphate in the buffer solution containing the functional molecule to allow the functional molecule to be adsorbed on the octacalcium phosphate; and A step of freeze-drying the substrate including the octacalcium phosphate adsorbed with the functional molecule.

9. The method for producing a bone regeneration material according to claim 8, wherein: The growth factor is at least one selected from stromal cell-derived factor 1, bone morphogenetic protein and vascular endothelial cell proliferation factor.

10. The method for producing a bone regeneration material according to claim 8, wherein: The serum-derived protein is at least one selected from fetuin, albumin and fibronectin.

11. A bone regeneration material manufacturing device, comprising: A reaction tank for receiving a slurry containing a base material including octacalcium phosphate; an acid / base supplying unit for supplying acid or base to the slurry in the reaction tank; a calcium ion supplying unit for supplying calcium ions to the slurry in the reaction tank; an inorganic phosphate ion supplying unit for supplying inorganic phosphate ions to the slurry in the reaction tank; a functional molecule supplying unit for supplying at least one functional molecule selected from growth factors and serum-derived proteins to the slurry in the reaction tank; a functional molecule concentrating section, which recovers and concentrates the functional molecules remaining in the supernatant of the slurry after the functional molecules are adsorbed on the substrate in the reaction tank, and supplies the functional molecules to the reaction tank again; A pH electrode for measuring the pH of the slurry in the reaction tank; A calcium ion electrode for measuring the concentration of calcium ions in the slurry in the reaction tank; as well as The control unit calculates the supersaturation of the slurry relative to octacalcium phosphate based on the pH of the slurry in the reaction tank measured by the pH electrode and the concentration of calcium ions in the slurry in the reaction tank measured by the calcium ion electrode, and based on the supersaturation, instructs the acid / base supply unit, the calcium ion supply unit, and the inorganic phosphate ion supply unit to supply acid or base, calcium ions, and inorganic phosphate ions to the slurry in the reaction tank in such a manner that the supersaturation of the slurry in the reaction tank becomes a set value.

12. The bone regeneration material manufacturing device according to claim 11, wherein: The growth factor is at least one selected from stromal cell-derived factor 1, bone morphogenetic protein and vascular endothelial cell proliferation factor.

13. The bone regeneration material manufacturing device according to claim 11, wherein: The serum-derived protein is at least one selected from fetuin, albumin and fibronectin.

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