JP6918326B2 - Composite porous scaffolding material containing dexamethasone and its manufacturing method - Google Patents

Description

The present invention relates to a composite porous scaffold material containing the compound dexamethasone having bone inducibility and a method for producing the same, in order to repair bone tissue damaged or lost due to a disease or accident. be.

When the bone tissue of humans and other vertebrates is deficient, if the deficiency is of a certain size, autologous bone tissue or allogeneic bone tissue that uses the patient's own ilium or splenic bone has been transplanted as a treatment. .. However, there is a limit to the size of autologous bone tissue that can be collected, and there is a problem of immune rejection when trying to transplant allogeneic bone tissue. It cannot be fully utilized. At present, these solutions are not available, and new treatments are being considered to replace them. As a specific method, attention is being paid to regeneration / reconstruction of bone tissue using a biotissue engineering method. In biotissue engineering, a material that serves as a scaffold for cells, that is, a scaffold material, is used when regenerating and reconstructing a tissue. The scaffold material maintains functions such as cell proliferation, differentiation, and extracellular matrix secretion, and also plays a role in maintaining the shape of new bone tissue. In order to enhance the therapeutic effect of bone defects, bone is formed in the scaffold material, and when implanted in natural bone in the living body, bone is formed along the surface of the scaffold material, and the scaffold material Bone conduction is important because the bone is united with the bone.

The scaffold material needs to be absorbed with tissue regeneration and eventually replaced by tissue. Bioabsorbable synthesis of polylactic acid (PLA), polyglycolic acid (PGA), copolymer of lactic acid and glycolic acid (PLGA), polycaprolactone (PCL), etc. is usually used as the base material for scaffolding materials. Examples include polymers and natural polymers such as collagen, gelatin, chitosan, and alginic acid. In addition, bioabsorbable ceramics such as tricalcium phosphate and octacalcium phosphate are also used. However, even if these materials are used alone, satisfactory results cannot be obtained in terms of bone formation. In order to promote bone formation, a mechanism that can continuously supply bone-inducible signals to cells in the scaffold material is required.

As examples of previous studies on scaffolding materials having bone inducibility, Non-Patent Documents 1 and 2 show porous scaffolding materials and nanofiber meshes prepared from a mixture of a bioabsorbable polymer and dexamethasone, respectively. A bioabsorbable polymer with excellent molding processability and handleability is used as the base material for the porous scaffolding material, but since it does not contain a component that promotes bone conduction, the surrounding tissue when transplanted into a living body It cannot be expected that it will be firmly fixed. Further, as a previous research example of a porous scaffold material having bone conductivity in addition to bone inducibility, Non-Patent Documents 3 and 4 describe a porous body made of hydroxyapatite in which dexamethasone is supported on fine particles of a bioabsorbable polymer. A porous material physically adsorbed on the surface of the pores of the body is shown. However, since it is made of ceramics as a base material, it is poor in molding processability and handleability as compared with a porous body made of a bioabsorbable polymer. Moreover, since hydroxyapatite does not have bioabsorbability, it does not replace the bone tissue of the living body. There is a demand for a scaffold material having a porous structure for cells to invade, having both bone inducibility and bone conductivity, and having high molding processability.

In addition, another biological tissue filling material containing dexamethasone has been developed (see, for example, Patent Document 1). According to Patent Document 1, a biological tissue filling material containing calcium phosphate fine particles and dexamethasone in a hydrogel such as collagen is provided. However, since the hydrogel of the present invention is spherical with a diameter of 1 to 1000 μm and they are not bound to each other, there is a concern that the hydrogel will leak even if it is implanted in a tissue defect site. Further, since the hydrogel does not have a porous structure in which cells can invade, cells can adhere only to the surface of the hydrogel. Therefore, a material that can retain its shape at the tissue-deficient site until the tissue is regenerated and has a porous structure for cells to invade is desired.

Japanese Unexamined Patent Publication No. 2008-279007

Yoon JJ et al. , Biomaterials, 24, 2323-2329 (2003). Martins A. et al. , Biomaterials, 31, 5875-5858 (2010). Jun Sik Son et al. , J. Control Release, 153, 133-140 (2011). Son JS, et al. , J. Biomed. Mater. Res. A. , 99, 638-647 (2011).

From the above, an object of the present invention is to provide a porous scaffold material having bioabsorbability, excellent biocompatibility, inducing bone formation, and excellent bone conductivity, and a method for producing the same. be.

The present inventors have the structure obtained by introducing dexamethasone-containing calcium phosphate nanoparticles into a polymer porous body, which can retain a specific shape, has biocompatibility, and forms bone. And found that it has a property that enables regeneration induction, and completed the present invention.

The composite porous scaffold material according to the present invention contains a porous body made of a bioabsorbable polymer and dexamethasone-containing nanoparticles located in the porous body, and the dexamethasone is contained in at least bioabsorbable calcium phosphate. This solves the above problem.
The calcium phosphate may contain the dexamethasone.
The particle size of the nanoparticles may be in the range of 1 nm or more and 1000 nm or less.
The nanoparticles may have a shape selected from the group consisting of spheres, rods, stars, spindles, triangular prisms, rectangular parallelepipeds and cubes.
The bioabsorbable polymer includes collagen, gelatin, hyaluronic acid, chitosanchondroitin sulfate, fibronectin, bitronectin, laminin, polylactic acid, polyglycolic acid, copolymer of lactic acid and glycolic acid, poly (ε-caprolactone), poly ( It may be selected from the group consisting of glycerol sebacic acid).
The porous body may have pores having a pore diameter in the range of 10 μm or more and 1000 μm or less.
The bioabsorbable polymer may be crosslinked.
^{The content of nanoparticles per 1 cm 3 of the} composite scaffold material may be in the range of 1 mg or more and 500 mg or less.
The content of dexamethasone in the nanoparticles may be in the range of 1 wt% or more and 70 wt% or less.
The content of dexamethasone in the nanoparticles may be in the range of 18 wt% or more and 50 wt% or less.
The nanoparticles and the bioabsorbable polymer may satisfy the range of 2: 1 or more and 1: 2 or less in terms of weight ratio.
The dexamethasone may be contained in a mixture of bioabsorbable calcium phosphate and hydroxyapatite.
The method for producing the above-mentioned composite porous scaffold material according to the present invention is a step of mixing dexamethasone-containing nanoparticles and a bioabsorbable polymer to make them porous, and the dexamethasone is contained in at least bioabsorbable calcium phosphate. The steps to be performed are included, thereby solving the above-mentioned problems.
In the step of making the porous material, the mixture of the nanoparticles and the bioabsorbable polymer may be freeze-dried.
Pore-forming agents may be used to form pores.
The pore-forming agent may be ice.
As the hardening step, a method consisting of a foam molding method, a pore forming agent removing method, and a phase separation method may be used.
Following the step of making the porosity, a step of cross-linking the bioabsorbable polymer may be further included.
The nanoparticles and the bioabsorbable polymer may be mixed so as to satisfy the range of 2: 1 or more and 1: 2 or less in terms of weight ratio.
The nanoparticles and the bioabsorbable polymer may be mixed so as to satisfy the range of 1.5: 1 or more and 1: 1.5 or less in terms of weight ratio.

