KR101178204B1 - Porous microsphere and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a porous microsphere and a method of manufacturing the same, and more particularly, porous pores are formed on the surface and inside of a spherical body including a bioactive ceramic, and the porous pores having an open pore structure in which the micropores are interconnected. It relates to a microsphere and a method of manufacturing the same.

The porous microspheres have an open pore structure that is well connected to each other, so that cell attachment and proliferation occur actively during transplantation, thereby effectively regenerating bone and periodontal tissue.

Microspheres, Bioactive Ceramics, Bone Tissue, Bone Conduction

Description

POROUS MICROSPHERE AND MANUFACTURING METHOD THEREOF

The present invention relates to a porous microsphere for bone tissue regeneration and a method of manufacturing the same, and more particularly, to have a large surface area and space to allow bone cells to be attached to bone conduction and bone regeneration having an open pore structure that is well connected to each other. It relates to a porous microsphere and a preparation method thereof.

In general, when bone tissue of a vertebrate is damaged by trauma, tumors, malformations, etc., severe pain and daily activities are limited. The treatment is performed to fill the damaged area with bone to generate new bone.

Treatments known to date include autologous bone grafts for transplanting their own bones, allogeneic bone grafts for transplanting bones of others, and xenograft grafts for treating and transplanting animal bones.

The autologous bone graft is a method of transplanting bone tissue, which has already been produced in the normal part of a patient, into a damaged tissue area, and has been successful in some patients. However, since it is a sacrifice of normal tissue, there are vulnerabilities such as new pain in the donor, and complications such as pain, fracture, bleeding, scars, and slow rehabilitation after surgery may occur. . Allogeneic bone grafts present a risk of transmission of diseases such as immune response and hepatitis. On the other hand, xenograft transplantation has the disadvantage of risk of immune response and mad cow disease. Accordingly, there is a demand for development of a bio-artificial material as a bone graft material having excellent biocompatibility.

As a representative material of artificial bone graft material, calcium phosphate-based ceramics such as hydroxyapatite, tricalcium phosphate, and bioactive glass are widely used. Dual calcium phosphate-based ceramics are not only excellent in biocompatibility but also excellent in safety, and are being actively researched as bone graft materials.

On the other hand, the microsphere (microsphere) has been conventionally used in radiodiagnostic drugs, and recently, has attracted wide attention as a drug carrier. Such microspheres generally refer to a particulate system of 1 to 2000 μm in which the drug is dispersed in a dissolved or microcrystalline state in the polymer matrix.

Microparticles, such as microspheres, are being developed for a variety of purposes, including delivery of drugs, proteins, cells, delayed release of drugs, delivery to target sites, and improvement of physical properties, including fluidity (S. Freiberg, XX Zhu, Int). J. Pharm. 282 , 1 (2004). Polymers, inorganic materials and mixtures thereof used as coating materials have been developed for the above-mentioned uses, and in vitro in vitro ) and feasibility and clinical performance (KJ Pekarek, JS Jacob, E. Mathiowitz, Nature 367 , 258 (1994); M. Borden, M. Attawia, Y. Khan, CT Laurencin, Biomaterials 23 , 551 (2002) HJ Gu, HH Lee, HW Kim, Tissue Eng. 13 , 965 (2007); QQ Qiu, P. Ducheyne, PS Ayyaswamy, J. Biomed. Mater.Res. 52 , 66 (2000); AM Rokstad, S. Holtan, B. Strand, B. Steinkjer, L. Ryan, B. Kulseng, G. Skjak-Braek, T. Espevik, Cell Transplant. 11 , 313 (2002); D. Kato, M. Takeuchi, T. Sakurai, S. Furukawa, H. Mizokami, M. Sakata, C. Hirayama, M. Kunitake, Biomaterials 24 , 4253 (2003); H. Maeda, T. Kasuga, Acta Biomater. 2 , 403 (2006); HW Kim, BH Yoon, HE Kim, J. Mater. Sci. Mater. Med. 16 , 1105 (2005); QQ Qiu, P. Ducheyne, PS Ayyaswamy, Biomaterials 20 , 989 (1999)). Many in vitro to be a micro-particle effective in maintaining a bioactive polymer and release in order to elicit a therapeutic effect in a gene, cell or tissue level (in vitro) and in vivo (in in vivo ).

