WO2020091035A1

WO2020091035A1 - Material for bone regeneration having surface including hydrophilized biodegradable fibers, and production method therefor

Info

Publication number: WO2020091035A1
Application number: PCT/JP2019/043010
Authority: WO
Inventors: 敏宏春日; 松原　孝至; 将央渡部; 直也大坂
Original assignee: 国立大学法人名古屋工業大学; Ｏｒｔｈｏｒｅｂｉｒｔｈ株式会社
Priority date: 2018-11-02
Filing date: 2019-11-01
Publication date: 2020-05-07
Also published as: JP7429391B2; JPWO2020091035A1

Abstract

The polymer surface of biodegradable fibers included in materials for bone regeneration has water repellency and hydrophobicity, and therefore, when such a material is implanted into an affected area, the material repels blood at a very early stage of being mixed with blood, and therefore it takes a long time for the material to be incorporated during an operation. This material for bone regeneration has a surface including hydrophilized biodegradable fibers, wherein the biodegradable fibers included in the material for bone regeneration contain osteogenic particles, and the surfaces of the biodegradable fibers are each substantially entirely covered by an amorphous calcium phosphate layer, and a portion of the calcium phosphate of the amorphous calcium phosphate layer includes carbonate apatite in which the crystal structure of calcium phosphate is replaced with calcium carbonate.

Description

Bone regeneration material consisting of biodegradable fiber having hydrophilic surface, and method for producing the same

TECHNICAL FIELD The present invention relates to a bone regenerating material comprising biodegradable fibers having hydrophilicity imparted by coating the surface of biodegradable fibers with an amorphous calcium phosphate layer, and a method for producing the same.

Recently, an artificial bone of a type in which a bone regenerating material composed of a biodegradable fiber is implanted in a living body to form a bone is used. The biodegradable fiber contains bone-forming particles such as calcium phosphate, and when the bone regeneration material is implanted in the body and comes into contact with body fluid and the biodegradable resin is hydrolyzed, the surface of the fiber causes bone formation. The effective ions are gradually released to promote bone formation in the affected area.

The biodegradable fiber can be spun using the melt spinning method or the electrospinning method. When the electrospinning method is used, it is possible to spin by adding inorganic particles to a spinning solution in which a resin is dissolved in a solvent, so that a bone regenerating material composed of biodegradable fibers containing osteogenic particles such as calcium phosphate can be used. It can be easily manufactured.

Since the polymer surface of biodegradable fiber has water repellency and hydrophobicity, when implanting the material in the affected area, it will be repelled at the earliest stage of mixing with blood, which will increase the time required for shaping during surgery. The problem has been pointed out. In order to deal with this problem, an attempt to make the surface of the biodegradable fiber hydrophilic has been proposed. As a hydrophilic treatment method, plasma treatment, hydrophilic polymer coating, inorganic film coating, calcium phosphate (CaP) coating are known. CaP coating is performed by immersing biodegradable fibers in an aqueous solution containing phosphate ions. Since the surface can be easily coated with calcium hydroxyphosphate (HA), it is a suitable method for hydrophilizing a fibrous bone regenerating material having a complicated three-dimensional structure.

As the CaP coating, alternate dipping method and biomimetic method are known. In the alternate immersion method, the material is alternately immersed in a solution containing calcium ions and a solution containing phosphate ions to form HA on the surface. It can be performed at room temperature and normal pressure, and can be applied to a material having a three-dimensional structure (Non-Patent Document 1). The biomimetic method forms HA on the surface of a material by immersing the material in a simulated body fluid (SBF) whose inorganic ion concentration is adjusted to be equal to that of human plasma. This method can form HA more easily than the alternate dipping method, and can also coat HA on a three-dimensional structure as in the alternate dipping method (Non-Patent Document 2).

The phase produced by the conventional alternate dipping method or biomimetic method has a high content of HA crystals, so it is difficult to dissolve in contact with water. Further, in order to coat the surface of the material fiber with HA using these methods, the material must be immersed in the solution for several hours to several days, so that the bone-forming particles contained in the biodegradable fiber during There is a problem that the ions are eluted into the solution, and the stimulating effect on the tissue due to the elution of the bone-forming particles is weakened.

The inventors of the present invention have studied the means for solving the above-mentioned problems, and have concluded that it is best to coat the surface of the biodegradable fiber with the amorphous calcium phosphate layer in a short time. Since amorphous calcium phosphate has a high solubility in water, it does not dissolve and disappear in a short time in contact with body fluid or blood, and does not prevent the elution of osteogenic particles. Further, since this amorphous phase has high ionicity, the hydrophilicity of the surface of the polymer fiber coated with it is significantly improved.

Based on the above idea, the inventors of the present invention have conducted extensive experiments and studies, and as a result, heat the biodegradable resin in a state where the vaterite phase calcium carbonate particles are attached to the surface of the biodegradable fiber by using the triboelectric charging method. By adhering and fixing the particles to the fiber surface by doing so, when the material is immersed in an aqueous solution containing phosphate ions of a predetermined concentration in that state, the amorphous calcium phosphate layer starts from the vaterite phase calcium carbonate particles in a short time. It was envisioned that they could grow and coat the entire surface of the fiber.