Since the composite porous scaffold material of the present invention contains a porous body made of a bioabsorbable polymer and dexametazone-containing nanoparticles located therein, cells transplanted to a defective site of bone tissue are placed in the porous body. It can invade and efficiently differentiate cells into bone. Further, since the dexamethasone-containing nanoparticles are located in the porous body, it is possible to suppress a decrease in the efficiency with which the nanoparticles are diffused and the dexamethasone is supplied to the bone defect site. In addition, dexamethasone can be sustained during bone tissue regeneration. In addition, the nanoparticles supporting dexamethasone contain bioabsorbable calcium phosphate, and thus have excellent bone conductivity. Further, both the porous body and the dexamethasone-containing nanoparticles have bioabsorbability and are excellent in biocompatibility.

The method for producing a composite porous scaffold material of the present invention is a step of mixing dexamethasone-containing nanoparticles and a bioabsorbable polymer to make them porous, in which dexamethasone is contained at least in bioabsorbable calcium phosphate. Including the steps, the above-mentioned composite porous scaffold material is obtained. According to the method of the present invention, a complicated process and a special device are not required, so that the method is practical.

The figure which shows typically the composite porous scaffolding material of this invention. The figure which shows the TEM image of the product of Reference Example 1. The figure which shows the SEM image of the scaffolding material 1 of Example 1. The figure which shows the SEM image of the scaffolding material 2 of Example 2. The figure which shows the SEM image of the scaffolding material 3 of Example 3. The figure which shows the time dependence of the sustained-release amount of dexamethasone which is sustained-release from the scaffolding material 1-3 of Examples 1-3 The figure which shows the appearance of the bone tissue regenerated using the scaffolding material 1-3 of Examples 1-3 The figure which shows the hematoxylin-eosin staining image of the bone tissue regenerated using the scaffolding material 1-3 of Examples 1-3 The figure which shows the immunostaining image of the bone tissue regenerated using the scaffolding material 1-3 of Examples 1-3 The figure which shows the immunostaining image of the bone tissue regenerated using the scaffolding material 4 of the comparative example 4.

Hereinafter, the composite porous scaffolding material of the present invention and a method for producing the same will be described with reference to the drawings.
FIG. 1 is a diagram schematically showing a composite porous scaffold material of the present invention.

The composite porous scaffolding material of the present invention (hereinafter, simply referred to as the scaffolding material of the present invention) 100 is a porous body 110 made of a bioabsorbable polymer and dexamethasone (DEX) -containing nanoparticles located in the porous body 110. Includes 120 and. Dexamethasone is contained in at least bioabsorbable calcium phosphate. Since the scaffolding material 100 of the present invention is made of a material having bioabsorbability in both the porous body 110 and the nanoparticles 120, it has excellent biocompatibility as a whole. Since the scaffold material 100 of the present invention contains dexamethasone, it is excellent in bone inducibility, but since dexamethasone is supported on nanoparticles 120 and is located in the porous body 110, it is transplanted to a defect site of bone tissue. The cells can be invaded into the porous body 110, and the cells can be efficiently differentiated into bones. In addition, since the nanoparticles 120 do not diffuse, it is possible to suppress a decrease in the efficiency of supplying dexamethasone to the bone defect site. In addition, dexamethasone is released slowly so that bone tissue can be regenerated over a long period of time. The nanoparticles 120 carrying DEX are composed of at least calcium phosphate and have excellent bone conductivity. That is, the scaffolding material 100 of the present invention is excellent in bioabsorbability and biocompatibility, induces bone formation, and is also excellent in bone conductivity.

Preferably, in the scaffolding material 100 of the present invention, the nanoparticles 120 are composed of calcium phosphate containing dexamethasone. This enables long-term sustained release. As used herein, inclusion is intended that calcium phosphate surrounds dexamethasone at least in part.

The external dimensions of the scaffolding material 100 of the present invention are not particularly limited and may be appropriately determined depending on the applicable portion. However, for example, the maximum side length is 0.1 mm or more and 200 mm or less, preferably 2 mm or more and 100 mm or less. Have.

Since the scaffolding material 100 of the present invention contains the porous body 110, it includes pores 130. The pore diameter of the pore 130 preferably has a range of 10 μm or more and 1000 μm or less. When the pore diameter is less than 10 μm, the seeded cells and the cells of the surrounding bone tissue cannot invade the pores. When the pore size exceeds 1000 μm, the efficiency of filling the pores with the extracellular matrix derived from cells is significantly reduced. More preferably, the pore diameter is 20 μm or more and 800 μm or less. This can promote bone differentiation. In FIG. 1, the hole 130 has a spherical shape, but the shape of the hole 130 is not limited to a spherical shape. Depending on the manufacturing method, it may have a shape other than a spherical shape.

The porosity of the scaffolding material 100 of the present invention is usually 5 to 99.9%, preferably 20 to 99.9%. If the porosity is less than 5%, the porosity is significantly reduced and the cells cannot be evenly distributed.

In the scaffold material 100 of the present invention, the bioabsorbable polymer (hereinafter, simply referred to as a polymer) constituting the porous body 110 is not particularly limited as long as it has bioabsorbability, but collagen, gelatin, and the like are preferable. Natural macromolecules such as chitin, hyaluronic acid, chitosan, chondroitin sulfate, fibronectin, hydronectin, laminin, alginic acid and keratin can be mentioned. In addition, polyglycolic acid, polylactic acid, copolymer of glycolic acid and lactic acid, poly (glycerol sebacic acid), poly (α-hydroxy acid) such as polyapple acid, poly-ε-caprolactone, poly-β-hydroxycarboxylic acid. Such as synthetic polymers can be mentioned.

Among them, preferably, the polymer is collagen, gelatin, hyaluronic acid, chitosanchondroitin sulfate, fibronectin, vitronectin, laminin, polylactic acid, polyglycolic acid, copolymer of lactic acid and glycolic acid, poly (ε-caprolactone), poly. Selected from the group consisting of (glycerol sebacic acid). These polymers are particularly excellent in biocompatibility.

More preferably, the polymer is a natural polymer selected from the group consisting of collagen, gelatin, fibronectin and laminin derived from animals such as cows, pigs, salmon and birds, and one kind or two of these polymers can be used. It can be used after mixing the above types. Among these, collagen or a mixture containing collagen as a main component is most desirable as a polymer having particularly excellent cell affinity. In the specification of the present application, the amount of the main component is one containing 50 wt% or more of collagen.

