KR20180054379A - Method for preparation of bone filler having cells and drug and bone filler prepared thereby - Google Patents

Abstract

The present invention relates to a method for manufacturing a bone filler comprising cells and drugs and composed of beads, which can differentiate or release the cells and drugs, and to a bone filler manufactured thereby. The method for manufacturing a bone filler according to the present invention can manufacture a bone filler in a bead form through an extrusion container having a dual nozzle, and can easily inject a bioactive material, which promotes bone generation, into a bone graft site without performing a sintering process. In addition, the bone filler according to the present invention can form pores by the coiling of ceramic fibers, thereby exhibiting excellent effects on osteogenesis induction and improving bone adhesion due to hydrogel surrounding coil structure ceramics. In addition, the bone filler according to the present invention can be widely used as a functional bone filler for realizing differential release of cells and drugs by enabling a drug contained in an internal structure to be released for a long period of time in a sustained release type and including a bioactive material, such as a cell or a growth factor, which capable of being discharged early, at the outside of a bead.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing a bone filler containing cells and a drug and a bone filler prepared thereby,

The present invention relates to a method for preparing a bone filler comprising beads capable of temporal release or sustained release of cells and drugs including cells and drugs, and a bone filler prepared thereby.

Generally, when a part of the bone tissue is deficient or needs reinforcement in a specific part of a living body, the bone is implanted into the part. Thus, when bone grafts are transplanted into a living body, the grafted bone induces the formation of new bone at the graft site, and the graft itself is gradually degraded, partly or wholly. When a certain period of time elapses, most of the graft site becomes filled with newly generated bone.

Here, it is common that a bone graft to be implanted in a living body is a mixture of an extracted portion of a bone tissue in a living body and an artificial bone artificially made of a material such as ceramics. At this time, the artificial bone is often referred to as a bone filler or a bone substitute.

The form of bone filler is simple granule type and granular type with surface irregularities and pores.

Simple granulation methods are representative of simple sintering methods, which are described in U.S. Patent No. 4,218,255, U.S. Patent No. 4,693,986, U.S. Patent No. 5,030,611, and U.S. Patent No. 5,064,436. The foregoing prior art discloses a simple sintering method for producing a bone filler by preparing granules using fine powder and then sintering the granules and introducing fine pores into the surface of the granules by controlling the degree of sintering in the sintering process It is described that the pore is hardly introduced. However, since the bone filler produced by the simple sintering method has a very high strength of the granule itself, but has a smaller specific surface area than that of the concavo-convex shape having irregularities formed on its surface, the area of attachment of the bone is relatively small. As a result, Is lowered.

On the other hand, the granular form into which surface irregularities and pores are introduced is classified into a porous form, a semi-porous form or a porous form by a porous formed body used in the manufacturing process. The most representative method for producing the granules of the present invention is a replica method using a porous polymer sponge, which is described in U.S. Patent No. 4,889,833. In the prior art, the porous sponge is impregnated into the bioactive ceramic slurry by the replica method to apply the slurry on the sponge skeleton, and then the porous body is formed by burning the sponge in the process of drying, degreasing and sintering the slurry, Followed by pulverization and sieving, thereby producing an intact granule having an appropriate size. The microporous granules prepared by this method have an advantage of controlling the size of pores generated according to the pore size of the porous sponge skeleton. However, due to the extremely low strength of the corpuscle filler in the form of a granule, the granule is not suitable for mass production due to the inconvenience of repeatedly impregnating and degreasing the slurry several times in order to obtain granules of desired strength So that the manufacturing cost is increased. In addition, the surface of the granules produced after grinding is very sharp, which is also inappropriate for adhesion of bone cells.

With the rapid development of medical technology, the average life expectancy has increased significantly compared to the past. As a result, the prevalence of several degenerative bone diseases including osteoporosis is increasing due to the rapid increase of the elderly population.

Therefore, studies for introducing drugs for treating bone diseases in bone fillers have been actively carried out. However, these drugs accumulate in the body and may be accompanied with serious side effects when they are administered for a long time or in large doses.

Further, in order to introduce a drug into a ceramic support, a high-temperature sintering process must be performed. Therefore, when a drug weak in heat is used, it must be introduced after sintering, thereby causing initial acute release of the drug and making it difficult to use it for a long time. In addition, the amount of drug introduced is limited.

Therefore, it is a common practice to mix ceramic with natural or synthetic polymer, or to introduce metal ions that are not affected by high-temperature heat treatment in the case of ceramics. However, in the former case, it is difficult to expect sufficient mechanical properties and bioactivity. In the latter case, it is pointed out that introduction of a sufficient amount of metal ion is difficult or adverse effect of metal ion is pointed out.

Prior Art 1. United States Patent No. 4,218,255 (registered on August 19, 1980) Prior art document 2. United States Patent No. 4,693,986 (registered on September 15, 1987) Prior art document 3. US Patent No. 5,030,611 (Registered on July 9, 1991) Prior Art Document 4. US Patent No. 5,064,436 (Registered on November 12, 1991) Prior Art Document 5. US Patent No. 4,889,833 (Registered on December 26, 1989)

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and it is an object of the present invention to provide a method of manufacturing a bead-shaped bone filler containing cells and drugs.