Small sizes (tens of tens to hundreds of micrometers) of granules prepared with the hydroxyapatite described above were prepared to fill and enhance defects in the periodontal pocket and alveolar bone (W. Paul, CP Sharma, J. Biomater. Appl. 17 , 253 (2003); KA Al Ruhaimi, Int. J. Oral. Maxillofac Implants. 16 , 105 (2001); PC Hobar, M. Pantaloni, HS Byrd, Clin.Plast.Surg. 27 , 557 (2000); JM Schmitt , DC Buck, SP Joh, SE Lynch, JO Hollinger, J. Periodontol. 68 , 1043 (1997)). These hydroxyapatite granules have been reported to have excellent bone formation ability in many experimental models because of the chemical composition similar to that of natural bone minerals. In vivo (in vivo) in vitro for therapy and tissue engineering (ex vivo) to induce specific biological responses including cell differentiation, an appropriate signal, such as a bioactive protein, antibiotics and growth factors have been introduced with hydroxyapatite granules and scaffold (BG Santoni, GE Pluhar, T. Motta, DL Wheeler, Biomed. Mater. Eng. 17 , 277 (2007); HW Kim, JC Knowles, HE Kim, J. Mater.Sci.Mate.Med. 16 , 189 (2005); I. Ono, T. Yamashita, HY Jin, Y. Ito, H. Hamada, Y. Akasaka, M. Nakasu, T. Ogawa, K. Jimbow, Biomaterials 25 , 4709 (2004); HW Kim, HE Kim, JC Knowles, Biomaterials 25 , 1279 (2004 )).

Compared to porous blocks, granular hydroxyapatite containing microspheres can be used as filler for filling missing defects of complex shape as well as injectable carriers (M. Borden, M. Attawia, Y. Khan). , CT Laurencin, Biomaterials 23 , 551 (2002); HJ Gu, HH Lee, HW Kim, Tissue Eng. 13 , 965 (2007)). Such microspheres can be used for the recruitment or ex vivo of osteogenic or stem cells when explanted directly into defective tissue. Designed in vivo , it is recognized as a useful vector for the delivery of characteristic cells (M. Borden, M. Attawia, Y. Khan, CT Laurencin, Biomaterials 23 , 551 (2002); HJ Gu, HH Lee, HW Kim, Tissue Eng. 13 , 965 (2007); QQ Qiu, P. Ducheyne, PS Ayyaswamy, J. Biomed.Mate.Res. 52 , 66 (2000); AM Rokstad, S. Holtan, B. Strand, B. Steinkjer, L. Ryan, B. Kulseng, G. Skjak-Braek, T. Espevik, Cell Transplant. 11 , 313 (2002); D. Kato, M. Takeuchi, T. Sakurai, S. Furukawa, H. Mizokami, M. Sakata, C. Hirayama, M. Kunitake, Biomaterials 24 , 4253 (2003)). Recent studies on cell culture and delivery using polymeric microparticles have shown that spherical materials are effective for cell growth and population (AM Rokstad, S. Holtan, B. Strand, B. Steinkjer, L. Ryan, B. Kulseng, G. Skjak-Braek, T. Espevik, Cell Transplant. 11 , 313 (2002); D. Kato, M. Takeuchi, T. Sakurai, S. Furukawa, H. Mizokami, M. Sakata, C. Hirayama, M. Kunitake, Biomaterials 24 , 4253 (2003)). Moreover, collagen-apatite microspheres have shown the potential of microspheres to help the growth of mouse-derived stem cells and the expression of bone-related genes (HJ Gu, HH Lee, HW Kim, Tissue Eng. 13 , 965 ( 2007)). However, while most of the research has focused on the control of drug delivery or sphere formation of bioceramic particles, there has not been much research on their usefulness as hydroxyapatite microspheres, especially cell delivery and tissue engineering matrices (QQ Qiu , P. Ducheyne, PS Ayyaswamy, Biomaterials 20 , 989 (1999); MP Ferraz, AY Mateus, JC Sousa, FJ Monteiro, J. Biomed. Mater.Res.A 81 , 994 (2007); SP Victor, TS Kumar, J. Mater. Sci. Mater. Med. 19 , 283 (2008)).

As an example of a study on the tissue engineering matrix of calcium phosphate-based ceramics, Korean Patent Registration No. 10-0498759 discloses dissolving a calcium phosphate-based compound in an acidic aqueous solution, adding a base to the aqueous solution to adjust neutral or alkaline, and adjusting the temperature. The manufacturing method of the hydroxyapatite granule which raises and reprecipitates hydroxyapatite is proposed.

In addition, Korean Patent Registration No. 10-0498759 discloses a method for producing spherical porous β-tricalcium phosphate spherical granules using tricalcium phosphate precursor, pore precursor, gelatin solution and dispersion medium.

However, the calcium phosphate-based granules manufactured according to the above-mentioned method are all closed structures, and have a fundamental problem in that they cannot provide a large surface area and space for bone conduction and bone regeneration. As it is difficult to promote bone formation there is a limit.

