KR101105285B1 - Porous microsphere and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a porous microsphere and a method for manufacturing the same, and more particularly, in a porous microsphere in which pores are formed in and on a spherical body surface, the microsphere comprises a polyester-based biodegradable polymer, The surface and the pore surface of the microspheres are related to a surface-modified porous microspheres and a method for producing the same coated with calcium phosphate-based crystals.

The porous microspheres have a pore structure that is well connected to each other to provide a large surface area and space for cell attachment and proliferation during implantation in the body as well as to be coated with calcium phosphate-based crystals and surface modified with hydrophilicity so that initial cell adhesion and proliferation is achieved. It can be promoted to effectively promote bone and periodontal tissue regeneration.

Porous Microspheres, Calcium Phosphate, Surface Modification

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. And a surface-modified hydrophilic to promote initial cell adhesion and proliferation, and a method for preparing the same.

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). In vitro and in vivo studies have shown that microparticles are effective at maintaining and releasing bioactive polymers to elicit therapeutic effects at the gene, cell or tissue level.

As such, microspheres are widely used to treat damaged tissue, including bone, cartilage and muscle. In many cases, biomolecules such as proteins and hormones have been loaded into the microparticles for therapeutic effects.

Recently, microspheres have been introduced as effective carriers of various tissue cells, ranging from stem cells to specialized tissue cells. Some microsphere materials Showed the same protein transport capability and cells in vivo (in vivo) experiments also In vitro (in vivo) experiments have shown the same tissue and cell capacity in (HJ Gu, HH Lee, HW Kim, Tissue Eng 2007, 13, 965;. QQ Qiu, P. Ducheyne, PS Ayyaswamy, J. Biomed. Mater. Res. 2000, 52 , 66; S. Frauenschuh, E. Reichmann, Y. Ibold, PM Goetz, M. Sittinger, J. Ringe, Biotech.Prog. 2007, 23 , 187; TK Kim, JJ Yoon, DS Lee, TG Park, Biomaterials 2006, 27 , 152; D. Kato, M. Takeuchi, T. Sakurai, S. Furukawa, H. Mizokami, M. Sakata, C. Hirayama, M. Kunitake, Biomaterials 2003, 24, 4253 ).

 When designing microspheres as effective carriers of cells, many variables must be considered. Above all, the composition and morphology of the microcarrier is the most important in determining cell and tissue responses such as initial cell adhesion, cell proliferation and differentiation, tissue formation and maintenance.

The composition of the experimentally implanted material modulates early cell adhesion and subsequent differentiation into specialized cells. In order to achieve improved biological reactivity of anchorage-dependent cells, surface chemistry must be properly considered. For osteogenic differentiation and new bone formation, bone bioreactive materials such as calcium phosphate-based ceramics and bioactive glasses are preferred. Furthermore, materials having adequate biodegradability are desirable for proper human reactivity and tissue formation.

In addition to chemical composition, the physical properties of fine particles such as surface topography, roughness and hydrophilicity are also important. From a macro perspective, it is desirable that the pores of the microspheres be largely designed in the case of porous scaffolds to promote angiogenesis, facilitate nutrition, and transplant and transport as many cells as possible. Unlike porous scaffolds in the block type, the microsphere shape can be effectively applied to the injection system during minimally invasive surgery. The utilization of microparticles of bio-inorganic particles together with the polymer matrix has attracted attention as an effective implantable bone substitute. Cell-mounted microspheres can also be an effective means of injection in tissue engineering where the size and shape of the defect site need not be considered.

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 to provide a large surface area and space for bone conduction and bone regeneration to be attached to the bone cells, surface-modified hydrophilic initial cells It is to provide a porous microsphere for bone regeneration that promotes adhesion and proliferation and a method of manufacturing the same.

In order to achieve the above object,

In porous microspheres with pores formed on and inside a spherical body,

The microsphere comprises a polyester-based polymer, the surface and the pore surface of the microsphere is coated with calcium phosphate-based crystals to provide a surface-modified porous microsphere.