Based on the above idea, the inventors of the present invention are a method for producing a bone regeneration material comprising a biodegradable fiber whose surface is hydrophilized,

Vaterite phase calcium carbonate particles are electrostatically attached to the surface of the biodegradable fiber containing the bone-forming particles by using a triboelectric charging method,

The vaterite phase calcium carbonate particles adhered to the biodegradable fiber, by heating at a temperature above the glass transition point of the biodegradable resin of the biodegradable fiber, the vaterite phase on the surface of the biodegradable fiber Adhere and fix calcium carbonate particles,

The vaterite phase calcium carbonate particles are adhered and fixed The biodegradable fiber is immersed in an aqueous solution containing a phosphate ion of a predetermined concentration for a predetermined time, whereby the vaterite phase is fixedly adhered to the surface of the biodegradable fiber. Starting from calcium carbonate particles, amorphous calcium phosphate is grown along the surface of the biodegradable fiber to cover the entire surface of the biodegradable fiber,

A bone regenerating material consisting of the biodegradable fiber whose surface is coated with the amorphous calcium phosphate is taken out from the aqueous solution containing the phosphate ion and dried,

The present invention has arrived at a method for producing a bone regeneration material comprising a biodegradable fiber having a hydrophilic surface.

The inventors of the present invention further provide a bone regeneration material comprising a biodegradable fiber whose surface is hydrophilized,

The biodegradable fiber constituting the bone regeneration material contains bone-forming particles,

The entire surface of the biodegradable fiber is covered with an amorphous calcium phosphate layer containing substantially no crystalline phase,

The present invention has reached the invention of a bone regeneration material comprising a biodegradable fiber having a hydrophilic surface.

Preferably, the amorphous calcium phosphate layer contains carbonate apatite containing carbonic acid in a part of calcium phosphate.

The inventors of the present invention further provide a method for producing a biodegradable fiber whose surface is hydrophilized,

Vaterite phase calcium carbonate particles are electrostatically attached to the surface of the biodegradable fiber containing the bone-forming particles by using a triboelectric charging method,

The vaterite phase calcium carbonate particles adhered to the biodegradable fiber, by heating at a temperature above the glass transition point of the biodegradable resin of the biodegradable fiber, the vaterite phase on the surface of the biodegradable fiber Adhere and fix calcium carbonate particles,

The vaterite phase calcium carbonate particles are adhered and fixed The biodegradable fiber is immersed in an aqueous solution containing a phosphate ion of a predetermined concentration for a predetermined time, whereby the vaterite phase is fixedly adhered to the surface of the biodegradable fiber. Starting from calcium carbonate particles, amorphous calcium phosphate is grown along the surface of the biodegradable fiber to cover the entire surface of the biodegradable fiber,

A bone regenerating material consisting of the biodegradable fiber whose surface is coated with the amorphous calcium phosphate is taken out from the aqueous solution containing the phosphate ion and dried,

The invention has arrived at a method for producing a biodegradable fiber having a hydrophilic surface.

The inventors of the present invention further have a biodegradable fiber whose surface is hydrophilized,

The biodegradable fiber contains bone-forming particles,

The entire surface of the biodegradable fiber is covered with an amorphous calcium phosphate layer containing substantially no crystalline phase,

The invention has been reached, which is a biodegradable fiber whose surface is hydrophilized.

Preferably, the amorphous calcium phosphate layer contains carbonate apatite containing carbonic acid in a crystal structure in a part of calcium phosphate.

Preferably, the vaterite phase calcium carbonate particles include siloxane, and the amorphous calcium phosphate layer grown from the vaterite phase calcium carbonate particles including siloxane includes silicon.

Preferably, the aqueous solution containing phosphate ions is a disodium hydrogen phosphate solution, and the bone regenerating material comprising biodegradable fibers having the vaterite phase calcium carbonate particles adhered and fixed to the surface thereof is used as the disodium hydrogen phosphate. Upon immersion in the solution, the amorphous calcium phosphate layer is formed containing sodium and coats the surface of the fiber.

Preferably, the diameter of the biodegradable fiber is 10 to 100 μm, and the diameter of the vaterite phase calcium carbonate particles is 0.5 to 4 μm. More preferably, the diameter of the biodegradable fiber is 20 to 60 μm, and the diameter of the vaterite phase calcium carbonate particles is 0.7 to 2.0 μm.

Preferably, the biodegradable resin of the biodegradable fiber is PLGA.

Preferably, the osteogenic particles are β-phase tricalcium phosphate particles.

Preferably, the bone-forming particles are silicon-eluting type vaterite phase calcium carbonate particles.

The inventors of the present invention further provide a method for hydrophilizing the surface of the biodegradable fiber constituting the cell culture substrate,

Vaterite phase calcium carbonate particles on the surface of the biodegradable fiber constituting the cell culture substrate, electrostatically attached using a triboelectric charging method,

By heating the biodegradable fiber to which the vaterite phase calcium carbonate particles are attached at a temperature equal to or higher than the glass transition point of the biodegradable resin of the biodegradable fiber, the vaterite phase carbonate is formed on the surface of the biodegradable fiber. Adhere and fix calcium particles,

The vaterite phase calcium carbonate particles are adhered and fixed The biodegradable fiber is immersed in an aqueous solution containing a phosphate ion of a predetermined concentration for a predetermined time, whereby the vaterite phase is fixedly adhered to the surface of the biodegradable fiber. Starting from calcium carbonate particles, amorphous calcium phosphate is grown along the surface of the biodegradable fiber to cover the entire surface of the biodegradable fiber,

Removing the biodegradable fiber whose surface is coated with the amorphous calcium phosphate from an aqueous solution containing the phosphate ion and drying the biodegradable fiber;

The invention has been reached, which is a method of hydrophilizing the surface of the biodegradable fiber constituting the cell culture substrate.

The inventors of the present invention further provide a cell culture substrate comprising a biodegradable fiber whose surface is hydrophilized,

The biodegradable fiber constituting the cell culture substrate contains an inorganic filler,

Substantially the entire surface of the biodegradable fiber is covered with an amorphous calcium phosphate layer,

The amorphous calcium phosphate layer may include carbonate apatite containing carbonic acid in a crystal structure in a part of calcium phosphate,

The invention has been reached, which is a cell culture substrate comprising a biodegradable fiber whose surface is hydrophilized.