In order to solve the above problems, an object of the present invention has an open pore structure that is well connected to each other, a porous micro for bone regeneration that can provide a large surface area and space for bone conduction and bone regeneration attached to bone cells It is to provide a sphere and a manufacturing method thereof.

In order to achieve the above object,

Provided is a porous microsphere having fine pores formed on the surface and inside of a spherical body including a bioactive ceramic, the micropores having an open pore structure interconnected.

Also,

S1) bioactive ceramics; Organic solvents; Dispersing agent; Pore formers; And preparing a ceramic slurry comprising a binder;

S2) preparing an aqueous phase comprising a hydrophilic solvent and a surfactant;

S3) dropping the ceramic slurry prepared in S1) to the aqueous phase prepared in S2);

S4) obtaining microspheres produced in the water phase;

S5) subliming the pore former to form pores in the microsphere body and inside; And

S6) heat-treating the microspheres with pores obtained in S5)

It provides a method for producing a porous microsphere comprising a.

Porous microspheres according to the present invention has open pores connected to the outside, these pores are well connected to each other to provide a large surface area and space that can be attached to bone cells and bone conduction and bone regeneration. Therefore, the porous microsphere according to the present invention can be usefully used as a bone graft material or bone cell support.

Hereinafter, the present invention will be described in more detail.

The porous microsphere of the present invention has micropores formed on the surface and inside of the spherical body including the bioactive ceramic, and has an open pore structure in which the micropores are interconnected.

At this time, the material of the microsphere includes a bioactive ceramic. Bioactive ceramic is an inorganic material that absorbs or remains in the body and conducts bone formation, like bone minerals. The adhesion and proliferation of tissue cells occur well, and the function of differentiated cells is preserved. It can be used as long as it has a biocompatibility that is compatible with surrounding tissues and does not cause an inflammatory response. Typically, calcium phosphate-based ceramics, calcium silicate-based ceramics, bioactive glass or mixtures thereof may be used. For example, hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ OH ₂ ), oxyapatite (Ca ₁₀ (PO ₄ ) ₆ O), monocalcium phosphate (Ca (H ₂ PO ₄ ) ₂ ), dicalcium phosphate (CaHPO ₄ -2H ₂ O), tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ), tetracalcium phosphate (Ca ₄ (PO ₄ ) ₂ O), calcium metaphosphate (Ca (PO ₃ ) ₂ ) and their Calcium phosphate ceramics selected from the group consisting of mixtures; Tricalcium silicate (Ca ₃ SiO ₅ ), calcium orthosilicate (Ca ₂ SiO ₄ ), calcium disilicate (CaSi ₂ O ₅ ), tricalcium disilicate (Ca ₃ Si ₂ O ₇ ), wollastonite (CaSiO ₃₎ Calcium silicate-based ceramics selected from the group consisting of; silica (SiO ₂ ), sodium oxide (Na ₂ O), calcium oxide (CaO), phosphorus pentoxide (P ₂ O ₅ ) and mixtures thereof Bioactive glass selected from the group; Or mixtures thereof.

In this case, the microspheres have an average particle diameter of preferably 220 to 260 μm, more preferably 230 to 250 μm, and micropore diameters of 20 to 50 μm in consideration of mechanical properties and tissue engineering uses.

Porous microspheres of the present invention has an open pore structure in which micropores are interconnected to allow bone tissue to be grown into the microspheres. Accordingly, a large surface area and space for attaching bone cells to conduct bone conduction and bone regeneration are provided.

1 is a schematic diagram showing a step of manufacturing a porous microsphere for bone tissue regeneration according to the present invention.

Referring to Figure 1, the porous microspheres for bone tissue regeneration as described above

S1) bioactive ceramics; Organic solvents; Dispersing agent; Pore formers; And preparing a ceramic slurry comprising a binder;

S2) preparing an aqueous phase comprising a hydrophilic solvent and a surfactant;

S3) dropping the ceramic slurry prepared in S1) to the aqueous phase prepared in S2);

S4) obtaining microspheres produced in the water phase;

S5) subliming the pore former to form pores in the microsphere body and inside; And

S6) heat-treating the microspheres with pores obtained in S5)

It is prepared through.

First, bioactivity in step S1); Organic solvents; A ceramic slurry is prepared comprising the dispersant and the binder.

Bioactive ceramics that can be used in the present invention are replaced by the foregoing. In this case, the content of the bioactive ceramic is preferably 5 to 50% by weight of the ceramic slurry so that the porous microspheres can be easily formed.