Also,

S1) polyester polymer; Organic solvents; And dispersing a polymer solution including a pore-forming agent in an aqueous phase containing a hydrophilic solvent and a surfactant to prepare an O / W emulsion.

S2) removing the organic solvent from the O / W emulsion to obtain a microsphere

S3) subliming the pore-forming agent to form pores in the microsphere body and inside;

S4) activating the surface by treating the microspheres formed pores with an alkaline solution; And

S5) immersing the surface activated microsphere in a mixed solution of calcium ions and phosphate ions

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 the bone cells to conduct bone conduction and bone regeneration. In addition, it is coated with calcium phosphate-based crystals and surface modified to hydrophilic to promote early cell adhesion and proliferation. Therefore, the porous microsphere according to the present invention can be usefully used as a bone graft material or bone cell support.

The present invention is described in more detail below.

Porous microspheres of the present invention is a porous microsphere in which pores are formed on the surface and the inside of the spherical body, the microsphere comprises a polyester-based polymer, the surface and the pore surface of the microspheres are calcium phosphate-based crystals Coated and surface modified.

The polyester-based polymer is a kind of biodegradable polymer, which spontaneously decomposes after a certain period of time in vivo, and refers to a polymer having one or more characteristics of blood affinity, cell affinity, and tissue affinity.

Such polyester-based polymers are not particularly limited in the present invention, typically polycaprolactone, polylactic acid, polyglycolic acid, polylactide-co-glycolide, polyvalerolactone, polyhydroxy butyrate, poly One kind selected from the group consisting of hydroxy valerate, copolymers thereof and blends thereof is possible. The polyester-based polymer may have a weight average molecular weight of 5,000 to 2,000,000 Daltons, more preferably 10,000 to 100,000 Daltons range, but is not necessarily limited thereto.

 Porous microspheres of the present invention are modified to have excellent biocompatibility, cell compatibility and osteoconductivity by coating the body surface and the pore surface with calcium phosphate-based crystals, which are hydrophilic, biocompatible minerals. As a result, the adhesion and proliferation of cells can be promoted, thereby effectively regenerating bone and periodontal tissue.

At this time, the calcium phosphate-based crystals are well adhered and proliferated in tissue cells, and the function of differentiated cells is preserved, and biocompatible calcium phosphate-based minerals are formed that do not induce an inflammatory response because they are compatible with surrounding tissues even after transplantation.

Such calcium phosphate-based crystals are not limited in the present invention, and calcium phosphate-based inorganic materials as known in the art are used. Typically, hydroxyapatite, oxyapatite, tricalcium phosphate, monocalcium phosphate, tetracalcium phosphate, calcium metaphosphate and the like are possible. The type of calcium phosphate crystals can be determined by adjusting the concentration of calcium ions and phosphate ions, treatment conditions or treatment time during surface modification.

Preferably the microsphere body surface and the pore surface are coated with hydroxyapatite crystals. The Ca / P molar ratio of hydroxyapatite having Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ structure is 1.67, similar to human bone. In the case of surface modification by coating the surface of microspheres and pores implanted in the human body with hydroxyapatite crystals that are chemically and crystallographically similar to inorganic tissues in bone or teeth, the adhesion to bones or tissues around the graft and There is an advantage of excellent stability.

In this case, the calcium phosphate-based crystals coated on the microspheres have a diameter of 3 nm to 100 nm. If the diameter of the calcium phosphate-based crystals is less than the above range, the crystals are separated or biodegraded before the cells are initially attached, so that they are difficult to act as hydrophilic ligands. On the contrary, if the diameters exceed the above ranges, the porosity of the microspheres is lowered.

The porous microspheres have an average particle diameter of preferably 100 to 1000 μm, more preferably 100 to 300 μm and micropore diameters of 1 to 100 μm in consideration of mechanical properties and histological use.

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) polyester biodegradable polymer; Organic solvents; And dispersing a polymer solution including a pore-forming agent in an aqueous phase containing a hydrophilic solvent and a surfactant to prepare an O / W emulsion.