The inventors of the present invention further have a biodegradable fiber whose surface is hydrophilized,

The biodegradable fiber consists essentially of biodegradable resin,

Substantially the entire surface of the biodegradable fiber is covered with an amorphous calcium phosphate layer,

The invention has been reached, which is a biodegradable fiber whose surface is hydrophilized.

Preferably, the aqueous solution containing phosphate ions is a disodium hydrogen phosphate solution, and the bone regeneration material comprising biodegradable fibers having the vaterite phase calcium carbonate particles adhered and fixed on the surface thereof is used as the disodium hydrogen phosphate. Upon immersion in the solution, the amorphous calcium phosphate layer is formed containing sodium and coats the surface of the fiber.

Preferably, the biodegradable resin of the biodegradable fiber is PLGA.

In one embodiment of the present invention, the vaterite phase calcium carbonate particles fixed on the surface of the biodegradable fiber are immersed in an aqueous solution containing phosphate ions and rapidly dissolved to supply carbonate ions to form a calcium phosphate layer. It can be formed on the fiber surface in a short time. The calcium phosphate layer thus formed is substantially entirely occupied by the amorphous phase and has no crystal structure (see FIG. 6).

In one embodiment of the present invention, in the process of covering and growing the surface of the biodegradable fiber, a large amount of carbonate ions are supplied from the calcium carbonate particles fixed on the surface of the fiber to form a highly soluble amorphous phase calcium phosphate. In some cases, soluble carbonate-containing apatite may be formed.

In one embodiment of the invention, the amorphous calcium phosphate coating the surface of the biodegradable fiber is bioabsorbable and the layer is so thin that it is absorbed shortly after implanting the material in vivo. It decomposes and disappears from the fiber surface. As a result, the elution of ions from the bone-forming particles contained in the biodegradable fiber is not impeded by the presence of the amorphous calcium phosphate layer.

In one embodiment of the present invention, the calcium phosphate phase coated on the surface of the biodegradable fiber has a large specific surface area and high ionicity, so that many proteins can be adsorbed. As a result, high initial adhesiveness of cells is obtained when the bone regeneration material is implanted in the body.

In one embodiment of the present invention, silicon is supported on the vaterite phase calcium carbonate particles fixed on the surface of the fiber, and the silicon is eluted in the course of dissolution of the vaterite phase calcium carbonate by the phosphoric acid treatment. It is incorporated in the amorphous calcium phosphate layer formed on the surface. When the bone regenerating material of the present invention is implanted into the body, silicon contained in the amorphous calcium phosphate layer is eluted and stimulates osteoblasts to promote bone formation.

In one embodiment of the present invention, a disodium hydrogen phosphate solution is used for the phosphoric acid treatment, and sodium is contained in the amorphous calcium phosphate layer during the process of forming the amorphous calcium phosphate layer on the surface of the fiber by the phosphoric acid treatment. Is taken into.

One embodiment of the present invention is a cell culture substrate comprising a biodegradable fiber whose surface is hydrophilized. The biodegradable fiber coated with amorphous calcium phosphate has high protein adsorption performance and good initial cell adhesion performance.

One embodiment of the present invention is a biodegradable fiber whose surface is hydrophilized, said biodegradable fiber consisting essentially of a biodegradable resin and containing no inorganic particles.