The organic solvent is not particularly limited in the present invention as long as it can uniformly dissolve the bioactive ceramic, the pore-forming agent, the dispersing agent and the binder. As preferred organic solvents, those having high hydrophobicity and high volatility can be used. For example, dichloromethane, chloroform, dimethylformamide, ethyl acetate, ethanol, methanol, acetone, acetonitrile, tetrahydrofuran, acetic acid, dimethyl sulfoxide and mixed solvents thereof are possible. At this time, the content of the organic solvent is preferably 10 to 93% by weight in the ceramic slurry.

The binder is added for the binding of the bioactive ceramic powder, and preferably polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl butyral and mixtures thereof. In this case, the content of the binder is preferably 0.1 to 10% by weight in the ceramic slurry.

The pore-forming agent of the present invention is added to impart an open pore structure to the finally prepared porous microsphere, and is not particularly limited as long as it can be removed by sublimation under atmospheric conditions. Preferably, those having a melting point of 20 to 60 ° C are used. Typically camphor, camphor, naphthalene, menthol, thymol, coumarin, vanillin, salicyamide, 2-aminopyridine, t -butanol, trichloro- t -butanol, imidazole, dimethylsulfone, urea, 2-amidopyridine This is possible. Preferably, camphor is used. At this time, the content of the pore-forming agent is preferably 1 to 70% by weight in the ceramic slurry.

A dispersant is added to suppress and evenly disperse the aggregation of the bioactive ceramic powder to produce a ceramic slurry having uniform properties. The dispersant is not particularly limited as long as the bioactive ceramic powder can be uniformly dispersed. However, oligomeric polyesters that give excellent dispersion of bioactive ceramic powders are preferred, and KD4 (Hypermer; UniQuema) may be more preferably used as the oligomeric polyester. At this time, the content of the dispersant is preferably 0.1 to 10% by weight in the ceramic slurry.

Next, in step S2), an aqueous phase containing a hydrophilic solvent and a surfactant is prepared.

The hydrophilic solvent is preferably used distilled water.

The aqueous phase of the present invention may include a surfactant to adjust the interface between the slurry droplet and the hydrophilic solvent. The surfactant may be used as long as HLB (hydrophilic-lipophilic balance) value is 10 or more. For example, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl butyral, polyvinyl methyl ether, polyvinyl ether and mixtures thereof may be used. The amount of the surfactant is preferably 0.1 to 10% by weight in the water phase in consideration of the particle size of the porous microspheres to be produced.

Subsequently, in step S3), the ceramic slurry prepared in S1) is added dropwise to the aqueous phase prepared in S2).

Dropping the ceramic slurry into the water phase dropwise causes the bioactive ceramics to combine to form spherical particles. Subsequently, solidification proceeds, and the spherical particles maintain a stable shape. At this time, the highly volatile organic solvent is volatilized by stirring.

The slurry is prepared by stirring sufficiently for 10 minutes to 24 hours at a stirring speed of 100 to 2,000 rpm to prepare a porous microsphere having a uniform structure. If the stirring speed is too fast, the formation of particles is difficult, there is a problem that is broken, on the contrary, if the stirring speed is too slow, there is a problem that the particle size of the microspheres due to agglomeration during the initial formation of the particles increases. The stirring time is a time required for the organic solvent to evaporate sufficiently and the microspheres to be solidified may be changed within the above ranges according to the material, the stirring speed, and the type of the organic solvent of the microspheres.

Next, in step S4), microspheres generated in the water phase are obtained.

Solidification is complete after evaporation of the organic solvent to give a cured microsphere. At this time, commonly known methods such as filtration, washing, drying and the like are performed.

Subsequently, in step S5), the pore-forming agent is sublimed to form pores in the microsphere body and the inside.

The pore-forming agent used in the present invention is easily removed by sublimation at atmospheric conditions without heating to a temperature above the melting point of the solution used in the manufacturing process. The microspheres have an open pore structure in which micropores are connected to each other by sublimation to such a pore-forming agent.

The pore forming step is performed for 10 minutes to 100 hours at ambient temperature. At this time, when the pore forming time is less than the above range, sufficient pores cannot be formed. On the contrary, even if the pore-forming time exceeds the above range, there is no further effect, which is uneconomical.

Finally, in step S6), the microspheres with pores are heat-treated.

Through such a heat treatment step, binders, surfactants, and unreacted substances remaining on the surface are removed, and the bioactive ceramics are densified and crystallized to have a stable structure.

At this time, the heat treatment is preferably performed for 10 minutes to 5 hours at 600 to 1500 ℃.

This heat treatment step is preferably carried out at a temperature increase rate of 0.1 to 5 ℃ / min.