The polymer may be crosslinked. This stabilizes the porous body 110. In particular, when the polymer is a water-soluble bioabsorbable polymer typified by gelatin or collagen, the effect of cross-linking is large. When a water-insoluble bioabsorbable polymer typified by polylactic acid or polyglycolic acid is used as the polymer, cross-linking is not always necessary.

In nanoparticles 120, dexamethasone is contained in at least bioabsorbable calcium phosphate, and specific examples thereof include tricalcium phosphate (TCP) and octacalcium phosphate (OCP). As tricalcium phosphate (TCP), one type of β, α, α', and γ phases, or one consisting of two or more types of phases can be used, but an appropriate one is selected according to the purpose. .. Dexamethasone may be contained in other basic calcium phosphates in addition to calcium phosphate. Illustratively, there is biphasic calcium phosphate with β-tricalcium phosphate and hydroxyapatite.

The shape of the nanoparticles 120 is not particularly limited as long as the nanoparticles 120 can be located in the porous body 110, but is exemplified by a group consisting of a spherical shape, a rod shape, a star shape, a spindle shape, a triangular prism, a rectangular parallelepiped, and a cube. It may be the shape of choice. With these shapes, the nanoparticles 120 can be dispersed and positioned in the porous body 110.

The particle size (average particle size) of the nanoparticles 120 has a range of 1 nm or more and 1000 nm or less. If the particle size is less than 1 nm, it may not be possible to contain dexamethasone. If the particle size exceeds 1000 nm, the uniform dispersibility of the particles is lowered when the particles are compounded with the porous body. Preferably, the particle size is in the range of 1 nm or more and 800 nm or less. As a result, the nanoparticles can be uniformly located in the porous body. More preferably, the particle size is in the range of 5 nm or more and 50 nm or less. As a result, sufficient dexamethasone can be contained and uniformly dispersed in the porous body.

In the specification of the present application, the particle size (average particle size) is a biaxial average diameter obtained from the major axis and the minor axis of a predetermined number (for example, 100) of particles. The major axis and the minor axis may be values geometrically obtained by an electron microscope, a sieve, or the like, or may be values indirectly obtained by a sedimentation method. Alternatively, depending on the shape of the particles, it may be obtained from the volume-based particle size distribution.

The content of dexamethasone in the nanoparticles 120 is preferably in the range of 1 wt% or more and 70 wt% or less. If the content of dexamethasone is less than 1 wt%, the supply of dexamethasone may be insufficient and the effect of inducing bone differentiation may be reduced. If the content of dexamethasone exceeds 70 wt%, calcium phosphate may not be able to surround dexamethasone. The content of dexamethasone is more preferably in the range of 18 wt% or more and 50 wt% or less. Within this range, cells can be continuously supplied with a sufficient amount of dexamethasone for bone formation, and high bone induction efficiency can be obtained.

^{The content of nanoparticles 120 per 1 cm 3} of the scaffolding material 100 of the present invention is preferably in the range of 1 mg or more and 500 mg or less. When the content of the nanoparticles 120 is less than 1 mg, the distribution density of the nanoparticles 120 in the scaffolding material 100 is too low, the supply of dexamethasone is insufficient, and the effect of inducing bone differentiation may be reduced. In addition, sufficient bone conduction may not be obtained. When the content of the nanoparticles 120 exceeds 500 mg, it is difficult to stably hold all the nanoparticles 120 in the porous body 110. Further, the moldability is also lowered, and the scaffolding material 100 having a predetermined pore may not be obtained.

^{The content of the nanoparticles 120 per 1 cm 3} of the scaffolding material 100 of the present invention is more preferably in the range of 5 mg or more and 50 mg or less. Within this range, the effect of bone differentiation is high. ^{The content of the nanoparticles 120 per 1 cm 3} of the scaffolding material 100 of the present invention is even more preferably in the range of 5 mg or more and 20 mg or less. Within this range, molding processability is good, cells can be reliably differentiated into bone, and sufficient bone conductivity can be obtained. Since it is difficult to determine the content of calcium phosphate nanoparticles in the composite porous scaffolding material with high accuracy, it is simply the same as the amount of calcium phosphate nanoparticles charged in the composite porous scaffolding material. You may assume.

In the scaffolding material 100 of the present invention, preferably, the nanoparticles 120 and the polymer 110 (nanoparticles 120: polymer 110) satisfy the range of 2: 1 or more and 1: 2 or less in terms of mass ratio. When the amount of the polymer 110 is more than 1: 2, the dexamethasone is insufficient and the effect of bone differentiation is reduced. If the number of nanoparticles 120 is larger than 2: 1, the nanoparticles 120 may not be uniformly positioned on the polymer 110. More preferably, the nanoparticles 120 and the polymer 110 satisfy a mass ratio of 1.5: 1 or more and 1: 1.5 or less. Within this range, cells can be reliably differentiated into bones and sufficient bone conductivity can be obtained. Since it is difficult to obtain the mass ratio of the nanoparticles 120 and the polymer 110 in the scaffolding material 100 with high accuracy, it can be simply regarded as the same as the amount of nanoparticles and polymers charged at the time of manufacture. can.

The composite porous scaffold material 100 according to the present invention has excellent biocompatibility and enables bone formation and induction of regeneration. By implanting the scaffold material 100 of the present invention in a site where human or animal bone tissue is defective and slowly releasing the contained dexamethasone, bone differentiation of various cells invading the pores of the scaffold material 100 is induced. Can be regenerated and rebuilt. Alternatively, bone tissue, bone cells, etc. are brought into contact with the pores of the scaffold material 100 of the present invention, and the contained dexamethasone is slowly released to induce bone differentiation of various cells invading the pores of the scaffold material 100. , Can be regenerated / reconstructed. In this way, by using the scaffolding material 100 of the present invention, it is possible to repair and reconstruct various bone tissues in vivo and in vitro.

Next, an exemplary production method of the composite porous scaffolding material of the present invention will be described.
The method for producing a scaffold material of the present invention includes a step of mixing nanoparticles made of bioabsorbable calcium phosphate containing dexamethasone and a bioabsorbable polymer (polymer) to make them porous. Here, the polymer is omitted because it is as described with reference to FIG.

Nanoparticles of bioabsorbable calcium phosphate containing dexamethasone may be prepared or commercially available if available. As an exemplary method for producing nanoparticles, a solution for dissolving dexamethasone, a calcium salt salt, and a phosphate salt may be mixed and heated to react. The solution for dissolving dexamethasone is a solution in which dexamethasone is dissolved in a protic polar solvent such as water or alcohol typified by ethanol, and is prepared, for example, at a concentration in the range of 0.05 M or more and 10 M or less. The calcium salt salt is, for example, calcium nitrate tetrahydrate, and is prepared in an aqueous solution having a concentration of 0.1 M or more and 1 M or less. The phosphate is, for example, disodium hydrogen phosphate, and is prepared in an aqueous solution having a concentration of 0.1 M or more and 1 M or less. These are mixed and heated and stirred at a temperature of 40 ° C. or higher and 80 ° C. or lower to react. Then, the slurry obtained by the reaction may be aged at room temperature for 10 hours or more and 48 hours or less. After mixing, the pH may be controlled to 8 or more and 12 or less with ammonium hydroxide or the like. The nanoparticles containing dexamethasone thus obtained are as described with reference to FIG. 1, and are therefore omitted.