S2) removing the organic solvent from the O / W emulsion to obtain a microsphere

S3) subliming the pore-forming agent to form pores in the microsphere body and inside;

S4) activating the surface by treating the microspheres formed pores with an alkaline solution; And

S5) immersing the surface activated microsphere in a mixed solution of calcium ions and phosphate ions

It is prepared through.

First, the polyester polymer in step S1); Organic solvents; And a polymer solution including a pore-forming agent is dispersed in an aqueous phase containing a hydrophilic solvent and a surfactant to prepare an O / W emulsion.

Polyester-based polymers that can be used in the present invention is replaced by the contents described above. At this time, the content of the polyester-based polymer is preferably 1 to 20% by weight of the polymer solution so that the porous microsphere can be easily formed.

The organic solvent is not particularly limited in the present invention as long as it can uniformly dissolve both the polyester-based polymer and the pore-forming agent. As preferred organic solvents, those having high hydrophobicity and high volatility can be used. For example, dichloromethane, chloroform, dimethylformamide, ethyl acetate, acetonitrile, tetrahydrofuran, dimethyl sulfoxide and mixed solvents thereof are possible. At this time, the content of the organic solvent is preferably 3 to 85% by weight in the polymer solution.

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 10 to 95% by weight in the polymer solution.

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 polymer solution 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.

At this time, the water phase is cooled below the melting point of the pore-forming agent to prevent sublimation and induce solidification under atmospheric conditions, and depends on the melting point of the pore-forming agent used, preferably below 60 ° C. Cool.

The emulsification method in preparing the emulsion is well known to those skilled in the art. For example, the emulsion may be prepared using an ultrasonic wave crusher, a homomixer, a stirrer, or the like.

When preparing the O / W emulsion, the volume ratio of the aqueous phase to the polymer solution is 1:10 to 1: 200, preferably 1:20 to 1:60. This is because when the volume ratio of the aqueous phase to the polymer solution (oil phase) is out of the range, it is difficult to expect stable O / W type emulsion formation due to phase transition under the same stirring conditions.

Subsequently, in step S2), the organic solvent is removed from the O / W emulsion to obtain microspheres.

Thereafter, the polyester-based polymer and the pore former may be cured through evaporation and removal of the organic solvent in the oil phase to obtain a solidified microsphere. The porous microspheres can be worked up by other commonly known methods. For example, methods such as centrifugation, filtration, washing, drying and the like can be performed.

At this time, for the preparation of the porous microsphere is prepared by stirring sufficiently for 10 minutes to 24 hours at a stirring speed of 100 to 2,000 rpm. 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 S3), the pore forming agent is sublimated 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 pore former, which has solidified with the polyester-based polymer by evaporation of an organic solvent in an aqueous phase cooled to below the melting point of the pore former, is sublimed at room temperature to form an open pore structure connected to the microspheres.

The pore forming step is performed for 10 minutes to 100 hours at room temperature atmospheric conditions. 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.

In particular, the production method of the present invention can easily control the pore size and pore size of the finally obtained porous microsphere by adjusting the weight ratio of the pore-forming agent and the polyester-based polymer.

Referring to Experiment 1 of the present invention, as the weight ratio of camphor / polycaprolactone increases, the average particle diameter of the microspheres decreases, and the pore diameter increases.

Next, in step S4), the microspheres formed with pores are treated with an alkaline solution to activate the surface.

The microspheres in which the pores are formed are immersed in an alkaline solution to surface-activate the microsphere body surface and the functional groups on the pore surface to be electrically negative carboxyl groups, thereby facilitating precipitation of calcium phosphate-based crystals in a subsequent immersion step.

At this time, it is preferable to use sodium hydroxide, potassium hydroxide or hydrogen peroxide as the alkaline solution.

The concentration, the treatment temperature and the treatment time of the alkaline solution should be determined within a range in which the microstructure of the porous microspheres is not destroyed and the mechanical properties of the porous microspheres are not degraded.

Preferably the concentration of the alkaline solution is 0.1 to 10 N.

Furthermore, the treatment time is 1 minute to 24 hours. At this time, when the concentration and the treatment time of the alkaline solution is less than the above range, the degree of surface activation is insignificant. On the contrary, when the concentration exceeds the above range, the physical properties of the microspheres are deteriorated.