It is also an object of the present invention to provide a bone filler in the form of a bead containing cell and drug.

According to a preferred embodiment of the present invention, a method of manufacturing a bone filler comprises the steps of: preparing a first paste containing calcium phosphate-based ceramics and a drug; Preparing a second paste comprising a hydrogel and cells; Placing the first paste in a container connected to an inner pipe of an extrusion container having a double nozzle and extruding the second paste into a container connected to an outer pipe of an extrusion container having a double nozzle to form a droplet; And immersing the droplet in a crosslinking solution to obtain beads containing cells and a drug.

The present invention is further characterized by further comprising the step of immersing the cells immersed in the crosslinking solution and the beads containing the drug in a curing liquid.

Further, the present invention is characterized by further comprising the step of culturing cells in the beads immersed in the curing solution.

Here, the nozzle size of the inner pipe for extruding the first paste is 100 to 1000 mu m.

Also, the hydrogel may be selected from the group consisting of alginate, gelatin, collagen, fibrinogen, chitosan, agar, Matrigel, starch, pectin, Hydroxyethyl ethyl cellulose, polyvinyl alcohol, polyurethane, poly (ethylene glycol), poly (propylene glycol), polypropylene glycol, And at least one polymer selected from the group consisting of methyl cellulose, carboxymethylcellulose, hyaluronan, and poly (vinylpyrrolidone).

Here, the polymer is contained in a concentration of 0.1 to 10% by weight in the second paste.

The drug may also be selected from the group consisting of Quercetein, Genistein, Curcumin, Saurolactam, Sauchinone, Baicalin, Daidzein, Rutin, Anthocyanidin, Fisetin, Icariin, Kaempferol, E. Korean Nakei, Equol, alendronate, ), Risedronate, etidronate, clodronate, neridronate, ibandronate, zoledronate, orphadronate (olpadronate) and a bisphosphonate-based compound.

The calcium phosphate-based ceramics may be selected from the group consisting of hydroxyapatite, dicalcium phosphate dihydrate (DCPD), monocalcium phosphate monohydrate (MCPM), dicalcium phosphate anhydrous (DCPA), biphasic calcium phosphate (BCP) phosphate, and β-TCP (β-Tricalcium phosphate), and more preferably α-TCP (α-Tricalcium phosphate), but not limited thereto, May be used.

In addition, the first paste may contain at least one selected from the group consisting of hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC), alginate, gelatin, collagen Fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, polyvinylpyrrolidone, A thickening agent of at least one of poly (ethylene glycol), poly (propylene glycol), hyaluronan and poly (vinylpyrrolidone) .

In addition, the solution containing the thickener may include 10 to 100 parts by weight based on 100 parts by weight of the calcium phosphate-based ceramic.

In addition, the cross-linking solution of calcium chloride (CaCl _2), preferably including at least one of magnesium chloride (MgCl _2), calcium phosphate (CaP) and calcium carbonate (CaCO _3), and the curing fluid is H ₂ O, PBS characterized by comprising at least one of phosphate buffer saline (MCPM), monocalcium phosphate monohydrate (MCPM), diammonium hydrogen phosphate (DAHP), NH ₄ H ₂ PO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ and NaH ₂ PO ₄ do.

According to another preferred embodiment of the present invention, the bead-shaped bracing material comprises an inner structure comprising a ceramic and a drug, wherein the single fiber is formed of a coil; And

And organic particles that surround the internal structure to form a surface and include a hydrogel and cells.

Here, the internal structure includes calcium-deficient hydroxyapatite (CDHA).

The drug may also be selected from the group consisting of Quercetein, Genistein, Curcumin, Saurolactam, Sauchinone, Baicalin, Daidzein, Such as Rutin, Anthocyanidin, Fisetin, Icariin, Kaempferol, E. Korean Nakei, Equol and the like, Based natural materials; But are not limited to, alendronate, risedronate, etidronate, clodronate, neridronate, ibandronate, zoledronate, Characterized in that it comprises at least one of an olpadronate and a bisphosphonate system.

The hydrogel may be selected from the group consisting of alginate, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC), gelatin, collagen, Fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, polyethylene, polyvinylpyrrolidone, A polymer comprising at least one polymer selected from the group consisting of poly (ethylene glycol), poly (propylene glycol), hyaluronan, and poly (vinylpyrrolidone) .

The method of manufacturing a bone filler according to the present invention can produce a bead-shaped bone filler through an extrusion container having a double nozzle, and can easily produce a bioactive material that promotes bone formation without sintering, It has the effect of injecting.

In addition, since the bone filler according to the present invention can form pores by the coiling of ceramic fibers, it exhibits an excellent effect in inducing bone formation, and bone adhesiveness can be improved due to the hydrogel surrounding the coil structure ceramic.