In this case, if the heat treatment temperature and time is less than the above range, sufficient crystallization of the bioactive ceramic is not performed. On the contrary, if the heat treatment temperature is exceeded, the diameter of the microsphere and the size of the pores formed therein are reduced. do.

Through the above heat treatment, the weight decrease and the bioactive ceramic powder are sintered by the disappearance of the binder, the unreacted material, etc., thereby reducing the size of the porous microspheres.

Optionally, the step of immersing the microspheres formed with pores obtained in S5) in a bio-like solution may be further performed. When this immersion step is performed, calcium phosphate precipitates are formed in the solids, thereby obtaining hydroxyapatite crystals after the final heat treatment. In particular, when calcium silicate-based ceramics are used as the bioactive ceramics, when the bio-oil solution is immersed and heat-treated, a porous microsphere having a hydroxyapatite-wollastonite double crystal structure can be obtained. At this time, the immersion step is performed for 1 to 1000 hours at room temperature. If the immersion time is less than the above range, the formation of calcium phosphate precipitate on the microsphere surface is insignificant, on the contrary, if it exceeds the above range, calcium ions and crystals are formed in the solution to form a precipitate and the precipitate cannot be formed on the solid surface. There is a problem.

The body Similar solutions can be prepared by dissolving a reagent-grade of _{NaCl, NaHCO 3, Na 2 SO} 4, KCl, K 2 HPO 4, MgCl 2? 6H 2 O, and CaCl _2? 2H ₂ O in de-ionized distilled water have. Most preferably 2.5 mM Ca ²⁺ , 142 mM Na ⁺ , 1.5 mM Mg ²⁺ , 5.0 mM K ⁺ , 125.0 mM Cl ⁻ , 7.0 mM HCO ₃ ⁻ , 1.0 mM HPO ₄ ^2- and 0.5 mM SO ₄ ^2- Use a bio-use solution of pH 7.4 containing (SBF).

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1: Preparation of hydroxyapatite porous microspheres

1.1 Preparation of hydroxyapatite powder

Ca (NO ₃ ) ₂ ˜4H ₂ O and (NH ₄ ) ₂ HPO ₄ as precursors The hydroxyapatite powder was manufactured by the wet reaction made into aqueous solution. Each compound was dissolved in distilled water and mixed stoichiometrically so that the molar ratio of Ca / P was 1.67. The reaction proceeded in air at 37 ° C. for 24 hours and the pH was adjusted to 9.0 using Tris-HCl buffer. After the reaction, the precipitate was filtered, washed and dried. The dried powder was calcined (calcine) at 700 ° C. for 3 hours to obtain hydroxyapatite powder.

1.2 Preparation of Ceramic Slurry

The obtained hydroxyapatite powder and camphor (C ₁₀ H ₁₆ , Sigma-Aldrich) were mixed at a weight ratio of 1: 8, and then ball milled at 50 ° C., to which a slurry mixture was added by adding 0.75 wt% of surfactant KD4. Prepared. A 10 wt% solution of 10 wt% polyvinyl butyral in dichloromethane was added to the slurry mixture. The slurry thus prepared was homogenized by ball milling for 24 hours to prepare a slurry.

1.3 Preparation of Porous Microspheres

The prepared slurry was dropped drop by drop while stirring at 750 rpm in an aqueous phase containing 2% PVA. The slurry was solidified for 30 minutes, filtered and washed with distilled water cooled with ice. The washed microspheres were stored at −20 ° C. for 10 minutes, collected in an alumina crucible, and dried overnight at atmospheric conditions to sublimate camphor. At this time, the weight change of the microsphere with the sublimation time was measured. The resulting microspheres were heated to 1400 ° C. at a rate of temperature increase of 2 ° C./min, heat treated for 3 hours, and cooled in air in a furnace.

Example 2: Preparation of Apatite-Wollastonite Porous Microspheres

2.1 Preparation of Calcium Silicate Powder

Calcium silicate powder was prepared as precursor for bioactive ceramic microspheres.

Specifically, Ca (NO ₃ ) _{2 to} 4H ₂ O and tetraethyl orthosilicate (TEOS) were mixed with ethanol at a molar fraction of 1: 1 using sodium hydroxide as a catalyst. The mixed solution was stirred and then left at 40 ° C. until the solution became gel, and then dried at 70 ° C. The dried powder was pulverized and then calcined at 500 ° C. for a time to obtain calcium silicate powder.

2.2 Preparation of Ceramic Slurry

A ceramic slurry was prepared in the same manner as in 1.2 except that the calcium silicate powder was used instead of the hydroxyapatite powder.