The nanoparticles and the polymer are mixed so as to have a weight ratio of 2: 1 or more and 1: 2 or less. As a result, the nanoparticles can be dispersed and positioned in the polymer. More preferably, the nanoparticles and the polymer are mixed so as to have a weight ratio of 1.5: 1 or more and 1: 1.5 or less. Thereby, a scaffolding material satisfying the content of nanoparticles per ^{1 cm 3} of the above-mentioned scaffolding material can be produced.

Porousization of a mixture of nanoparticles and a polymer is carried out by using a foam molding method using a foaming agent, a pore forming agent removing method, a phase separation method or the like. At this time, the polymer may be used as it is, or may be dissolved in an arbitrary solvent in which the polymer is uniformly dissolved. For example, when the polymer is a natural polymer such as collagen, a protic polar solvent typified by an alcohol such as ethanol can be used. When the pore-forming agent removing method is adopted, it is preferable to dissolve it in a solvent because the pore-forming agent described later is uniformly mixed with the polymer. Further, when the phase separation method is adopted, it is preferable to dissolve the polymer in a solvent because phase separation can be easily caused.

(1) Foaming formation method In the foaming molding method using a foaming agent, a foaming agent is added to a mixture of nanoparticles and a polymer (hereinafter, simply referred to as a mixture), and if necessary, the mixture is heated under pressure. do. Air bubbles are formed by the action of the foaming agent with heating, and the composite porous scaffold material of the present invention can be obtained.

The foaming agent can be any solid or gas that produces a foaming action when mixed with the mixture described above (which may be referred to as a melt or mixed solution), although solid foaming agents are exemplary exemplary. , A particle or crystal of a salt such as ammonium carbonate, sodium carbonate, magnesium carbonate, sodium bicarbonate, and the gas foaming agent is, for example, an inert gas such as carbon dioxide, nitrogen, hydrogen, argon, helium. Is. When a gas foaming agent is used, it may be directly blown into the mixture to foam it.

When the foaming agent is added, the foaming agent and its amount can be appropriately determined according to the degree of foaming, the size of the obtained air foam, and the like. At the time of foaming, pressurization can be appropriately applied if necessary. As the foaming temperature, when a solid foaming agent is used, a temperature at which gas can be generated from the foaming agent is adopted.

(2) Pore-forming and removing method In the pore-forming and removing method, water-soluble sugar or salt powder or crystals are used as a porosity-forming agent, which is mixed with a mixture and usually dried and subjected to high molecular weight. By incorporating the pore-forming agent into the molecule and then removing the pore-forming agent, the composite porous scaffold material of the present invention can be obtained. When the pore-forming agent is added, the size and shape of the obtained air bubbles can be controlled by appropriately selecting the size and shape of the pore-forming agent.

Water when the pore-forming agent is a neutral salt composed of sodium chloride or potassium chloride, a water-soluble sugar such as butou sugar or sugar, or an organic acid salt such as sodium tartrate, sodium aspartate or sodium citrate. If it is washed with a solvent such as, it will be dissolved and removed. The solvent dissolves the pore-forming agent, but is not particularly limited as long as it does not dissolve the polymer. When the pore-forming agent is a water-soluble salt or a water-soluble sugar, it is easily dissolved and removed with water.

As the pore forming agent, ice fine particles made of ice can also be used. In this case as well, if the ice fine particles are mixed with the mixture and freeze-dried, the ice fine particles are removed, and the composite porous scaffold material of the present invention can be obtained. Freeze-drying is preferably carried out under vacuum vacuum.

(3) Phase Separation Method In the phase separation method, a solution of a polymer dissolved in a solvent is phase-separated into a concentrated phase and a dilute phase by changing the temperature, and the phase-separated solution is freeze-dried to obtain the composite porous scaffold of the present invention. The material is obtained. The conditions for phase separation differ depending on the type of polymer, the type of solvent, and the concentration of the polymer solution, and those skilled in the art will set the conditions in consideration of these. At this time, there are a method of gradually lowering the temperature, a method of suddenly lowering the temperature, and a method of holding the cloudy temperature of the polymer solution for a certain period of time and then suddenly lowering the temperature. By appropriately setting these conditions, the pore diameter and pore shape of the obtained composite porous scaffold material can be controlled.

In the above operation, a composite porous scaffold material containing a porous body made of a bioabsorbable polymer and nanoparticles containing dexamethasone located therein and containing at least bioabsorbable calcium phosphate is obtained. Can be manufactured.

Following the step of making the polymer porous, a step of cross-linking the polymer may be performed. Thereby, the structure of the porous body can be stabilized and a strong scaffolding material can be provided. As described above, when a water-soluble bioabsorbable polymer represented by gelatin or collagen is used, cross-linking is necessary, but a water-insoluble bioabsorbable polymer represented by polylactic acid or polyglycolic acid is used. If so, cross-linking is not always necessary. As the cross-linking method used, any conventionally known cross-linking method can be used. Generally, the vapor method or the solution method can be used.

When the vapor method is adopted as the cross-linking method, it is preferable to use the above-mentioned cross-linking agent in the form of a gas. Specifically, the scaffolding material is crosslinked at a constant temperature at a constant concentration or in an atmosphere of a crosslinking agent vapor saturated with an aqueous solution thereof for a certain period of time. As the cross-linking agent used in the steam method, any conventionally known cross-linking agent can be used. Preferred cross-linking agents are aldehydes such as glutaraldehyde, formaldehyde, paraformaldehyde, especially glutaraldehyde. The cross-linking temperature may be within a range in which the scaffolding material does not melt and vapor of the cross-linking agent can be formed, and is usually set to 20 ° C. to 50 ° C. The cross-linking time depends on the type of the cross-linking agent and the cross-linking temperature, but it is carried out within a range in which the cross-linking immobilization is performed so as not to inhibit the bioabsorbability of the scaffold material and to prevent the scaffold material from being dissolved at the time of transplantation into a living body. Is desirable. The preferred cross-linking time is about 10 minutes to 12 hours.

When the solution method is adopted as the cross-linking method, the cross-linking agent such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and other carbodiimides, aldehydes and epoxies and the activity of N-hydroxysuccinimide and the like are used. Crosslink with an agent. Since the uncrosslinked scaffold material is soluble in water, these crosslinkers are dissolved in a mixed solvent of ethanol and water to crosslink. The volume ratio of ethanol to water is from 99/1 to 1/99. The cross-linking temperature is 4 ° C to 40 ° C, preferably room temperature. The concentration of the cross-linking agent is 5 mM to 500 mM, preferably 10 mM to 100 mM. The concentration of the activator is 5 mM to 500 mM, preferably 10 mM to 100 mM.