In addition, the bone filler according to the present invention can release the drug contained in the internal structure in a sustained release for a long time, and the outside of the beads can contain a bioactive substance that can be released early such as a cell or a growth factor, And can be widely used as a functional bone filler that realizes a time lag release of the bone.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a method for manufacturing a bone filler composed of beads containing a drug and cells of the present invention. FIG.
2 is a photograph showing cell growth according to Experimental Example 1 of the present invention.
3 is a photograph showing cell growth according to Experimental Example 2 of the present invention.
FIG. 4 is a graph and photographs showing the supporting and quenching effect of quercetin according to Experimental Example 3 of the present invention. FIG.
5 is a photograph showing cell growth according to Experimental Example 4 of the present invention.
6 is a result of an XRD analysis in which the change of the structure according to Experimental Example 4 of the present invention was confirmed.
7 is a photograph showing cell growth according to Experimental Example 5 of the present invention.
8 is a result of an XRD analysis in which the change of the structure according to Experimental Example 5 of the present invention was confirmed.

Hereinafter, the present invention will be described in detail. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately It should be interpreted in accordance with the meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined. Therefore, the constitutions described in the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the technical ideas of the present invention. Therefore, various equivalents which can be substituted at the time of application It should be understood that variations can be made.

In the present invention, the term " bone filler " is also referred to as a " bone substitute ", " bone graft material ", and is defined as implantation when a part of bone tissue in a living body is deficient or needs reinforcement.

The term " beads " used in the present invention is also referred to as " beads ", and is defined as spherical particles.

A method for producing a bone filler according to the present invention comprises:

Preparing a first paste containing calcium phosphate-based ceramics and a drug;

Preparing a second paste comprising a hydrogel and cells;

Placing the first paste in a container connected to an inner pipe of an extrusion container having a double nozzle and extruding the second paste into a container connected to an outer pipe of an extrusion container having a double nozzle to form a droplet; And

And immersing the droplet in a crosslinking solution to obtain beads (see Fig. 1).

Hereinafter, the method for producing a bone filler of the present invention will be described in more detail.

First, a first paste containing calcium phosphate-based ceramics is prepared.

In the first paste preparing step, it is preferable that the step of preparing a paste usable in the extrusion step is made of calcium phosphate-based ceramics.

The calcium phosphate-based ceramics are not particularly limited in their kind but may be selected from the group consisting of hydroxyapatite, dicalcium phosphate dihydrate (DCPD), monocalcium phosphate monohydrate (MCPM), dicalcium phosphate anhydrous (DCPA), biphasic calcium phosphate , α-TCP (α-Tricalcium phosphate), and β-TCP (β-Tricalcium phosphate), and more preferably α-TCP (α-Tricalcium phosphate).

For example, when α-TCP (α-Tricalcium phosphate) is used as the calcium phosphate-based ceramic, it reacts with water to form a calcium-deficient hydroxyapatite (CDHA) It is possible to improve the mechanical strength and maintain the stable shape of the structure. In addition, it is more advantageous in introducing cells and other functional materials than β-TCP (β-Tricalcium phosphate) which requires acidic conditions in the curing process.

[Reaction Scheme 1]

_{3αCa 3 (PO 4) 2 +} H 2 O → Ca 9 (HPO 4) (PO 4) 5 (OH)

The drug may also be selected from the group consisting of Quercetein, Genistein, Curcumin, Saurolactam, Sauchinone, Baicalin, Daidzein, Such as Rutin, Anthocyanidin, Fisetin, Icariin, Kaempferol, E. Korean Nakei, Equol and the like, Based natural materials; But are not limited to, alendronate, risedronate, etidronate, clodronate, neridronate, ibandronate, zoledronate, It is preferable to include at least one of synthetic compounds such as olpadronate and bisphosphonate, and more preferably, it may include at least one of Quercetein and Genistein. In particular, quercetin can be used most preferably because it can affect both osteoblasts and osteoclasts without affecting the phase change in the curing process of ceramics. On the other hand, antibiotics known in the art such as vancomycin, centamycin, rifampin, penicillin and the like may be included in the drug.

Here, the drug may contain 0.01 to 20 parts by weight, more preferably 0.1 to 10 parts by weight, and most preferably 1 to 5 parts by weight, based on 100 parts by weight of the calcium phosphate- Section. When used in the weight parts described above, it is effective for promoting osteocyte behavior and inhibiting osteoclast behavior, thereby improving the bone regeneration efficacy of the bone filler.

In addition, the first paste may further comprise an additive which is an organic material having excellent biocompatibility in order to impart fluidity and moldability of the calcium phosphate-based ceramic. The type of the thickening agent is not particularly limited, and examples thereof include hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC), alginate, gelatin Gelatin, collagen, fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, Polyurethane, poly (ethylene glycol), poly (propylene glycol), hyaluronan, and poly (vinylpyrrolidone). But it is preferably at least one, and more preferably, it may contain hydroxypropylmethylcellulose (HPMC).

In addition, the thickener may include a solvent, and the type of the solvent is not particularly limited, but is preferably distilled water, tetrahydrofuran, dioxane, ethyl ether, 1,2-dimethoxyethane, dimethylformamide, dimethyl Sulfoxides, dichloromethane, dichloroethane and C ₁ to C ₄ alcohols, and more preferably ethanol. Here, the viscosity can be controlled so that the first paste can be easily extruded and the coils can be formed well by using the coagulant containing the solvent. The viscosity of the first paste may be adjusted to a range of preferably 10,000 to 1,000,000 cP, preferably 50,000 to 500,000 cP, more preferably 100,000 to 300,000 cP.