2.3 Preparation of Porous Microspheres

The prepared slurry was dropped drop by drop while stirring at 750 rpm in an aqueous phase containing 2% PVA. The slurry was solidified for 30 minutes, filtered and washed with distilled water cooled with ice. The washed microspheres were stored at −20 ° C. for 10 minutes, collected in an alumina crucible, and dried overnight at atmospheric conditions to sublimate camphor. Subsequently, bioavailable SBF solution (2.5 mM Ca ²⁺ , 142 mM Na ⁺ , 1.5 mM Mg ²⁺ , 5.0 mM K ⁺ , 147.8 mM Cl ⁻ , 4.2 mM HCO ₃ ⁻ , 1.0 mM HPO ₄ ^2- and 0.5 After soaking in mM SO ₄ ^2- ) at room temperature for 24 hours, the microspheres were heated to 1400 ° C. at an elevated rate of 2 ° C./min for heat treatment for 3 hours, and cooled in air in a furnace.

Experimental Example 1: Scanning Electron Microscope Analysis

In order to determine the surface shape and microstructure of the porous microspheres prepared in Example 2, the porous microspheres after Pt coating were analyzed by scanning electron microscopy, and the obtained results are illustrated in FIGS. 2A and 2B. ).

2 (A) is a scanning electron micrograph of 300 times the surface of the porous microsphere prepared in Example 2, (B) is enlarged 1500 times.

Referring to FIG. 2, it can be seen that micropores having a diameter of 20 to 50 μm are well formed through the entire microspheres, and the formed micropores have an open structure.

Experimental Example 2: Analysis of Average Particle Size of Porous Microspheres

To determine the size distribution of the porous microspheres prepared in Example 2 Hazard particle size was analyzed and the results are shown in FIG. 3.

Referring to Figure 3, the porous microsphere is a size having a particle size of several tens to several hundred micrometers, it can be seen that the average particle diameter is 241 ㎛. These average particle diameters are judged to be appropriate sizes for cell delivery and tissue engineering applications.

Experimental Example 3: Analysis of Weight Change

The weight change of the microspheres according to the sublimation of camphor used as a pore-forming agent in the production method of the present invention was measured, which is shown in FIG.

4 is a graph showing the weight change of the porous microspheres with the sublimation time in Example 2.

Referring to FIG. 4, it can be seen that the sublimation of camphor proceeds rapidly and is completed within almost 6 hours.

Experimental Example 4: XRD Analysis

X-ray diffraction of the hydroxyapatite powder used as the starting material in Example 1 and the porous microspheres prepared in Example 1 to determine the effect of the method of the present invention on the crystallization of the starting material calcium phosphate ceramic Measured using an analyzer (X-Ray Diffraction, XRD), the results obtained are shown in FIG.

 5 (A) is an X-ray diffraction spectrum of the hydroxyapatite porous microsphere of Example 1, (B) is an X-ray diffraction of the apatite-walla stonite porous microsphere of Example 2 according to the heat treatment temperature Analysis spectrum.

Referring to Figure 5 (A), unlike the starting material hydroxyapatite powder (1) in the case of porous microspheres (2), the peak of the characteristic hydroxyapatite crystals are observed, through the production method of the present invention It can be confirmed that the starting material hydroxyapatite is crystallized.

Referring to FIG. 5B, heat treatment is required at a temperature of 1200 ° C. or higher to provide a mechanical strength level associated with the densification of porous microspheres. It can be seen that the crystal structure of hydroxyapatite (A) and wollastonite (W) has been changed. This apatite-wallaston double phase is the major crystalline phase commonly observed in AW glass ceramics. Furthermore, in vitro and in vivo Formation of bone mineral-like phases on the surface has been reported to have bone activity (Yamamuro T, World Scientific ; 1993. p. 89.103; Nakamura T, J Biomed Mater Res 1985 ; 19: 685.9). Using a bone graft material with a similar phase can control the osteogenic response and directly bind to hard tissue.

Experimental Example 5: in vitro  Bioactivity confirmation experiment

To verify the in vitro activity, in vivo similar to the solution in SBF solution ^{(2.5 mM Ca 2+, 142 mM} Na +, 1.5 mM Mg 2+, 5.0 mM K +, 147.8 mM Cl -, 4.2 mM HCO 3 -, 1.0 mM The porous microspheres prepared in Example 2 were incubated in HPO ₄ ^2- and 0.5 mM SO ₄ ^2- ) (Kokubo T. Biomaterials 1991 ; 12: 155.63).