FIG. 1 illustrates a method of depositing calcium carbonate particles on the surface of polymer fibers using the triboelectric charging method of the present invention. FIG. 2 shows a method of embedding calcium carbonate particles attached to the surface of a polymer fiber by using the triboelectric charging method of the present invention by utilizing the residual stress of the fiber. FIG. 3 shows a method of immersing a cotton-like bone regeneration material in an aqueous solution containing phosphate ions to perform phosphoric acid treatment. FIG. 4 (A) is a phosphoric acid treatment in which a biodegradable fiber having calcium carbonate particles adhered to the surface of a polymer fiber is immersed in a 0.02 M disodium hydrogen phosphate solution using the triboelectric charging method of the present invention and treated with phosphoric acid The state of the surface of the fiber after applying is shown. FIG. 4B is a phosphoric acid treatment in which a biodegradable fiber having calcium carbonate particles adhered to the surface of a polymer fiber by the triboelectric charging method of the present invention is immersed in a 0.2 M disodium hydrogen phosphate solution. The state of the surface of the fiber after applying is shown. FIG. 4C is a phosphoric acid treatment in which a biodegradable fiber having calcium carbonate particles adhered to the surface of a polymer fiber by the triboelectric charging method of the present invention is immersed in a 2.0 M concentration disodium hydrogen phosphate solution. The state of the surface of the fiber after applying is shown. FIG. 5 shows the hydrophilicity of the phosphorous-treated cotton-shaped bone regeneration material. FIG. 6 shows the results of STEM observation of a cotton-shaped bone regeneration material that has been treated with phosphoric acid. From the vague image in the lower left part a, it can be seen that the calcium phosphate layer is substantially entirely occupied by the amorphous phase. FIG. 7 (A) shows the surface of a biodegradable fiber obtained by immersing a sample of the amorphous calcium phosphate coating experiment in a disodium hydrogen phosphate solution having a concentration of 19.2 mM for 5 minutes to grow an amorphous calcium phosphate layer. .. FIG. 7 (B) shows the surface of a biodegradable fiber obtained by immersing the sample of the amorphous calcium phosphate coating experiment in a disodium hydrogen phosphate solution having a concentration of 19.2 mM for 10 minutes to grow an amorphous calcium phosphate layer. .. FIG. 8 (A) shows the surface of a biodegradable fiber in which an amorphous calcium phosphate coating experiment sample was immersed in a disodium hydrogen phosphate solution having a concentration of 192 mM for 5 minutes to grow an amorphous calcium phosphate layer. FIG. 8 (B) shows the surface of a biodegradable fiber obtained by immersing a sample of the amorphous calcium phosphate coating experiment in a disodium hydrogen phosphate solution having a concentration of 192 mM for 10 minutes to grow an amorphous calcium phosphate layer. FIG. 9 (A) shows the surface of a biodegradable fiber in which an amorphous calcium phosphate coating experiment sample was immersed in a disodium hydrogen phosphate solution having a concentration of 1.92 M for 5 minutes to grow an amorphous calcium phosphate layer. .. FIG. 9 (B) shows the surface of a biodegradable fiber obtained by immersing a sample of the amorphous calcium phosphate coating experiment in a disodium hydrogen phosphate solution having a concentration of 1.92 M for 10 minutes to grow an amorphous calcium phosphate layer. .. In Fig. 10 (A), 20 mg of the sample (19.2 mM) was set aside and placed in a test tube, and a weight (56.5 mg) was placed and pressed for 2 min. Then, the weight was removed, and 5 μl of a red solution prepared by dissolving Rhodamine B in distilled water was dropped on the sample, and after 5 minutes, the red solution permeated the sample. In FIG. 10 (B), 20 mg of the sample (19.2 mM) was set aside and placed in a test tube, and a weight (56.5 mg) was placed and pressed for 2 min. Then, the weight was removed, and 5 μl of a red solution prepared by dissolving Rhodamine B in distilled water was dropped on the sample, and after 10 minutes, the red solution permeated the sample. In Fig. 11 (A), 20 mg of the sample (192 mM) was set aside and placed in a test tube, and a weight (56.5 mg) was placed and pressed for 2 min. Then, the weight was removed, and 5 μl of a red solution prepared by dissolving Rhodamine B in distilled water was dropped on the sample, and after 5 minutes, the red solution permeated the sample. In Fig. 11 (B), 20 mg of the sample (192 mM) was set aside and placed in a test tube, and a weight (56.5 mg) was placed and pressed for 2 min. Then, the weight was removed, and 5 μl of a red solution prepared by dissolving Rhodamine B in distilled water was dropped on the sample, and after 10 minutes, the red solution permeated the sample. In Fig. 12 (A), 20 mg of the sample (1.92M) was set aside and placed in a test tube, and a weight (56.5 mg) was placed and pressed for 2 min. Then, the weight was removed, and 5 μl of a red solution prepared by dissolving Rhodamine B in distilled water was dropped on the sample, and after 5 minutes, the red solution permeated the sample. In FIG. 12 (B), 20 mg of the sample (1.92 M) was set aside and placed in a test tube, and a weight (56.5 mg) was placed and pressed for 2 min. Then, the weight was removed, and 5 μl of a red solution prepared by dissolving Rhodamine B in distilled water was dropped on the sample, and after 10 minutes, the red solution permeated the sample. FIG. 13 shows the results of an adsorption test of bovine serum albumin (BSA) on the surface of the biodegradable fiber coated with the amorphous calcium phosphate of the present invention. FIG. 14 shows an evaluation method for evaluating the cell adhesion / proliferation property of the bone regeneration material comprising the biodegradable fiber coated with the amorphous calcium phosphate of the present invention. FIG. 15 shows a diagram of metabolic activity values of cells by Alamar Blue. FIG. 16 shows the morphology of the fiber surface by FE-SEM and the state after the cells have grown. The part surrounded by the line is the part where cells exist. FIG. 17 shows that the CaP phase is confirmed by morphology of the fiber surface by FE-SEM and strong expansion of the fiber surface after 24 hours of culture. FIG. 18 shows the elution behavior of ions from the sample measured by ICP-AES. FIG. 19 shows an example of usage of the cotton-like bone filling material of the present invention.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.
<Biodegradable fiber>
The bone regeneration material of the present invention is composed of biodegradable fibers spun by electrospinning or melt spinning. The biodegradable fiber used in the present invention contains bone-forming particles such as calcium phosphate, and when the material is implanted in the body and the biodegradable resin is decomposed, the bone-forming particles contained in the fiber are changed in the process. Ions are eluted and promote bone formation in the affected area. By collecting the spun biodegradable fibers in the form of cotton or nonwoven fabric, a fibrous bone regeneration material is formed.

The thickness of the biodegradable fiber used in the present invention is preferably 10 to 100 μm in diameter, more preferably 20 to 60 μm in diameter. In the present invention, since the inorganic particles having a diameter of about 1 to several μm must be evenly distributed and adhered / fixed on the surface of the biodegradable fiber, the diameter of the biodegradable fiber should be 10 μm or more. preferable. When the fiber diameter is 10 μm or less, it becomes difficult to uniformly distribute and fix the particles on the surface of the fiber.

Biodegradable fibers having a diameter of 10 μm or more can be easily spun by using the melt spinning method. However, in the melt spinning method, since the amount of inorganic particles that can be mixed with the molten resin is limited, it is difficult to include the bone-forming particles in an amount exceeding 5% by weight. On the other hand, in the electrospinning method, since the spinning solution is prepared by dissolving the resin or the composite of the resin and the inorganic particles with the solvent, it is possible to include a larger amount of the bone-forming particles. The inventors of the present invention succeeded in spinning a biodegradable fiber containing 70% by weight of inorganic particles by using a thermal kneading method together (Japanese Patent No. 6251462).
However, in the electrospinning method, the solvent is volatilized during the winding process due to the unstable flight trajectory (bending instability) of the spinning solution discharged from the nozzle of the device before reaching the collector. Since the phenomenon of making ultrafine fibers is utilized, the diameter of the fiber spun by it is usually several tens nm to several μm, and it is difficult to make the diameter 10 μm or more. The inventors of the present invention installed a nozzle having a large diameter downward, sent the spinning solution filled in a syringe to the exit of the nozzle at a high speed, and ejected the spinning solution vertically downward to obtain a diameter of 10 μm or more. Have succeeded in stably spinning the biodegradable fiber (Japanese Patent Application No. 2019-73453).