Further, by using the thickening agent, it is possible to facilitate mixing of the drug and the calcium phosphate ceramic, thereby enabling uniform dispersion of the drug in the ceramic structure.

The first paste according to the present invention is preferably prepared by pulverizing and mixing the calcium phosphate type ceramic, the drug and the thickening agent using a ball mill device.

As a preferred example, the first paste may be prepared by using a ball mill apparatus using the gradual solution containing the calcium phosphate-based ceramic and the solvent. The first paste may contain 10 to 100 parts by weight, more preferably 30 to 80 parts by weight, of the incrementing agent solution based on 100 parts by weight of the calcium phosphate-based ceramic, Preferably 50 to 70 parts by weight. If the content of the thickening agent is less than the above range, the viscosity may become high and the fluidity may be insufficient, so that formation of the coil may be difficult. If the content of the thickening agent exceeds the above range, the mechanical properties may be greatly deteriorated.

The average particle size of the calcium phosphate ceramic powder is preferably 1 to 50 탆, more preferably 2 to 25 탆, and most preferably 3 to 6 탆. The particle size is related to the size of the inner tube nozzle, and as the size of the nozzle is reduced, the particle size must be reduced so that the paste can be extruded without clogging. In addition, when the drug is mixed, it is preferable to use particles having a similar particle size to uniformly mix the drug and the ceramic powder. Specifically, considering the average particle size of the drug to be mixed, the average particle size of the ceramic powder is most preferably 3 to 6 mu m.

In addition, since the drug may be thermally denatured at a high temperature, it is preferable that the mixing is performed at room temperature to prevent changes in physical properties of the drug.

Next, a second paste containing a hydrogel is prepared.

In the second paste preparing step, a paste usable in the extrusion process is prepared including the hydrogel and the cell made of the polymer.

Here, the type of the polymer is not particularly limited, but may be selected from the group consisting of alginate, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC) Such as gelatin, collagen, fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, At least one of polyurethane, poly (ethylene glycol), poly (propylene glycol), hyaluronan, and poly (vinylpyrrolidone) It is preferable to include one, and more preferably, it may include alginate.

The second paste may preferably contain the polymer in a concentration of 0.1 to 10% by weight, more preferably 0.5 to 5% by weight, and most preferably 1 to 2% % ≪ / RTI > by weight. Here, when the polymer is contained below the defined concentration, it is difficult to form droplets in the extrusion process, and the surface tension of the formed droplets may flow into the hydrogel because the weight of the calcium phosphate-based ceramics formed in the droplets can not be maintained. The viscosity of the second paste is increased and it becomes difficult to control the shape of the filler.

The cells may include osteoblasts. Specifically, MC3T3-E1, ST-2, UAMS32, MG-63 and the like may be used. Preferably, MC3T3-E1 may be used. The cells are located on the outside of the bone filler, and grow rapidly in the initial stage after transplantation so that bone regeneration can be more effectively performed.

Further, the second paste may further include a gelling agent having a biostability. When the gelling agent is included, there is an advantage that the paste can easily control the physical properties so as to have fluidity suitable for extrusion and molding. The type of the gelling agent is not limited, but preferably includes at least one of H ₂ O, PBS, tris (hydroxymethyl) aminomethane, and N- [tris (hydroxymethyl) methyl] glycine And more preferably, PBS.

In addition, the hydrogel may induce crosslinking by physical (ionic, stereocomplex, thermal, and / or chemical) (UV, depending on the physical properties thereof) and physical crosslinking is advantageous.

Next, the first paste and the second paste prepared above are put into an extrusion container having a double nozzle and extruded to form droplets.

Here, the first paste is placed in a container connected to the inner tube of the extrusion container, and the second paste is put into a container connected to the outer tube of the extrusion container and extruded.

The extrusion container may be composed of two containers connected to the inner pipe and the outer pipe. The first paste material for forming the coil structure ceramic is inserted into the container connected to the inner pipe and is extruded. A second paste material for forming the outer surface is put in the container and extruded to form an organic-inorganic composite body.

In addition, the nozzle size of the inner tube of the extrusion container is preferably 100 to 1000 占 퐉, more preferably 200 to 700 占 퐉, and most preferably 300 to 500 占 퐉. If the size of the inner pipe nozzle is less than the above defined range, the size of the beads becomes large to control the shape, the content of the ceramic in the beads is relatively small and the bony filling effect is not sufficient. The size of the beads is shortened, the coils are not formed well, the internal pores are not formed sufficiently, and the ceramic is protruded to the outside of the hydrogel. It is possible.

In the extrusion process, pressure can be applied by various methods. However, in the case of extruding a first paste containing a ceramic paste, a screw pressure is preferably used because a high pressure is required, and a second paste containing a hydrogel In the case of extrusion, pneumatic pressure is preferably used because it requires fine pressure control, but it is not limited thereto.