Specifically, 100 mg of porous microspheres were put in a polyethylene tube, immersed in 10 ml of SBF solution, and incubated with stirring at 120 rpm at 37 ° C. After incubation, washed with distilled water and ethanol, dried in an oven and the weight change was measured. Surface properties of the porous microspheres were observed by scanning electron microscopy to confirm the precipitation induced on the surface of the microspheres, and the elements of the deposits were analyzed by energy dispersive spectroscopy (EDS).

6 (A) is a scanning electron micrograph of the surface of the porous microsphere prepared in Example 2 in SBF solution for 1 day, (B) and (C) after 7 days incubated.

Referring to (A) of FIG. 6, it can be seen that a small precipitate is formed on most of the surfaces of the porous microspheres after 1 day precipitation. Referring to (B), the nanocrystals formed after 1 day precipitation are more turned on. Under the microscope, no significant change in pore size and microsphere formation was observed.

7 (A) and (B) are graphs showing the energy dispersion spectrum, and (C) is a graph showing the weight change according to the incubation time.

Referring to FIG. 7, the phosphorus (P) is present after 7 days of incubation, and the strength of calcium (Ca) is higher than that of silicon (Si), as compared with the case before the deposition (S) in the SBF solution. It means that phosphate precipitated out. The Ca / P ratio was measured to be less than 1.53, which is similar to hydroxyapatite (1.67) but stoichiometrically, indicating that hydroxyapatite lacking calcium was formed.

In addition, referring to FIG. 7C, as the deposition time increases, the weight of the microspheres increases, indicating that calcium phosphate is precipitated. Although the ions dissolve (calcium, silicon) from the microspheres, the precipitation of calcium phosphate predominates and the microspheres appear to have increased in weight.

Experimental Example 6: Progenitor Bone Cell Culture Experiment

In order to determine the bioactivity of the porous microsphere prepared in Example 2, mouse-derived MC3T3-E1 cells, which are preosteoblasts, were cultured to observe the shape of the cells grown on the surface and the inside of the microspheres. Cell proliferation was measured using CellTiter 96 AQueous One solution cell proliferation assay (promega) assay.

Microspheres were washed twice with serum free medium before seeding the cells and placed in each well of 10 mg 96-well plates. Thereafter, 70 μl cell suspension (cell suspension, cell concentration 6 × 10 ⁴ cells / mL (low density), 5 × 10 ⁵ cells / mL (high density)) was seeded into each well. After 6 hours of incubation in a 95% CO ₂ incubator, 50 μg / ml sodium ascorbate, 10 mM β-glycerol phosphate and 10 nM dexamethasone were added per 125 μl medium to induce osteoblastic differentiation. . After 6 h incubation, 150 μl medium was added to each well and then incubated for 3, 7, 10 days.

Cell morphology grown on the surface and inside the microspheres was fixed with glutaraldehyde, dehydrated with ethanol (50%, 70%, 90%, 95% and 100%) and then treated with hexamethyl disilazane and Pt It was coated with and analyzed by scanning electron microscopy (Scanning electron microscopy), the results obtained are shown in Figure 8 (A) to (C).

Cell proliferation was quantified by MTS ((3- (4,5-dimethylthiazol-2-yl) -5 (3-carboxymethonyphenol) -2- (4-sulfophenyl) -2H-tetrazolium)) assay and the results are shown. 9 is shown.

For each incubation period (1, 5, and 15 days), the medium was separated, and CellTitero 96 AQueous One Solution Reagent (Promega, Madison) was added to each sample and reacted at 37 ° C. for 2 hours. 490 nm absorbance was measured by Elisa Plate Reader. Five samples were analyzed for each condition, and the experimental results were expressed as mean ± SD.

Figure 8 (A) using the porous microspheres prepared in Example 2 MC3T3-E1 osteogenic cells 5 days (high density), (B) 15 days (high density) (C) 15 days (low density) Scanning electron micrograph showing the surface enlarged after culturing.

Referring to FIG. 8A, when the cells were seeded at a high density, the cells adhered well to the microsphere surface, and thus, the empty spaces of the surfaces were crosslinked with each other for 5 days. Referring to FIG. 8B, when the incubation time was extended and cultured for 15 days, the cells were more activated to almost cover the surface of the porous microspheres. Referring to FIG. 8C, when cells were seeded at a low density, the cells adhered well to the surface, and the cells increased as the incubation time passed, but did not cover the entire microsphere surface even after being cultured for 15 days. .

9 is a graph showing the results of analyzing the proliferation rate of osteoblasts on the surface of the porous microsphere prepared in Example 2 of the present invention by the MTS method.