After the step of cross-linking by the evaporation method or the solution method, the obtained scaffold material may be washed and the cross-linking may be stopped. Specifically, the scaffolding material after the final cross-linking reaction is washed by immersing it in pure water at room temperature, and this is repeated three times as one washing. After washing, the composite porous scaffold material is immersed in an aqueous glycine solution to inactivate unreacted active functional groups. The concentration of the aqueous glycine solution is 0.01M to 1.0M, preferably 0.05M to 0.3M. The immersion temperature is 4 ° C. to 37 ° C., preferably 4 to 30 ° C. The immersion time is 1 to 24 hours, preferably 4 to 12 hours.

After the above-mentioned porosification step or cross-linking step, the obtained scaffold material may be washed and dried. Illustratively, washing with pure water for 30 minutes is repeated 3 times or more. After washing, freeze-drying is performed for 48 hours under a reduced pressure of 5 Pa or less.

Next, the present invention will be described in detail with reference to specific examples, but it should be noted that the present invention is not limited to these examples.

[Reference example 1]
Prior to the examples, three types of nanoparticles having different contents of dexamethasone used in the examples were produced. Here, the nanoparticles contained at least bioabsorbable calcium phosphate.

1.5 mg, 3.0 mg and 4.5 mg of dexamethasone (Sigma-Aldrich LLC) were dissolved in 2 mL of ethanol (Wako Pure Chemical Industries, Ltd.), respectively, to prepare an ethanol solution of dexamethasone. The prepared ethanol solution of dexamethasone was added to a calcium nitrate tetrahydrate solution (Sigma Aldrich LLC) to prepare a calcium nitrate tetrahydrate concentration of 0.5 M and a total volume of the solution of 50.0 mL. .. 50.0 mL of a 0.5 M calcium nitrate tetrahydrate solution containing dexamethasone was added dropwise to 33.4 mL of a 0.5 M diammonium hydrogen phosphate solution (Sigma-Aldrich LLC) at a rate of 150 mL / h. While stirring this mixed solution at 55 ° C., an ammonium hydroxide solution (Sigma-Aldrich LLC) was added dropwise to adjust the pH of the reaction solution to 9.5. Then, with stirring, the reaction was carried out in a water bath at 55 ° C. for 30 minutes. The slurry of the product obtained by the reaction was held at room temperature for 36 hours and aged. The solution was centrifuged at 8000 rpm for 5 minutes and washed with Milli-Q water 5 times or more.

The morphology of the obtained product was observed with a transmission electron microscope (TEM, model number JEM-2100F, JEOL Ltd.). The results are shown in FIG.

FIG. 2 is a diagram showing a TEM image of the product of Reference Example 1.

FIG. 2 shows a low-magnification TEM image (A) and a high-magnification TEM image (B) of the product obtained using 4.5 mg of dexamethasone. According to FIG. 2, the product is nanoparticles having a rod-like shape, with a major axis in the range of 25 nm or more and 70 nm or less, a minor axis in the range of 4 nm or more and 20 nm or less, and a major axis and a minor axis. It was found that the biaxial average diameter calculated from the above ranged from 13.5 nm to 45 nm. In addition, the surface morphology of the nanoparticles is uniform, suggesting that they are covered with the same material. Although not shown, it was confirmed that the products obtained with 1.5 mg and 3.0 mg of dexamethasone had similar shapes. Hereinafter, the products obtained by using 1.5 mg, 3.0 mg and 4.5 mg of dexamethasone, respectively, were referred to as nanoparticles (DEX1.5), nanoparticles (DEX3.0) and nanoparticles (DEX4.5). Refer to.

Further, when the composition of the surface of the nanoparticles (DEX4.5) was analyzed by the powder X-ray diffraction (XRD) method, the surface of the nanoparticles (DEX4.5) had a main phase of β-tricalcium phosphate. The second phase was hydroxyapatite.

Furthermore, in order to confirm that the nanoparticles contained dexamethasone, evaluation was performed from the calibration curve of the dexamethasone solution having a known concentration at 242 nm using an ultraviolet-visible spectrophotometer. Specifically, nanoparticles (DEX4.5) from which dexamethasone on the surface was removed by washing with ethanol were decomposed with hydrochloric acid, and the peak intensity at 242 nm of this solution was measured. On the other hand, for comparison, dexamethasone was adsorbed on the surface of nanoparticles containing no dexamethasone (used in Comparative Example 4 described later), the surface was washed with ethanol and then decomposed with hydrochloric acid, and the peak intensity at 242 nm of this solution was measured. did. As a result, the absorption peak intensity of the solution obtained by decomposing the nanoparticles (DEX4.5) was significantly higher than that of the nanoparticles having dexamethasone adsorbed on the surface. This suggests that the biphasic calcium phosphate of β-tricalcium phosphate and hydroxyapatite contains dexamethasone in the nanoparticles (DEX4.5).

The content and content of dexamethasone in nanoparticles (DEX1.5), nanoparticles (DEX3.0) and nanoparticles (DEX4.5) were measured. 10 mg of each nanoparticle was dissolved in 0.3 mL of 1M HCl (Wako Pure Chemical Industries, Ltd.). Then, 1.7 mL of phosphate buffered saline (PBS, Nacalai Tesque, Inc.) was added to these solutions to dilute the solutions. The amount of dexamethasone in the sample of the diluted solution was measured using an ultraviolet / visible spectrophotometer at 242 nm using a calibration curve of a dexamethasone solution having a known concentration. From this, the content of dexamethasone was calculated. The content and content of dexamethasone in nanoparticles (DEX1.5), nanoparticles (DEX3.0) and nanoparticles (DEX4.5) are 225 ± 26, 382 ± 21 and 460 ± 35 ng / mg, respectively. And it was 22.5, 38.2 and 46.0 wt%. These results are summarized in Table 1 for simplicity.

[Example 1]
In Example 1, a composite porous scaffold material using the nanoparticles (DEX1.5) in Reference Example 1 and collagen as a bioabsorbable polymer was produced.

Pig-derived type I collagen (Nitta Gelatin Co., Ltd.) was dissolved in a 10 (v / v)% ethanol aqueous solution to prepare a 2.5 (wt / v)% collagen aqueous solution. The prepared collagen was allowed to stand in a low temperature chamber at −5 ° C. for 2 hours, and the temperature of the collagen solution was equilibrated at −5 ° C. Next, nanoparticles (DEX1.5) were dispersed in pure water, ethanol was added so that the volume concentration of ethanol was 10 (v / v)%, and nanoparticles (DEX1.5) having a particle concentration of 100 mg / mL were added. ) Was prepared. An aqueous ethanol solution of nanoparticles (DEX1.5) was moved to a temperature in a low temperature chamber of −5 ° C. and allowed to stand for 2 hours to adjust the temperature to −5 ° C.