As the resin of the biodegradable fiber used in the present invention, biodegradable resin such as PLA, PLGA, PCL can be used. PLGA, which is an amorphous resin and has a glass transition point of about 40 ° C., is particularly preferably used. The biodegradable fiber used in the present invention contains a considerable amount of inorganic particles that are bone-forming particles such as calcium phosphate and silicon-eluting calcium carbonate, and the bone regeneration material implanted in the living body is in contact with body fluid. When the biodegradable resin is decomposed, the bone-forming particles contained in the process are eluted to promote bone formation.

<Material for bone regeneration>
In the present invention, the bone regeneration material includes an implant material in which biodegradable fibers are collected in a non-woven fabric or cotton form. As a preferred method of using the bone regenerating material of the present invention, the operator fills the bone regenerating material with the bone regenerating material as it is, or mixes the material with blood in advance and embeds it in the bone defective portion (see FIG. 19). ).

<Osteogenic particles>
In the present invention, the bone-forming particles refer to inorganic particles that are contained in a bone regenerating material and are implanted into the human body together with the material to elute ions that promote bone formation by coming into contact with body fluid or blood. Bone-forming particles include, but are not limited to, tricalcium phosphate, hydroxyapatite, calcium carbonate, and the like.

<Vaterite phase calcium carbonate particles>
In the present invention, vaterite phase calcium carbonate particles are preferably used as the inorganic particles to be adhered and fixed on the surface of the biodegradable fiber by using the triboelectric charging method. Calcium carbonate has the most stable calcite phase at room temperature and normal pressure, the metastable aragonite phase, and the metastable vaterite phase.The calcium phosphate phase starts from the particles when immersed in an aqueous solution containing phosphate ions. The vaterite phase, which has a high solubility in water, is most suitable for the production of

The vaterite phase calcium carbonate particles used in the present invention preferably have a diameter of 0.5 μm to 4.0 μm.
It is important that the range of the particle size of the vaterite phase calcium carbonate recovered in the form of particles is smaller than the thickness of the biodegradable fiber to which the particles are attached. Furthermore, it is preferable that the particle size is smaller than the larger particle size because the reaction with the aqueous solution containing phosphate ions becomes faster.

Siloxane can be supported on the surface of the vaterite phase calcium carbonate particles by the carbonation process (Japanese Patent No. 5131724). When the amount of siloxane supported on the surface of the vaterite phase calcium carbonate particles by the carbonation process is increased, the particle diameter increases accordingly, and the maximum particle diameter increases to 4 μm. If the amount of siloxane is further increased from the level, it becomes difficult to recover the vaterite phase calcium carbonate as particles.

<Friction charging method>
In the present invention, the triboelectric charging method refers to a method for adhering and fixing inorganic particles on the surface of polymer fibers. Specifically, when the powder of inorganic particles and the fiber material are housed in a container such as a plastic bag and the container is vibrated up and down or front and back and left and right at high speed with the container in that state, the inorganic particles and the fibers are The resin surface is rubbed to generate static electricity, and the generated electrostatic attraction uniformly attaches the inorganic particles to the entire surface of the fiber material (see FIG. 1). Alternatively, the material can be put into a ball mill and rotated without adding a ball (only by the pot) to uniformly attach the material.

By heating the fibrous material having the inorganic particles attached to the surface thereof by the triboelectric charging method at a temperature equal to or higher than the glass transition point of the resin, the inorganic particles attached to the surface of the fibers by electrostatic attraction can be fixed. The biodegradable fiber is pulled during spinning flight in electrospinning, and in melt spinning, the fiber is pulled when it is wound on a rotating drum and residual stress is generated inside the fiber. When heat is applied to the fiber, the fiber shrinks in the longitudinal direction, and during the shrinking process, the inorganic particles adhering to the polymer surface are embedded inside at the contact part with the fiber and firmly fixed at that position (Fig. 2).

<Phosphoric acid treatment>
A fibrous material having inorganic particles adhered and fixed on the surface is immersed in an aqueous solution containing phosphate ions, and allowed to stand for a fixed time under a predetermined temperature condition. Then, the fiber material is taken out from the aqueous solution containing phosphate ions and dried (see FIG. 3).

As the aqueous solution containing a phosphate ion used for the phosphoric acid treatment of the present invention, an aqueous solution containing a phosphate ion such as a disodium hydrogen phosphate aqueous solution or a diammonium hydrogen phosphate aqueous solution is preferable. By using an aqueous solution of disodium hydrogen phosphate, it is possible to add sodium (Na) to the calcium phosphate phase formed on the fiber surface.

When observing the fiber surface of the material dried by immersing it in a 0.02M, 0.2M, 2.0M disodium hydrogen phosphate aqueous solution for 5 minutes, at a concentration of 0.02M, calcium phosphate was found near the vaterite phase calcium carbonate particles. In the concentration of 0.2 M, calcium phosphate was formed on the entire fiber surface. At 2.0 M, the particles fixed on the surface of the fiber fell off and small pores were formed (Fig. 4 (A), (B), (C)). In order for calcium phosphate to cover the entire fiber surface, it is important to adjust the concentration of the aqueous solution containing phosphate ions in which the material is immersed.