Referring to Figure 9, it shows that the amount of cells continuously increased as the incubation time is longer, which means that the microsphere of the present invention is a preferred support for the cells to attach and proliferate. In addition, according to the culture time, the cell proliferation rate showed a significant difference (P <0.05). Initial differences in cell adhesion at day 1 were found to persist throughout subsequent cell proliferation. Based on the rate of cell growth, when inoculated at low concentrations, longer incubation times may be required to cover the entire microsphere. In view of these results, when microspheres are used as cell delivery means and tissue engineering matrices, the initial cell inoculation concentration must be carefully controlled. Cell density and confluence time on three-dimensional substrates are very important in finding appropriate conditions for controlling osteogenic differentiation and for controlling the properties of detailed external matrices. Based on preliminary cell culture studies, porous bioactive microspheres showed good response to osteoblasts, provided adequate scaffold conditions for initial conjugation and cell growth.

Porous microspheres according to the present invention is excellent in cell affinity and bone conductivity, and can be effectively used in the field of dental and orthopedic surgery as a support for tissue engineering for regeneration of bone and periodontal tissue.

1 is a schematic diagram showing a manufacturing step of a microsphere according to the present invention.

2 (A) is a scanning electron micrograph of 300 times the surface of the porous microsphere prepared in Example 2, (B) is enlarged 1500 times.

Figure 3 shows the size distribution of the porous microspheres prepared in Example 2 of the present invention.

4 is a graph showing the weight change of the porous microspheres with sublimation time in Example 2 of the present invention.

 Figure 5 (A) X-Ray diffraction analysis spectrum of the hydroxyapatite porous microspheres of Example 1, (B) X-Ray diffraction of the apatite- wollastonite porous microspheres of Example 2 according to the heat treatment temperature Analysis spectrum.

6 (A) is a scanning electron micrograph of the surface of the porous microsphere prepared in Example 2 in SBF solution for 1 day, (B) and (C) after 7 days incubated.

7 (A) and (B) are graphs showing the energy dispersion spectrum, and (C) is a graph showing the weight change according to the incubation time.

Figure 8 (A) using the porous microspheres prepared in Example 2 MC3T3-E1 osteogenic cells 5 days (high density), (B) 15 days (high density) (C) 15 days (low density) Scanning electron micrograph showing the surface enlarged after culturing.

9 is a graph showing the results of analyzing the proliferation rate of bone forming cells on the surface of the porous microsphere prepared in Example 2 of the present invention by the MTS method.

Claims (13)

delete delete delete delete S1) bioactive ceramics; Organic solvents; Dispersing agent; Pore formers that can be removed by sublimation at atmospheric conditions; And preparing a ceramic slurry comprising a binder; S2) preparing an aqueous phase comprising a hydrophilic solvent and a surfactant; S3) dropping the ceramic slurry prepared in S1) to the aqueous phase prepared in S2); S4) obtaining microspheres produced in the water phase; S5) subliming the pore former to form pores in the microsphere body and inside; And S6) heat-treating the microspheres with pores obtained in S5) Method for producing a porous microsphere comprising a. The method of claim 5, The method of claim 5, further comprising the step of immersing the microspheres formed with pores obtained in the bio-use solution. The method of claim 5, wherein the bioactive ceramic is Calcium phosphate ceramics selected from the group consisting of hydroxyapatite, oxyapatite, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate, calcium metaphosphate and mixtures thereof; Calcium silicate-based ceramics selected from the group consisting of tricalcium silicate, calcium orthosilicate, calcium disilicate, tricalcium disilicate, wollastonite and mixtures thereof; Bioactive glass selected from the group consisting of silica, sodium oxide, calcium oxide, phosphorus pentoxide and mixtures thereof; And a mixture selected from the group consisting of these. The method of claim 5, wherein the organic solvent Dichloromethane, chloroform, dimethylformamide, ethyl acetate, ethanol, methanol, acetone, acetonitrile, tetrahydrofuran, acetic acid, dimethyl sulfoxide, and a mixed solvent thereof. The method of claim 5, wherein the dispersing agent Method for producing an oligomeric polyester (oligomeric polyester). The method of claim 5, wherein the pore-forming agent Campen, camphor, naphthalene, menthol, thymol, coumarin, vanillin, salicyamide, 2-aminopyridine, t -butanol, trichloro- t -butanol, imidazole, dimethylsulfone, urea, 2-amidopyridine Production method characterized in that the one selected from the group. The method of claim 5, wherein the binder A method of producing a polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl butyral and mixtures thereof. The method of claim 5, wherein the surfactant The polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl ether, polyvinyl alcohol, polyvinylpyrrolidone and the production method characterized in that one selected from the group consisting of. The method of claim 5, wherein the heat treatment Method for producing a 10 minutes to 5 hours at 600 to 1500 ℃.

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