In order to prepare ice fine particles as a pore-forming agent, pure water was sprayed on a container containing liquid nitrogen to freeze water droplets. The frozen product was transferred to a low temperature chamber at −15 ° C. together with the container. Liquid nitrogen was vaporized by allowing it to stand in a low temperature chamber for about 2 hours. Next, ice having a size of 425 μm to 500 μm was sieved using a sieve having a nominal opening of 500 μm and a sieve having a nominal opening of 425 μm in a low temperature chamber. The temperature in the low temperature chamber was changed to −5 ° C., and the prepared ice fine particles were allowed to stand for 2 hours to adjust the temperature of the ice fine particles to −5 ° C.

11 mL of a 2.5 (w / v)% collagen solution and 3 mL of an aqueous ethanol solution of 100 mg / mL nanoparticles (DEX1.5) were mixed at -5 ° C, and the nanoparticles were uniformly suspended. A mixed solution of DEX1.5) / collagen was prepared. Here, the nanoparticles (DEX1.5) and collagen were mixed so as to have a weight ratio of 1: 1. 12.5 g (corresponding to 15.4 mL) of ice fine particles were added to this mixed solution, and the mixture was mixed in a low temperature chamber at −5 ° C. at a ratio of 1: 1 (volume mL to weight g). The ice particles were stirred well so that they were uniformly dispersed in the mixed solution.

Subsequently, the mixture of this mixed solution and ice fine particles was filled in a pre-cooled silicone mold (internal dimensions 65 mm × 35 mm × thickness 5 mm). The mixture was allowed to stand at −12 ° C. for 12 hours and then at −80 ° C. for 6 hours to freeze the mixture. The frozen product was freeze-dried at room temperature for 2 days to obtain a composite porous scaffold material.

Collagen was then crosslinked in the resulting composite porous scaffold material. As the cross-linking reaction solution, 50 mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, Peptide Institute, Ltd.) and 20 mM N-hydroxysuccinimide (NHS, Wako Pure Chemical Industries, Ltd.) 80% aqueous ethanol solution was used. The composite porous scaffold material was immersed in the cross-linking solution, and the cross-linking reaction was carried out for 8 hours while shaking at room temperature (25 ° C.) at a speed of 20 rpm.

The composite porous scaffold material after the cross-linking reaction was immersed in ultrapure water for 1 hour at room temperature, and this was repeated 3 times as one washing. Subsequently, the obtained product was immersed in a 0.1 M aqueous solution of glycine at room temperature for 8 hours, and then washed with pure water for 1 hour was repeated 3 times. After washing, freeze-drying was carried out for 2 days to obtain a composite porous scaffold material of the final product. Hereinafter, for the sake of clarity, the composite porous scaffolding material produced in Example 1 will be referred to as scaffolding material 1.

The scaffolding material 1 was observed with a scanning electron microscope (SEM, S-4800, Hitachi High-Technologies Corporation). The results are shown in FIG. In addition, the pore size was determined from the SEM image, and the content (mg) of nanoparticles per ^{1 cm 3 of the scaffolding material was determined.} The results are shown in Table 2.

The amount of dexamethasone released slowly from scaffolding material 1 was examined. For the measurement of the sustained release amount, six scaffolding materials 1 hollowed out in a columnar shape having a diameter of 6 mm and a height of 3 mm by a biopsy punch were used. The sustained release of dexamethasone was measured as follows. Six scaffolding materials 1 were placed in one test tube, 6 mL of PBS was added, and the mixture was immersed. This test tube was shaken at 37 ° C. and 60 rpm, and 100 μL of the supernatant was sampled after a lapse of a predetermined time, and this was used for the sustained release amount measurement. The remaining scaffold material / solution was supplemented with 100 μL of PBS and subsequently shaken. The shaking time was 0.5, 1, 2, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 35 days. The absorbance of the sampled solution was measured with an ultraviolet / visible spectrophotometer (V-660, JASCO Corporation), and the concentration of sustained release dexamethasone was determined from a calibration curve prepared from a dexamethasone solution having a known concentration. The weight of dexamethasone was calculated from the concentration of dexamethasone and the volume of the solution, and expressed as the cumulative sustained release amount. The cumulative sustained release amount of dexamethasone was divided by the amount of dexamethasone contained in the calcium phosphate nanoparticles to determine the proportion of sustained release dexamethasone. This series of experiments was repeated 3 times, and the average value was calculated. The results are shown in FIG. 6 and Table 3.

Next, the bone tissue was regenerated using the scaffold material 1. For the regeneration of bone tissue, scaffold material 1 was used, which was hollowed out into a cylinder having a diameter of 6 mm and a height of 3 mm with a biopsy punch and sterilized with ethylene oxide gas for 22 hours. Sterilized scaffold material 1 was immersed in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Japan GK) and equilibrated by incubating at 37 ° C. for 4 hours. Human mesenchymal stem cells (Lonza Japan Co., Ltd.) were dispersed in DMEM ^{to prepare a suspension of 2 × 10 6} cells / mL, and 85 μL of this cell suspension (number of cells 1.7 × 10 ⁵ cells). Was dropped on the upper surface of the scaffold material 1. After culturing at 5% CO ₂ , 37 ° C. for 3 hours, the composite porous scaffold material was turned upside down, and 85 μL of the cell suspension was added dropwise on the upper surface thereof. Number of seeded cells totaled 3.4 × 10 ⁵ cells. _{Scaffold material 1 seeded with cells was cultured in vitro at 37 ° C. and 5% CO 2} for 1 week in DMEM supplemented with 10 mM β-glycerophosphate (Sigma-Aldrich Japan GK), and then 6-week-old nude. The mice were implanted subcutaneously in the back of the mice for 6 to 12 weeks. The appearance after transplantation was observed. The results are shown in FIGS. 7 (A) and 7 (B).

In order to observe the cells that invaded the scaffold material 1, hematoxylin and eosin staining was performed and observed. The results are shown in FIGS. 8A to 8C. Furthermore, in order to examine the formation of bone tissue at the protein level, immunostaining of type I collagen (Col I) and osteocalcin (OCN), which are bone differentiation-related proteins, was performed on the transplanted product 12 weeks after transplantation. The results are shown in FIGS. 9 (A) and 9 (B).

Furthermore, in order to examine the formation of bone tissue at the genetic level, the bone differentiation marker genes alkaline phosphatase (ALP), bone sialoprotein 2 (IBSP), and runt-related translation factoror were used for the transplanted product 12 weeks after the transplantation. The expression levels of -2 (RUNX2) and bone-forming factor 2 (BMP-2) were analyzed by the real-time RT-PCR method. The expression of the bone differentiation marker gene of the scaffold material 1 was compared with the scaffold material containing no dexamethasone (Comparative Example 4 described later). The results are shown in Table 4.