Finally, in step S5), the surface-activated microspheres are immersed in a mixed solution of calcium ions and phosphate ions.

Calcium phosphate-based crystals are uniformly coated on the body surface and pore surface of the microsphere according to the present invention prepared through this step.

At this time, the immersion step is preferably carried out under conditions similar to the temperature and pH of the body, that is, by a biomimetic manufacturing process.

Specifically, the concentration of calcium ions in the mixed solution is 1 mM to 10 mM, the concentration of phosphate ions is 0.5 mM to 5 mM, the pH is 7.35 to 7.45 using a buffer solution of 36.5 to 37.5 ℃ for 1 to 28 days To perform. 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 bio-use solution may be prepared by dissolving NaCl, NaHCO ₃ , Na ₂ SO ₄ , KCl, K ₂ HPO ₄ , MgCl ₂ · 6H ₂ O, and CaCl ₂ · 2H ₂ O in the reaction reagent grade in deionized distilled water. have.

To the mixed solution is 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 _Use a bioavailable solution (SBF) at pH 7.4 containing ₄ ^2- .

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.

Examples 1-5: Preparation of Porous Microspheres

Poly (ε-caprolactone) (Sigma, weight average molecular weight 80,000, hereinafter 'PCL') was dissolved in a vial containing chloroform at a concentration of 10% by weight to prepare a PCL solution. A camphor solution was prepared by dissolving camphor (C ₁₀ H ₁₆ , Sigma-Aldrich) as a pore-forming agent in another vial containing chloroform at the concentration according to Table 1 below. The PCL solution was stirred and mixed with the camphor solution for 3 hours, and then the mixed solution was dropped at 4 ° C. while stirring at 600 rpm in an aqueous phase containing 1 wt% PVA containing distilled water. At this time, the volume ratio of the aqueous phase to the mixed solution was 30. The mixed solution solidified rapidly as it dropped into the ice-cooled water phase, forming microspheres. After solidification the microspheres were further stirred for 6 hours to harden and induce pore channels. The solidified porous microspheres were filtered and washed five times with distilled water cooled with ice. The washed PCL microspheres were lyophilized and stored at 4 ° C.

100 mg of the obtained PCL porous microsphere was immersed in 2N NaOH solution at 25 ° C. for 6 hours with gentle stirring at 100 rpm to activate the surface. After washing with distilled water and dried. Microspheres having an activated surface 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 HPO ₄ ^2- and 0.5 mM SO ₄ ^2- ) were soaked at 100 rpm for 5 days with gentle stirring to produce apatite precipitate on the surface.

division PCL content
(weight%) Kamfen Content
(weight%) Kampen / PCL weight ratio Comparative Example 1 10 0 0 Example 1 10 10 One Example 2 10 20 2 Example 3 10 40 4 Example 4 10 60 6 Example 5 10 80 8

Comparative Example 1: Preparation of Solid Microspheres

In the same manner as in Example 1 except that no camphor was used as the pore-forming agent. Solid microspheres were prepared by performing no pores.

Experimental Example 1: Scanning Electron Microscope Analysis

In order to examine the microsphere surface shapes and microstructures of Examples 1 to 5 and Comparative Example 1 prepared above, porous microspheres after Pt coating were analyzed by scanning electron microscopy, and the obtained results are shown in FIG. 2. Indicated.

2 (a) to 2 (f) are scanning electron micrographs in which the surfaces of the microspheres of Comparative Example 1 and Examples 1 to 5 are enlarged.

Referring to FIG. 2, in Comparative Example 1 without using a camphor as a pore-forming agent (a), a solid microsphere having no pores formed only of PCL was prepared, and pores were formed in the PCL microsphere by using a camphor. It can be seen that (b to f).