<Amorphous calcium phosphate layer>
In the present invention, amorphous calcium phosphate includes carbonate apatite (Ca _10-x (HPO ₄ , CO ₃ ) _x (PO ₄ ) _6-x (OH, CO ₃ ) _2-x · nH ₂ O) and / or calcium. Deficient calcium phosphate (Ca _10-x (HPO ₄ ) _x (PO ₄ ) _6-x (OH) _2-x・ nH ₂ O may be included. Amorphous calcium phosphate is soluble at around neutral pH Is high and dissolves in a short time on contact with water.
In the present invention, when the bone regeneration material is immersed in an aqueous solution containing phosphate ions, an amorphous calcium phosphate layer is formed along the surface of the fiber from the vaterite phase calcium carbonate particles fixed on the surface of the fiber by the triboelectric charging method. And cover the entire surface of the fiber.

FIG. 6 shows that, using the method of the present invention, SiV (silicon-eluting calcium carbonate) particles are used as the vaterite phase calcium carbonate to be adhered and fixed on the fiber surface, and disodium hydrogen phosphate (concentration 0) as an aqueous solution containing phosphate ions. SEM observation image of the surface of the biodegradable fiber coated with calcium phosphate by using S. The presence of Na and Si was identified. The biodegradable fiber did not chemically react with the resin surface and was fixed via the vaterite phase CaCO ₃ particles embedded in the fiber surface.

<Cell culture substrate>
In the present invention, the cell culture substrate is composed of a non-woven fabric made of biodegradable fibers spun by the electrospinning method. By coating the surface of the biodegradable fiber constituting the cell culture substrate with amorphous calcium phosphate, protein adsorption on the fiber surface is improved, and thus high cell initial adhesion can be obtained.

Amorphous Calcium Phosphate Coating Experiment Samples used in the experiment were prepared using the materials shown below.
ReBOSSIS (registered trademark) (cotton-like bone regeneration material consisting of biodegradable fibers with an outer diameter of 10 to 60 μm. Distributor ORTHOREBIRTH Co., LTD) Composition of ReBOSSIS (registered trademark): PLGA 30 wt% / β-TCP 40 wt % / SiV30 wt%)
・ PLLA: PGA (85:15) Evonik LG855S (not including D body)
Β-TCP: β-TCP-100 manufactured by Taihei Chemical Industry Co., Ltd. A β-TCP crushed product obtained by crushing 1.7 mm in particle size to about 4 μm was used.
・ SiV (silicon elution type vaterite phase calcium carbonate): calcium hydroxide (reagent special grade purity of 96% or more Wako Pure Chemical Industries, Ltd.), methanol (reagent special grade purity of 99.8% or more Wako Pure Chemical Industries, Ltd.), γ -Aminopropyltriethoxysilane (SILQUEST A-1100 with a purity of 98.5% or more Momentive Performance Materials Japan LLC), carbon dioxide (high purity liquefied carbon dioxide purity 99.9% Taiyo Chemical Co., Ltd.) Was used to prepare a vaterite phase calcium carbonate having a Si content of 2.9% by weight. SiV particle size: 1 to 1.5 μm. Details of the method for producing SiV are disclosed in Japanese Patent Laid-Open No. 2008-100878 (silicon-eluting calcium carbonate and method for producing the same).

1. Step of Adhering SiV Particles to Biodegradable Fibers Constituting ReBOSSIS® 1. Adhesion of SiV particles to the fiber surface
1.0 g of SiV powder and 0.1 g of ReBOSSIS (registered trademark) were put in a glass bottle (5 cmφ × 10 cm), rotated (264 rpm, 2 min.) For temporary adhesion and heat treatment (60 ° C., 10 min.). Then, electrostatically non-adhering SiV particles existing on the sample surface were removed by a compressor to prepare a sample.

Step 2. Dip the sample in an aqueous solution containing phosphate ions (ReBOSSIS® with SiV powder adhered) 50 ml of disodium hydrogen phosphate aqueous solution with different concentrations (19.2, 192 mM, 1.92 M) at 37 ° C It was soaked for 5 and 10 min respectively.

Step 3. The dried sample was removed from the aqueous disodium hydrogen phosphate solution and then dried in a drier (50 ° C., 2 h). SEM images of the surface of the fiber of the sample obtained as a sample (19.2 mM), a sample (192 mM), and a sample (1.92 M) for each concentration of the disodium hydrogen phosphate aqueous solution are shown in FIG. 7 (A) ( B), FIGS. 8A and 8B, and FIGS. 9A and 9B.

2. 20 mg each of the hydrophilic evaluation sample (19.2 mM), sample (192 mM) and sample (1.92 M) was placed in a test tube, and a weight (56.5 mg) was placed thereon and pressed for 2 min. Then, the weight was removed, and 5 μl of a red solution prepared by dissolving Rhodamine B in distilled water was added dropwise to the sample to evaluate hydrophilicity. The results are shown in FIGS. 10 (A) and (B), FIGS. 11 (A) and (B), and FIGS. 12 (A) and 12 (B).

2. Throughout the hydrophilicity evaluation result (1), it was observed that the time until the red solution permeated became shorter as the concentration of the phosphoric acid solution was increased.
(2) Throughout the whole, it was observed that the sample soaked in the aqueous solution containing phosphate ions for 10 minutes had a shorter time until the red solution soaked in the sample for 10 minutes.
(3) Regarding the sample treated with phosphoric acid at 19.2 mM, although calcium phosphate or other calcium phosphate salt was partially formed, it was said that it took time until the red solution permeated because the amount of adhesion was uneven. Seem. As the treatment time was lengthened, the time until soaking became shorter.
(4) Regarding the sample treated with phosphoric acid at 1.92 M, it was observed that pores were formed on the fiber surface. This hole is considered to be a portion where SiV particles were embedded. It is considered that the treatment with the high concentration of 1.92 M caused the PLGA to erode and the SiV particles to fall off. Regarding the sample treated with phosphoric acid at 1.92 M, it was observed that the sample partly collapsed and the shape could not be maintained. This is considered to indicate that the fibers of ReBOSSIS (registered trademark) were eroded by the aqueous disodium hydrogen phosphate solution (alkaline).