[Example 2]
In Example 2, a composite porous scaffold material using the nanoparticles (DEX3.0) in Reference Example 1 and collagen as a bioabsorbable polymer was produced. Since the procedure is the same as that of the first embodiment except that the nanoparticles (DEX3.0) are used instead of the nanoparticles (DEX1.5), the description thereof will be omitted. The composite porous scaffolding material produced in Example 2 is referred to as scaffolding material 2.

For scaffold 2, in the same manner as in Example 1, subjected to SEM observation it was determined the content of the pore size and the scaffold 1 cm ³ per nanoparticles (mg). The results are shown in FIG. 4 and Table 2. Similar to Example 1, the amount of dexamethasone released slowly from the scaffolding material 2 was examined. The results are shown in FIG. Similar to Example 1, the scaffolding material 2 was used to regenerate the bone tissue. The appearance after transplantation was observed, and hematoxylin / eosin staining and immunostaining were performed. The results are shown in FIGS. 7 (C) and 7 (D), FIGS. 8 (D) to (F) and FIGS. 9 (C) and 9 (D). Similar to Example 1, the expression of the bone differentiation marker gene using the scaffolding material 2 was examined. The results are shown in Table 4.

[Example 3]
In Example 3, a composite porous scaffold material using the nanoparticles (DEX4.5) in Reference Example 1 and collagen as a bioabsorbable polymer was produced. Since the procedure is the same as that of the first embodiment except that the nanoparticles (DEX4.5) are used instead of the nanoparticles (DEX1.5), the description thereof will be omitted. The composite porous scaffolding material produced in Example 3 is referred to as scaffolding material 3.

For scaffold 3, in the same manner as in Example 1, subjected to SEM observation it was determined the content of the pore size and the scaffold 1 cm ³ per nanoparticles (mg). The results are shown in FIG. 5 and Table 2. Similar to Example 1, the amount of dexamethasone released slowly from the scaffolding material 3 was examined. The results are shown in FIG. Similar to Example 1, the scaffolding material 3 was used to regenerate the bone tissue. The appearance after transplantation was observed, and hematoxylin / eosin staining and immunostaining were performed. The results are shown in FIGS. 7 (E) and 7 (F), FIGS. 8 (G) to (I) and FIGS. 9 (E) and 9 (F). Similar to Example 1, the expression of the bone differentiation marker gene using the scaffolding material 3 was examined. The results are shown in Table 4.

[Comparative Example 4]
In Comparative Example 4, a composite porous scaffold material using nanoparticles containing no dexamethasone and collagen as a bioabsorbable polymer was produced. The nanoparticles containing no dexamethasone were produced in the same manner in Reference Example 1 except that dexamethasone was not used. Since the procedure is the same as that of Example 1 except that nanoparticles containing no dexamethasone are used instead of nanoparticles (DEX1.5), the description thereof will be omitted. The composite porous scaffolding material produced in Comparative Example 4 is referred to as scaffolding material 4.

Similar to Example 1, the scaffolding material 4 was used to regenerate the bone tissue. The appearance after transplantation was observed, and immunostaining was performed. The results are shown in FIG. Similar to Example 1, the expression of the bone differentiation marker gene using the scaffolding material 4 was examined.

The above results will be described.
FIG. 3 is a diagram showing an SEM image of the scaffolding material 1 of the first embodiment.
FIG. 4 is a diagram showing an SEM image of the scaffolding material 2 of the second embodiment.
FIG. 5 is a diagram showing an SEM image of the scaffolding material 3 of Example 3.

According to FIGS. 3 (A), 4 (A) and 5 (A), the scaffolding materials 1 to 3 of the present invention all have a spherical pore structure reflecting the size of ice fine particles and their communication. It was found that it had a porous structure consisting of the voids. 3 (B), 4 (B) and 5 (B) are high-magnification observation results, and particulate matter corresponding to nanoparticles was observed on the pore wall surface.

According to Table 2, it was confirmed that the pore diameters in the scaffolding materials 1 to 3 reflected the size and shape of the ice fine particles used. The content of the nanoparticles of the outer volume 1 cm ³ per scaffold 1-3 was calculated as follows. The volume of the mixed solution (14 mL) of the aqueous collagen solution (11 mL) and the ethanol solution of nanoparticles (3 mL) after freezing is 15.4 mL (because the volume increases 1.1 times when the water changes to ice). Become. The total volume of the frozen mixed solution (15.4 mL) and the ice fine particles (15.4 mL) is 30.8 mL, and the outer volume of the scaffold material after freeze-drying can be regarded as ^{30.8 cm 3.} Since using nanoparticles 300mg, nanoparticles per outer volume 1 cm ³ becomes 300 / 30.8~9.7mg / cm ^3.

FIG. 6 is a diagram showing the time dependence of the sustained release amount of dexamethasone released from the scaffolding materials 1 to 3 of Examples 1 to 3.

According to FIG. 6, sustained release of dexamethasone was observed for 35 days in all of the scaffolding materials 1 to 3 of Examples 1 to 3, indicating that sustained release of dexamethasone was possible. In addition, the sustained release amount of dexamethasone increased as the concentration of dexamethasone in the nanoparticles contained in the scaffolding material increased.

As shown in Table 3, the cumulative dexamethasone releases from the scaffolding materials 1 to 3 after incubation were 339.9 ± 35.8 ng, 553.5 ± 38.3 ng, and 712.5 ± 57.1 ng, respectively. The above is the amount per scaffolding material). The cumulative ratio (%) of sustained-release dexamethasone was calculated with respect to the content of dexamethasone in the scaffolding materials 1-3. After 35 days, the cumulative percentages of dexamethasone released from scaffolding materials 1-3 were 76.2 ± 6.8%, 72.4 ± 5.7%, and 79.1 ± 7.0%, respectively. From this, it was shown that the scaffold material of the present invention can release dexamethasone slowly for at least one month, which is advantageous for the regeneration of bone tissue. These results are shown in Table 3 for readability.

FIG. 7 is a diagram showing the appearance of bone tissue regenerated using the scaffolding materials 1 to 3 of Examples 1 to 3.

7 (A) and 7 (B) are appearances of bone tissue transplanted for 12 weeks using scaffold material 1, and FIGS. 7 (C) and 7 (D) show bone transplanted for 12 weeks using scaffold material 2. The appearance of the tissue, FIGS. 7 (E) and 7 (F), is the appearance of the bone tissue transplanted for 12 weeks using the scaffolding material 3. 7 (A), (C), and (E) show scaffolding materials 1 to 3 taken from above, and FIGS. 7 (B), (D), and (F) show scaffolding materials 1 to 3. It looks like it was taken from the side.

Although shown in gray scale in FIG. 7, the transplant (bone tissue) was entirely reddish, confirming that bone tissue was formed. In particular, the part where the lightness is dark is the capillaries, and it was found that the scaffold material of the present invention is effective for the regeneration of bone tissue. It was also found that the dimensions before transplantation were maintained even 12 weeks after transplantation.

FIG. 8 is a diagram showing hematoxylin / eosin-stained images of bone tissue regenerated using the scaffolding materials 1-3 of Examples 1 to 3.