It is preferable that the first paste has an extrusion pressure of 100 to 1000 U and the second paste has an extrusion pressure of 1 to 300 U. The extrusion pressure is a pressure for moving a distance of 1 mm per minute. The higher the extrusion pressure, the more the amount of the paste to be discharged increases. If the extrusion pressure of the first paste is too low, the content of the ceramic in the beads is relatively small and the filling effect of the trowel is not sufficient. If the extrusion pressure of the first paste is too high, the ceramic in the first paste is contained in the second paste And the grains fall vertically due to gravity, so that a bead-shaped bone filler can not be manufactured.

Here, when extrusion is performed by the pressure according to the present invention, a first paste to be extruded from the inner tube is formed in the form of a coil, and a second paste is extruded from the outer tube to form a droplet while surrounding the coil. At this time, when the surface tension of the extruded droplet is smaller than the weight of the droplet due to gravity, the droplet shape is maintained. As the time for maintaining the shape of the droplet is lengthened, the extrusion amount of the first and second pastes increases, The size of the beads to be formed becomes larger.

Next, the liquid droplets prepared above are immersed in a crosslinking liquid to obtain beads containing coil structure ceramics.

As the weight of the droplet due to the gravity increases, the surface tension of the extruded second paste becomes larger than the surface tension of the second paste while the extrusion of the previous step continues, and the droplet is dripped by the gravity into the crosslinking solution located below .

When the droplet is immersed in the crosslinking solution, it is preferable to maintain the crosslinking reaction for 30 minutes to 3 hours, more preferably 30 to 2 hours for maintaining the crosslinking reaction.

The crosslinking solution is not limited in its kind but may contain at least one of calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ ), calcium phosphate (CaP) and calcium carbonate (CaCO ₃ ) And more preferably calcium chloride (CaCl ₂ ).

When the dropwise droplet crosslinking reaction of the polymer is performed by the crosslinking liquid, a coil structure ceramic containing a drug is located inside, and beads having an organic substance including cells are formed on the outside.

The beads after the cross-linking reaction are not subjected to a separate sintering process. The beads of the present invention which have not undergone the sintering process have an advantage of being easily injected irrespective of the type of bone graft site, and thus being excellent in bone adhesion and bone induction.

Next, a step of immersing the beads immersed in the cross-linking solution in the hardening liquid and curing may be further roughened.

The curing liquid is not limited in its kind but may be selected from the group consisting of H ₂ O, phosphate buffer saline (PBS), monocalcium phosphate monohydrate (MCPM), diammonium hydrogen phosphate (DAHP), NH ₄ H ₂ PO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ and NaH ₂ PO ₄ , And most preferably PBS (phosphate buffer saline).

The concentration of the curing liquid is preferably 0.1 to 5.0 M. If the concentration of the curing liquid is less than 0.1 M, there is a problem that the curing reaction takes a long time. If the concentration exceeds 5.0 M, the curing reaction occurs too quickly, causing a nonuniform reaction.

Further, in the curing step, the beads may preferably be immersed in a curing solution for 6 to 24 hours to induce a self-curing reaction, more preferably to immerse in a curing solution for 6 to 12 hours to induce a self-curing reaction , The immersing time may be suitably adjusted in consideration of the size and reactivity of the beads.

Here, in order to further crosslink the hydrogel in the curing liquid, the curing liquid and the above-mentioned crosslinking liquid may be further included. It is preferable that the added amount of the crosslinking liquid is so low that it reacts with the curing liquid and does not cause precipitation and does not cause pH change of the curing liquid.

As a preferred example, a first paste containing calcium phosphate-based ceramics, a second paste formed of a hydrogel containing alginate, a calcium chloride (CaCl ₂ ) crosslinking solution, and a hardening solution containing calcium chloride (CaCl ₂ ) and PBS A bone filler can be produced. The first paste and the second when the paste is respectively injected into the inner tube and outer tube of the extrusion container droplets formed by the extrusion step the crosslinking liquid immersion, a divalent cation of the alginate in the hydrogel (Ca ^{2 +,} Ba ^{2 +,} Sr ^{2 +,} Etc.) are substituted with Na + ions to form a crosslinked network of Alg-Ca. Among them, Ca ^{2 +} can be expected to have the highest crosslinking effect. Then, the bead-shaped bone filler is taken out of the cross-linking solution and immersed in a curing solution containing PBS and calcium chloride (CaCl ₂ ) (step 2), whereby the alginate can additionally induce cross-linking. Therefore, in step 1, alginate is mixed with calcium chloride (CaCl ₂ ) at a low concentration to induce primary crosslinking. After molding, secondary crosslinking is performed by further immersing the alginate in a curing solution containing calcium chloride (CaCl ₂ ) .

In the crosslinking step and the curing step, curing may proceed after crosslinking as described above, but it is also possible to perform crosslinking and curing simultaneously in a monocalcium phosphate monohydrate (MCPM) solution.

The calcium ions of the MCPM can induce crosslinking of the hydrogel and induce curing of the calcium phosphate, but the MCPM is a solution of a low pH condition and thus has a great influence on the cell survival rate. Therefore, it is preferable to conduct the reaction for 3 minutes to 30 minutes, more preferably 5 minutes to 20 minutes, and most preferably 5 minutes to 10 minutes, and the cell viability is significantly lowered .