In addition, the pore structure was changed according to the amount of camphor. Specifically, in the porous microsphere of Example 1 (camphor / PCL weight ratio: 1), micropores having a diameter of several micrometers were formed (b), and Example 2 (camphor / PCL weight ratio: 2) and Example 3 (camphor / PCL). In the porous microspheres having a weight ratio of 4), micropores having a diameter of 10 and 20 μm were formed (c, d), respectively. For Example 4 (camphor / PCL weight ratio: 6), large pores with diameters of 50 μm or less were formed along the small pores (e). It can be seen that pores of a similar aspect to Example 4 were formed in the porous microspheres of Example 5 (Kampen / PCL weight ratio: 8) in which the amount of camphor was further increased (f).

In the microspheres of Example 3, the pores were observed to be well connected to each other, and the microspheres of Examples 4 and 5 were empty inside. This is clearly distinguished from Comparative Example 1 which is a known microsphere.

These morphology of microspheres with empty spaces inside and large pores on the outside show that they can be used as cell carriers and tissue engineering matrices in that they provide steric spaces and channels for cell migration and proliferation.

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

Finding the size distribution of the microspheres prepared in Examples 1 to 5 and Comparative Example 1 Hazard particle size was analyzed, and the results are shown in Table 2 and FIG. 3 below.

Referring to Figure 3, the porous microspheres prepared in Example 4 has a particle size of several tens to several hundred micrometers, it can be seen that the average particle diameter is 183 ㎛. These average particle diameters are judged to be appropriate sizes for cell delivery and tissue engineering applications. In addition, as shown in the optical image, it can be seen that large pores are well connected to each other, as contrasted with bright areas.

division pore Average particle size (standard deviation) Comparative Example 1 - 261 (71) Example 1 Several μm 220 (73) Example 2 About 10 ㎛ 224 (67) Example 3  About 20 ㎛ 191 (43) Example 4 Several microns and about 50 to 100 microns 183 (85) Example 5 Several microns and about 50 to 100 microns 168 (71)

As shown in Table 2, the prepared microspheres showed a similar range of average particle diameters, and the average particle diameter tended to decrease slightly as the amount of camphor, the pore-forming agent, increased. This is because the PCL is replaced with camphor as the camphor / PCL weight ratio increases, thus reducing the viscosity of the PCL / camphor mixed solution.

As illustrated in FIG. 2, large pores having a size of at least 50 μm or more may be prepared when a weight ratio of camphor / PCL is used at 6 or more. The size of the microspheres is preferably within a range suitable for injectable cell delivery means. Given the tissue cell size in the range of tens of micrometers (mostly 100 micrometers or less), microspheres of several hundred micrometers in size can preferably be used as cell support matrix. In addition, the pore size of the microspheres is also preferably adjusted so as not to interfere with the cell migration within the range not larger than the microspheres. The average particle diameter and pore size of the microspheres shown in Table 2 above are determined to be suitable for cell delivery and tissue engineering applications.

Experimental Example 3: Confirming Surface Modification of Calcium Phosphate-Based Crystals

3-1. Scanning electron microscope

The porous microsphere surface of the present invention was modified with an apatite similar to the bone mineral component. Much research is underway to improve the low hydrophilicity and cellular interactivity of biodegradable polymer surfaces such as PCL. In the present invention, in order to improve the surface activity of the microspheres, the microspheres were immersed in the bioavailable solution to induce calcium phosphate deposition on the surface.

Because of this, the microspheres are immersed in alkaline solvent for surface activity and then in SBF to induce precipitation of calcium phosphate.

Figure 4 (a) is a scanning electron micrograph taken after 6 hours treatment with 2N NaOH in Example 4, (b) is a scanning electron microscope picture taken after immersion in SBF solution for 5 days, (c) is SBF Scanning electron micrograph taken at high magnification of the inside of the microspheres after immersion in solution, (d) is scanning electron micrograph taken at high magnification outside the microspheres after immersion in BF solution.

Referring to FIG. 4, mineral precipitation is induced after being immersed in the SBF solution, and it can be seen that apatite nano crystallites exist on the inner and outer surfaces of the microspheres.

3-2. Determination of Dispersion in Cell Culture Media

5 is a photograph of the dispersion state (a) and the dispersion state (b) after surface modification in the microspheres of the microspheres of Example 4 before surface modification.