Under the same conditions as the above experiment, except that 1.0g of SiV powder and 0.5g of ReBOSSIS registered trademark were put in a glass bottle (5cm × 10cm) and rotated (264rpm, 2min.) To adhere and fix the particles to the fiber surface. The same result was obtained in the case of heat treatment. ‥

Under the same conditions as the above experiment, except that 5.0g of SiV powder and 0.5g of ReBOSSIS registered trademark are put in a glass bottle (5cm x 10cm) and rotated (264rpm, 2min) to adhere and fix the particles to the fiber surface. The same result was obtained in the case of heat treatment.

Under the same conditions as the above experiment, except that the disodium hydrogen phosphate solution concentration was set to 100 mM / L, 150 mM / L for 5 minutes to form a calcium phosphate layer on the surface of the fiber, the disodium hydrogen phosphate solution At a concentration of 100 mM / L, the red solution was not soaked instantly, but at 150 mM / L water was soaked instantly. It is considered that 150 mM / L is more suitable than 100 mM / L as the optimum concentration of the disodium hydrogen phosphate aqueous solution for the water to soak into the water instantly.
For the purpose of precipitating calcium phosphate from vaterite phase calcium carbonate, a higher concentration of disodium hydrogen phosphate is better (eg 192 mM / L), but if the concentration is too high, PLGA will be attacked and SiV particles will fall off. there's a possibility that.

Under the same conditions as in the above experiment, except that the fibers were finely broken when they were immersed in an aqueous solution of disodium hydrogen phosphate and dried with a dryer at a temperature of 37 ° C.
The reason is that after drying at 37 ° C, it could not be dried sufficiently even after 4 hours, and it was left for 4 hours with the phosphoric acid solution, as a result, the reaction proceeded and the cracks in the fiber became high. The phosphoric acid solution with a high concentration was infiltrated, and calcium phosphate was deposited there, which is considered to have caused the destruction.
In order to avoid such a phenomenon, it is considered necessary to dry at 50 ° C. or higher as soon as possible after immersing in an aqueous solution of disodium hydrogen phosphate. The upper limit of the temperature may be limited to 110 ° C, but if it is 60 ° C or higher, the precipitated calcium phosphate phase will be buried again in PLGA, so 50 to 60 ° C is considered optimal.

Protein Adsorption Measurement on Bone Regenerating Material Coated with Amorphous Calcium Phosphate In order to see the effect of the amorphous calcium phosphate coating of the present invention on protein adsorption, BSA adsorption on a sample (192 mM) coated with amorphous calcium phosphate The amount was compared with the amount of BSA adsorbed on the sample not coated with calcium phosphate. The result is shown in FIG.

The following considerations were obtained from the results of the BSA adsorption experiment.
(1) It is considered that since the sample hydrophilized with the amorphous calcium phosphate coating has a large specific surface area, the amount of adsorbed protein becomes large.
(2) In the sample not coated with calcium phosphate, since the bone-forming particles contained in the fibers, SiV, are also present near the surface, it is considered that they are adsorbed by COO − of BSA molecule and electrostatic interaction.
(3) In the sample hydrophilized with the amorphous calcium phosphate coating, Ca ^{2+ on} the surface of the amorphous calcium phosphate present on the fiber surface, NH ⁴⁺ in SiV and COO ^{− of the} BSA molecule are adsorbed by electrostatic interaction. It is considered to have contributed.
(4) It is considered that the reason why the adsorption amount became almost constant after the elapse of a certain time is because the adsorption sites disappeared.

Cell Proliferation Test Using Bone Regeneration Material Coated with Amorphous Calcium Phosphate A culture test was performed using osteoblast-like cells MC3T3-E1 strain. The outline is shown in FIG. 80 mg of bone regeneration material was added per well of a 24-well plate, 2 mL of α-MEM medium was injected, cells were seeded, and a culture test was performed at 37 ° C. in the presence of 5% CO ₂ . The cell seeding density was 90,000 cells / mL, and the culture time points were 3 hr, 24 hr, and 72 hr, respectively. After assessing the amount of ions in the medium by ICP-AES, transfer the solution to an unused well plate and allow Alamar Blue to react for 4 hours to assess metabolic activity, and for bone regeneration after cell proliferation. Regarding the material, the morphology of the sample surface was observed by FE-SEM.

The metabolic activity was significantly higher in the bone regenerating material-injected sample than in the control glass plate sample alone, and was significantly higher in the hydrophilized complex than in the untreated complex. In addition, the metabolic activity was higher in 24 hr than in 3 hr and in 72 hr than in 24 hr in each sample. The result is shown in FIG.
From the morphological observation of the sample surface by FE-SEM, it was found that the hydrophilized composite had a rough surface with larger irregularities than the untreated composite. The cells were covered more with the hydrophilized complex, and the fiber surface was entirely covered with the cells. The result is shown in FIG. The CaP phase was produced on both the untreated complex and the hydrophilized complex on the fiber surface after 24 hours of culturing, and in the latter, the CaP phase was proceeding. The CaP phase was partially formed in the untreated complex, which is probably due to the mechanism similar to that of the biomimetic method. The results are shown in FIG.