Meanwhile, it is preferable in terms of structural stability and cell viability to perform additional curing after performing the crosslinking and curing at the same time, and a curing solution such as H ₂ O, phosphate buffered saline (PBS) or diammonium hydrogen phosphate (DAHP) And is preferably reacted in the curing liquid for 6 to 12 hours.

Next, a step of culturing the cells in the beads immersed in the hardening solution may be further carried out.

Here, the beads immersed in the curing solution are transferred to a culture medium after washing, and the cells contained in the beads are cultured.

The culture medium is preferably? -MEM.

Since the bone filler according to the present invention has a drug introduced into a ceramic structure and a hydrogel is formed on the outside thereof, the drug can be released in a sustained release form for a long period of time without acute release of an initial drug, , Allowing cells located outside to perform bone regeneration more rapidly. In addition, it is possible to release the externally located cells first, and then to allow for a time lag release that can later release the drug.

In the bead type bone filler according to the present invention,

An inner structure in which a single ceramic fiber is formed of a coil and comprises a drug; And organic particles that surround the internal structure and form a surface and include a hydrogel and cells.

Hereinafter, the bead type bone filler of the present invention will be described in more detail.

It is preferable that the internal structure includes phase-changed calcium-deficient hydroxyapatite (CDHA) through hydration reaction of? -TCP.

The drug may also be selected from the group consisting of Quercetein, Genistein, Curcumin, Saurolactam, Sauchinone, Baicalin, Daidzein, Such as Rutin, Anthocyanidin, Fisetin, Icariin, Kaempferol, E. Korean Nakei, Equol and the like, Based natural materials; But are not limited to, alendronate, risedronate, etidronate, clodronate, neridronate, ibandronate, zoledronate, It is preferable to include at least one of synthetic compounds such as olpadronate and bisphosphonate, and more preferably, it may include at least one of Quercetein and Genistein. In particular, quercetin can be used most preferably because it can affect both osteoblasts and osteoclasts without affecting the phase change in the curing process of ceramics. On the other hand, antibiotics known in the art such as vancomycin, centamycin, rifampin, penicillin and the like may be included in the drug.

In addition, the internal structure may be formed of at least one selected from the group consisting of hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC), alginate, gelatin, collagen Fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, polyvinylpyrrolidone, A thickening agent of at least one of poly (ethylene glycol), poly (propylene glycol), hyaluronan and poly (vinylpyrrolidone) .

The hydrogel may be selected from the group consisting of alginate, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC), gelatin, collagen, Fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, polyethylene, polyvinylpyrrolidone, It is preferable to include a polymer which is at least one of poly (ethylene glycol), poly (propylene glycol), hyaluronan, and poly (vinylpyrrolidone) Do.

In addition, the organic particles may further include a gelling agent having a biostability. When the gelling agent is included, there is an advantage that the paste can easily control the physical properties so as to have fluidity suitable for extrusion and molding. The type of the gelling agent is not limited, but preferably includes at least one of H ₂ O, PBS, tris (hydroxymethyl) aminomethane, and N- [tris (hydroxymethyl) methyl] glycine And more preferably, PBS.

Here, the content of the composition of the internal structure and the organic particles is the same as that described above.

The beads in the form of beads including the drug and cells preferably have a diameter of 1 to 10 mm, more preferably 2 to 5 mm.

The foregoing description is merely illustrative of the technical idea of the present invention, and various changes and modifications may be made by those skilled in the art without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are intended to illustrate rather than limit the scope of the present invention, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas within the scope of equivalents should be construed as falling within the scope of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1. Drugs and cells containing Beads  Produce

Step 1: First Paste  Preparation phase

Calcium carbonate (CaCO ₃ ) and dicalcium phosphate (CaHPO ₄ ) were reacted at a molar ratio of 1: 2 and physically mixed to prepare an α-TCP powder mixture. The α-TCP powder mixture and ethanol were mixed and milled for 24 hours. After 24 hours, the resulting slurry was dried and sieved with a 100 [mu] M sieve. The powder was sintered at 1400 ° C for 12 hours and quenched at 1400 ° C. In order to reduce the particle size of the powder, a ball mill was performed in ethanol at a speed of 250 rpm for 4 hours. Then, the powder was dried and sieved with a sieve of 25 mu m to prepare an alpha-TCP powder having an average particle size of 4.8 mu m.

Then, 1 part by weight of quercetin was added to 100 parts by weight of? -TCP powder, followed by ball milling to prepare a mixed powder for uniform dispersion. 1% by weight of hydroxypropyl methyl cellulose (HPMC) dissolved in 30% by weight of ethanol was separately added thereto at a liquid separation ratio of 1: 0.6 and uniformly mixed to obtain a first paste having a viscosity of 140,000 cP Prepared.

Step 2. The second Paste  Preparation phase

MC3T3-E1 cells (MC3T3-E1 Subclone 4, ATCC CRL-2593, obtained from ATCC) were cultured and dissolved in PBS solution. Then, alginate was mixed with a hydrate having a concentration of 1 wt% to prepare a second paste containing cells Prepared.