8 (A) to 8 (C) are stained images of bone tissue transplanted for 12 weeks using scaffold material 1, and FIGS. 8 (D) to 8 (F) are transplanted for 12 weeks using scaffold material 2. It is a stained image of the bone tissue, and FIGS. 8 (G) to 8 (I) are stained images of the bone tissue transplanted for 12 weeks using the scaffolding material 3. 8 (A), (D), and (G) are the entire vertical and horizontal cross sections of the scaffolding materials 1 to 3, and FIGS. 8 (B), (E), and (H) are FIGS. 8 (A) and 8 (D). ), (G), and FIGS. 8 (C), (F), and (I) are further enlarged images of FIGS. 8 (B), (E), and (H).

According to FIGS. 8 (A), (D), and (G), the whole is stained (corresponding to the region where the lightness is darkened on the gray scale), and the cells have invaded the pores. It was shown that the invading cells were uniformly distributed in the scaffold material and that the extracellular matrix was uniformly deposited.

In FIGS. 8C, 8F, and I, the arrow (→) indicates a new blood vessel, and the asterisk (*) indicates a new bone. From this, it was shown that the scaffolding material of the present invention can be used to regenerate bone tissue.

FIG. 9 is a diagram showing immunostaining images of bone tissue regenerated using the scaffolding materials 1 to 3 of Examples 1 to 3.
FIG. 10 is a diagram showing an immunostaining image of bone tissue regenerated using the scaffolding material 4 of Comparative Example 4.

9 (A) and 9 (B) are immunostained images of type I collagen (Col I) and osteocalcin (OCN) of bone tissue transplanted for 12 weeks using scaffold material 1, respectively, and FIG. 9 (C). , (D) are immunostained images of Col I and OCN of bone tissue transplanted for 12 weeks using scaffold material 2, and FIGS. 9 (E) and 9 (F) are transplanted for 12 weeks using scaffold material 3. It is an immunostained image of Coll I and OCN of the bone tissue (implant). 10 (A) and 10 (B) are immunostained images of Col I and OCN of bone tissue transplanted for 12 weeks using scaffold material 4.

9 and 10 show in grayscale, but the darkened areas were stained with anti-Col I antibody, anti-OCN antibody. According to FIG. 9, the transplants using the scaffolding materials 1 to 3 were stained with the anti-Col I antibody and the anti-OCN antibody. In particular, when FIG. 9 and FIG. 10 were compared, the stained images of the scaffolding materials 1 to 3 were dyed deeper than those of the scaffolding material 4. From this, it was shown that the degree of bone differentiation of the implant of the scaffold material 1 of the present invention was significantly higher than that of the scaffold material 4.

Table 4 shows the results of comparing the expression levels of the bone differentiation marker genes in the transplants using the scaffold materials 1 to 3 with those using the scaffold materials 4.

According to Table 4, it was shown that the expression of the bone differentiation marker gene was increased at least about 2 times in the transplants of the scaffold materials 1 to 3 as compared with those of the scaffold materials 4. This also indicates that the scaffolding material of the present invention regenerates bone tissue with higher efficiency.

Since the composite porous scaffold material of the present invention can allow cells to invade into the porous material to be transplanted to a defective site of bone tissue, cells can be efficiently differentiated into bone. Moreover, since the dexamethasone-containing nanoparticles are supported on the porous body, it not only prevents the nanoparticles from diffusing quickly and reducing the efficiency of supplying dexamethasone to the bone defect site, but also regenerates the bone tissue. During this period, the dexamethasone supported on the nanoparticles can be released slowly. By shaping the composite porous scaffold material into the shape of the defect site, the bone tissue having a shape corresponding to the defect site is regenerated. Therefore, the composite porous scaffold material of the present invention is extremely effective in bone regenerative medicine.

100 Composite Porous Scaffolding Material 110 Porous Body 120 Nanoparticles 130 Voids

Claims (17)

A porous body made of a bioabsorbable polymer and
Containing dexamethasone-containing nanoparticles located in the porous body,
The dexamethasone is contained in at least bioabsorbable calcium phosphate ,
The calcium phosphate is a composite porous scaffold material containing the dexamethasone. The composite porous scaffold material according to claim 1 , wherein the nanoparticles have a particle size in the range of 1 nm or more and 1000 nm or less. The composite porous scaffold material according to claim 1 or 2 , wherein the nanoparticles have a shape selected from the group consisting of spherical, rod-shaped, star-shaped, spindle-shaped, triangular prism, rectangular parallelepiped and cube. The bioabsorbable polymer includes collagen, gelatin, hyaluronic acid, chitosanchondroitin sulfate, fibronectin, vitronectin, laminin, polylactic acid, polyglycolic acid, copolymer of lactic acid and glycolic acid, poly (ε-caprolactone), poly ( The composite porous scaffold material according to any one of claims 1 to 3 , which is selected from the group consisting of glycerol sebacic acid). The composite porous scaffold material according to any one of claims 1 to 4 , wherein the porous body has pores having a pore diameter in the range of 10 μm or more and 1000 μm or less. The composite porous scaffold material according to any one of claims 1 to 5 , wherein the bioabsorbable polymer is crosslinked. The composite porous scaffold material according to any one of claims 1 to 6 ^{, wherein the content of nanoparticles per 1 cm 3} of the composite porous scaffold material is in the range of 1 mg or more and 500 mg or less. The composite porous scaffold material according to any one of claims 1 to 7 , wherein the nanoparticles and the bioabsorbable polymer satisfy the range of 2: 1 or more and 1: 2 or less in weight ratio. The composite porous scaffold material according to any one of claims 1 to 8 , wherein the dexamethasone is contained in a mixture of bioabsorbable calcium phosphate and hydroxyapatite. Any of claims 1 to 9 , which is a step of mixing dexamethasone-containing nanoparticles and a bioabsorbable polymer to make it porous, wherein the dexamethasone is contained in at least bioabsorbable calcium phosphate, which comprises the step. A method for producing a composite porous scaffold material described in Crab. The porous to step lyophilizing the mixture of the bioabsorbable polymer and the nanoparticles, the method of claim 1 0. Using the pore-forming agent to form the pores, the method according to claim 1 0 or 1 1. The pore-forming agent is glacial method of claim 1 2. The porous to process foam molding method, using a method from the group consisting of pore-forming agent removing method and a phase separation method, Method according to claim 1 2. The Following porous to step, further comprising the step of crosslinking the bioabsorbable polymer, A method according to any one of claims 10 to 1 4. Wherein the bioabsorbable polymer and nanoparticles in a weight ratio of 2: 1 or more 1: 2 are mixed so as to satisfy the following range, method according to any one of claims 1 0 to 1 5. The method according to claim 16 , wherein the nanoparticles and the bioabsorbable polymer are mixed so as to satisfy the range of 1.5: 1 or more and 1: 1.5 or less in terms of weight ratio.

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