Regarding the ion elution behavior from the biodegradable fiber coated with calcium phosphate of the present invention, the elution of Ca ion and P ion was almost the same between the hydrophilized complex and the untreated complex, but the elution of Si ion Only at the 24-hour and 72-hour time points, the hydrophilized complex tended to be less. It is considered that Si was eluted from SiV when it was immersed in the solution in the phosphoric acid treatment, and that it was due to the coating with the amorphous calcium phosphate layer. Note that the amount of elution was the same at the time point of 3 hours. It is considered that this is due to elution from the SiV particles / amorphous phase on the surface of the hydrophilized fiber. The results are shown in FIG.

Although the present invention has been described with reference to the examples of the bone regeneration material using the biodegradable fiber containing the bone-forming particles, the present invention is not limited to the examples, and the bone regeneration is not limited thereto. A biodegradable fiber containing a biodegradable fiber constituting a material for use, and further, a biodegradable fiber containing no inorganic particles and / or a cell culture substrate using such a biodegradable fiber is also coated with an amorphous calcium phosphate layer. As long as it is a material that has been removed, it is similarly applicable.

Claims

A method for producing a bone regeneration material comprising a biodegradable fiber whose surface is hydrophilized,

Vaterite phase calcium carbonate particles are electrostatically attached to the surface of the biodegradable fiber containing the bone-forming particles by using a triboelectric charging method,

The biodegradability to which the vaterite phase calcium carbonate particles are attached, by heating at a temperature not lower than the glass transition point of the biodegradable resin of the biodegradable fiber, the vaterite phase carbonate on the surface of the biodegradable fiber. Adhere and fix calcium particles,

The vaterite phase calcium carbonate particles are adhered and fixed The biodegradable fiber is immersed in an aqueous solution containing a phosphate ion of a predetermined concentration for a predetermined time, whereby the vaterite phase is fixedly adhered to the surface of the biodegradable fiber. Starting from calcium carbonate particles, amorphous calcium phosphate is grown along the surface of the biodegradable fiber to cover the entire surface of the biodegradable fiber,

A bone regenerating material consisting of the biodegradable fiber whose surface is coated with the amorphous calcium phosphate is taken out from the aqueous solution containing the phosphate ion and dried,

A method for producing a bone regeneration material comprising a biodegradable fiber having a hydrophilic surface.
The method according to claim 1, wherein the biodegradable fiber has a diameter of 10 μm to 100 μm.
The method according to claim 1 or 2, wherein the vaterite phase calcium carbonate particles have a diameter of 0.5 µm to 4 µm.
The method according to any one of claims 1 to 3, wherein the aqueous solution containing phosphate ions is a disodium hydrogen phosphate solution.
The method according to any one of claims 1 to 4, wherein the biodegradable fiber comprises PLGA resin.
A bone regenerating material comprising a biodegradable fiber having a hydrophilic surface,

The biodegradable fiber constituting the bone regeneration material contains bone-forming particles,

The entire surface of the biodegradable fiber is covered with an amorphous calcium phosphate layer containing substantially no crystal structure,

A bone regeneration material comprising a biodegradable fiber whose surface is hydrophilized.
The bone regenerating material according to claim 6, wherein the amorphous calcium phosphate layer contains carbonate apatite containing carbonic acid in a crystal structure in a part of calcium phosphate.
The bone regeneration material according to claim 6 or 7, wherein the biodegradable fiber has a diameter of 10 µm to 100 µm.
The bone regeneration material according to any one of claims 6 to 8, wherein the biodegradable fiber contains PLGA resin.
A method for producing a biodegradable fiber whose surface is hydrophilized,

Vaterite phase calcium carbonate particles on the surface of the biodegradable fiber containing bone-forming particles, electrostatically attached using a triboelectric charging method,

The vaterite phase calcium carbonate particles adhered to the biodegradable fiber, by heating at a temperature above the glass transition point of the biodegradable resin of the biodegradable fiber, the vaterite phase on the surface of the biodegradable fiber Adhere and fix calcium carbonate particles,

The vaterite phase calcium carbonate particles are adhered and fixed The biodegradable fiber is immersed in an aqueous solution containing a phosphate ion of a predetermined concentration for a predetermined time, whereby the vaterite phase is fixedly adhered to the surface of the biodegradable fiber. Starting from calcium carbonate particles, amorphous calcium phosphate is grown along the surface of the biodegradable fiber to cover the entire surface of the biodegradable fiber,

Removing the biodegradable fiber whose surface is coated with the amorphous calcium phosphate from an aqueous solution containing the phosphate ion and drying the biodegradable fiber;

A method for producing a biodegradable fiber having a hydrophilic surface.
The method according to claim 10, wherein the diameter of the biodegradable fiber is 10 μm to 100 μm.
The method according to claim 10 or 11, wherein the vaterite phase calcium carbonate particles have a diameter of 0.5 µm to 4 µm.
The method according to any one of claims 10 to 12, wherein the aqueous solution containing phosphate ions is a disodium hydrogen phosphate solution.
The method according to any one of claims 10 to 13, wherein the biodegradable fiber comprises PLGA resin.
A biodegradable fiber whose surface is hydrophilized,

The biodegradable fiber contains bone-forming particles,

The entire surface of the biodegradable fiber is covered with an amorphous calcium phosphate layer containing substantially no crystal structure,

A biodegradable fiber whose surface is hydrophilized.
The surface-hydrophilized biodegradable fiber according to claim 15, wherein the amorphous calcium phosphate layer contains carbonate apatite containing carbonic acid in a crystal structure in a part of calcium phosphate.
The biodegradable fiber according to claim 15 or 16, wherein the diameter of the biodegradable fiber is 10 μm to 100 μm.
The biodegradable fiber according to any one of claims 15 to 17, wherein the biodegradable fiber contains PLGA resin.