Step 3. Droplet  Forming step

The first paste prepared in step 1 was placed in a container containing an inner tube having a diameter of 400 m in the extrusion container of the double nozzle and the second paste prepared in step 2 was put into a container containing an outer tube and extruded to prepare a droplet . Here, the extrusion pressure of the first paste was adjusted to 600 U and the extrusion pressure of the second paste was adjusted to 100 U.

Step 4. Cross-linking step

The droplets prepared in step 3 were immersed in 2.5 M calcium chloride (CaCl ₂ ) to induce crosslinking for 30 minutes to prepare a bead type filler.

Step 5. Curing step

The bead type bone filler prepared in step 4 was washed and immersed in PBS for 12 hours to induce a curing reaction.

Step 6. Culture step

1 to from a bead form of the bone filling material containing intracellular cured in step 5, the standard culture conditions _{(37 ℃, 5% CO 2} ) with α-MEM (α-Minimum Essential Medium, GIBCO) filled with penicillin / streptomycin in And cultured for 50 days.

Experimental Example  1. Cells Survival rate  observe

The cell viability in the crosslinking step, the curing step and the culturing step of the bone filler cultured by Example 1 was observed, and the results are shown in FIG.

FIG. 2 (a) shows the cell viability at the curing step, (b) shows the cell viability at the crosslinking step, and (c) shows the cell viability at the culturing step.

It can be seen from FIG. 2 (a) that the cell viability is maintained during crosslinking of the hydrogel, and (b) maintains high cell viability during curing of the coil shape of the inner ceramic. c), it was confirmed that the cell culture was well performed in the medium.

Experimental Example  2. Observation of cell growth by cell culture period

The cell growth rate was observed by culturing the bone filler cultured according to Example 1 for up to 50 days, and the cell growth rate was measured at 3 days, 5 days, 28 days, 35 days, 42 days, and 50 days The results are shown in Fig.

3, cell growth was observed from the third day of cell culture, cell growth was observed in alginate at the fifth day, and cell growth was observed from the alginate region to the ceramic region on the 28th, 35th, And it was confirmed that it could be applied as a bone filling material containing cells.

Experimental Example  3. Drug Release Test

The drug release characteristics of quercetin were examined in the bone filler prepared in Example 1, and the results are shown in FIG.

4 shows the beads containing no drug and the right photograph shows the beads containing quercetin of Example 1. It was confirmed that the yellow color of quercetin powder was totally transferred to the ceramic structure. As a result, quercetin was uniformly distributed in the structure As shown in Fig.

Also, the amount of quercetin released from the beads can be confirmed through the graph of FIG. 4, and it was confirmed that quercetin can be sustained release for 40 days.

Example  2. MCPM  Simultaneous induction of crosslinking and curing by

Step 1 to Step 3 of Example 1 were carried out in the same manner, and in Step 4, the following crosslinking step and curing step were carried out simultaneously.

Bond type filler was prepared by simultaneous crosslinking of hydrogel and hardening of ceramics in 0.1M monocalcium phosphate monohydrate (MCPM) solution. Cells were then cultured in the same manner as in step 6 of Example 1.

Experimental Example  4. Identification of cell survival / structural changes by curing reaction time

In the bone filler cultured according to Example 2, the curing reaction time was adjusted to 5 minutes, 15 minutes, 30 minutes, and 60 minutes, respectively, and cell viability and structural modification were confirmed. The results are shown in FIGS. 5 and 6 Respectively.

As shown in FIG. 5, although the cells did not die and showed a high survival rate until the reaction time of 5 minutes, it was confirmed that the cells rapidly died after 15 minutes. Therefore, it can be confirmed that when the cross-linking and the curing are simultaneously performed, the reaction time adversely affects cell survival if the reaction time is long.

On the other hand, FIG. 6 shows that after 5 minutes, the α-TCP was transformed into a calcium-deficient hydroxyapatite (CDHA) phase. When the cell viability was considered, It was confirmed that the reaction time during induction was preferably within 5 minutes.

Example  3. Performing additional curing

Steps 1 to 3 of Example 1 were carried out in the same manner, and the droplets formed in Step 3 were immersed in 0.1 M of MCPM (Monocalcium phosphate monohydrate) for 5 minutes to induce a curing reaction, followed by curing in PBS for 6 hours Lt; / RTI > The cured beads were then incubated for 18 hours in α-MEM (α-Minimum Essential Medium, GIBCO) filled with penicillin / streptomycin under standard culture conditions (37 ° C., 5% CO 2).

Example  4. Omission of additional hardening

The procedure of Example 3 was followed except that the incubation was carried out in? -MEM (GIBCO) immediately except for the curing reaction in PBS.

Experimental Example  5. Identification of cell survival / structural deformation with or without additional curing

The cell viability and structural modification of the bone filler prepared by Examples 3 and 4 were confirmed, and the results are shown in FIGS. 7 and 8, respectively.

7, when the curing reaction was further induced in the PBS after the curing reaction in the monocalcium phosphate monohydrate (MCPM) as in Example 3, the cell viability could be increased without cell death. In addition, as in Example 4, it was confirmed that the cell survival rate was higher when cultured in? -MEM after monoculturing reaction with monocalcium phosphate monohydrate (MCPM), and it is expected that the cell culture medium induces a similar effect to PBS.

However, as shown in FIG. 8, in Examples 3 and 4, it was confirmed that α-TCP was phase-transformed into a calcium-deficient hydroxyapatite (CDHA) phase, (Fig. 8 (b)), the degree of phase transformation into calcium-deficient hydroxyapatite (CDHA) phase is low. In order to secure structural stability, it is preferable to further cure .

Claims (17)

Preparing a first paste containing calcium phosphate-based ceramics and a drug;
Preparing a second paste comprising a hydrogel and cells;
Placing the first paste in a container connected to an inner pipe of an extrusion container having a double nozzle and extruding the second paste into a container connected to an outer pipe of an extrusion container having a double nozzle to form a droplet; And
And immersing the droplets in a crosslinking solution to obtain beads containing drugs and cells.
The method according to claim 1,
Further comprising the step of immersing the beads immersed in the crosslinking solution in a curing liquid.
3. The method of claim 2,
And culturing the cells in the beads immersed in the hardening liquid.
The method according to claim 1,
Wherein a nozzle size of the inner pipe for extruding the first paste is 100 to 1000 占 퐉.
The method according to claim 1,
The hydrogel may be selected from the group consisting of alginate, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC), gelatin, collagen, Fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, polyethylene glycol, And at least one polymer selected from the group consisting of poly (ethylene glycol), poly (propylene glycol), hyaluronan, and poly (vinylpyrrolidone) By weight.
6. The method of claim 5,
Wherein the polymer is contained in a concentration of 0.1 to 10% by weight in the second paste.
The method according to claim 1,
The drug may be selected from the group consisting of Quercetein, Genistein, Curcumin, Saurolactam, Sauchinone, Baicalin, Daidzein, Rutin, Anthocyanidin, fisetin, icariin, kaempferol, E. Koreanum Nakei, equol, alendronate, But are not limited to, risedronate, etidronate, clodronate, neridronate, ibandronate, zoledronate, olpadronate, ) And a bisphosphonate-based compound. ≪ RTI ID = 0.0 > 11. < / RTI >
The method according to claim 1,
The calcium phosphate-based ceramics may be selected from the group consisting of hydroxyapatite, dicalcium phosphate dihydrate (DCPD), monocalcium phosphate monohydrate (MCPM), dicalcium phosphate anhydrous (DCPA), biphasic calcium phosphate, And β-TCP (β-Tricalcium phosphate).
The method according to claim 1,
The first paste may contain at least one selected from the group consisting of hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC), alginate, gelatin, collagen, Fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, polyethylene wax, A thickening agent of at least one of poly (ethylene glycol), poly (propylene glycol), hyaluronan and poly (vinylpyrrolidone) ≪ / RTI >
10. The method of claim 9,
And 10 to 100 parts by weight of a solution containing the thickening agent, based on 100 parts by weight of the calcium phosphate-based ceramic.
The method according to claim 1,
Wherein the cross-linking liquid comprises at least one of calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ ), calcium phosphate (CaP) and calcium carbonate (CaCO ₃ ).
3. The method of claim 2,
The curing solution may be selected from the group consisting of H ₂ O, phosphate buffer saline (PBS), monocalcium phosphate monohydrate (MCPM), diammonium hydrogen phosphate (DAHP), NH ₄ H ₂ PO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ and NaH ₂ PO ₄ The method comprising the steps of:
An inner structure in which a single fiber is formed of a coil and includes a ceramic and a drug; And
Forming a surface surrounding the internal structure, forming an organic particle
Wherein the bead-shaped bone filler is in the form of a bead.
14. The method of claim 13,
Wherein the internal structure comprises calcium-deficient hydroxyapatite (CDHA).
14. The method of claim 13,
The drug may be selected from the group consisting of Quercetein, Genistein, Curcumin, Saurolactam, Sauchinone, Baicalin, Daidzein, Rutin, Polyphenolic natural materials such as polyvinylpyrrolidone, anthocyanidin, fisetin, icariin, Kaempferol, E. Koreanum Nakei and Equol. Derived material; But are not limited to, alendronate, risedronate, etidronate, clodronate, neridronate, ibandronate, zoledronate, Characterized in that it comprises at least one of olpadronate and bisphosphonate.
14. The method of claim 13,
The internal structure may be formed of at least one selected from the group consisting of hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), methyl cellulose, carboxymethylcellulose (CMC), alginate, gelatin, collagen, Fibrinogen, chitosan, agar, matrigel, starch, pectin, polyvinyl alcohol, polyurethane, polyethylene wax, A thickening agent of at least one of poly (ethylene glycol), poly (propylene glycol), hyaluronan and poly (vinylpyrrolidone) Wherein the bone filler is a bone filler.
14. The method of claim 13,
The hydrogel may be selected from the group consisting of alginate, gelatin, collagen, fibrinogen, chitosan, agar, Matrigel, starch, pectin, Polyvinyl alcohol, polyurethane, poly (ethylene glycol), poly (propylene glycol), methyl cellulose (hydroxyethyl ethyl cellulose), polyvinyl alcohol, Wherein the filler comprises at least one polymer selected from the group consisting of methyl cellulose, carboxymethylcellulose, hyaluronan and poly (vinylpyrrolidone).

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