As shown in FIG. 5, PCL microspheres prior to surface modification do not precipitate upon dispersion in culture. This is because the density of microspheres decreases as the hydrophobicity and pores of PCL are formed. In contrast, it can be seen that the surface modified PCL microspheres completely settle to the bottom of the vial. Therefore, the modification of the microsphere surface with apatite is considered to be a prerequisite for application in the biomedical field.

In addition, bone mineral-like apatite phases play an important role in cell proliferation and osteogenic induction. Adhesion proteins and their cells present in serum prefer PCA surface-treated with apatite rather than pure PCL with no sites for specific proteins and cells to adhere to. Furthermore, further cellular treatments such as osteoblast differentiation and mineralization are made easier by the presence of apartments on the surface.

3-3. EDS Analysis

The calcium phosphate-based crystals formed on the surface of the porous microspheres prepared in Example 4 were measured for molar ratio of Ca / P by using energy dispersive spectroscopy (EDS). 6 shows the obtained EDS spectrum.

Referring to FIG. 6, the molar ratio of Ca / P of the calcium phosphate-based crystals formed on the surface of the nanofiber film was about 1.71. These results indirectly mean that the crystal formed is apatite hydroxide considering that the molar ratio of apatite hydroxide Ca / P is 1.67.

Experimental Example 4: Bone Marrow Stem Cell Culture Experiment

Rat bone-marrow derived stromal cells (hereinafter referred to as 'rBMSCs') were cultured to examine the bioactivity of the prepared porous microspheres, and the shape of the cells grown on the surface and the inside of the microspheres was observed. Cell proliferation was measured using the CellTiter 96 AQueous One solution cell proliferation assay (promega) assay.

4-1. in vitro  rBMSCs culture

Rat bone-marrow derived stromal cells (“rBMSCs”) were obtained by making minor modifications to the information previously published (HW Kim, HE Kim, JC Knowles, Adv. Funct. Mater. 2006, 16, 1529).

Briefly, the bone marrow extracted from the distal and distal femur and tibia of the cut rat (4-8 weeks old) was centrifuged to collect suspended solids with α-MEM. Then, the cell suspension was incubated for one day in total culture medium (α-MEM, 2 × 10 ^-3 M glutamine, 100 U · mL ⁻¹ ) at 37 ° C. and humidification with a carbon dioxide concentration of 5%. The remaining cells were incubated for 7 days until the non-adhesive cells were discarded to form a monolayer. The cells were then trypsinized and then again suspension treated at a total concentration of ¹ × 10 ⁻⁶ cells per mL in a total culture medium. The microspheres prior to cell transplantation were 30 mg and injected into each well of a 96-well plate surface modified with 1% Pluronic-127 so that the cells did not adhere to the well surface. Since 50 μl of the cell suspension was added to each well, and then cultured for 6 hours to facilitate cell adhesion. At this time, 150 μl of osteogenic medium (total culture medium + 50 μg · mL ⁻¹ sodium ascorbate, 10 ⁻² M β-glycerol phosphate sodium, 10 ⁻⁸ M dexamethasone) was added and then cultured for 10 days. The culture medium was changed every 2-3 days.

 4-2. Cell proliferation observation

Cell proliferation was performed on the porous microspheres of Example 4 and the solid microspheres of Comparative Example 1, and a culture dish was used as a control. Each culture day (3, 7, 10 days), MTS / PMS reagent was added and incubated for 2 hours at 37 ℃.

After the solution was separated by a pipette, the 490 nm absorbance was measured by an Elisa Plate Reader. For cell growth image observation, confocal laser scanning microscopy (CSLM, Carl Zeiss 510L) was used. Cells grown in each sample were fixed with 4% formaldehyde and osmotic with 0.2% Triton X-100. After washing with PBS, 1% of BSA was added for 20 minutes to prevent nonspecific protein binding. PBS diluted 25 mL of Alexa Fluor 546 Phalloidin (invitrogen A22283) was added to stain F-actin. The nuclei were also stained with DAPI (invitrogen P36935) with the addition of Prolong ^® gold discoloration inhibitors. Fluorescence images were analyzed with a z-axis tomographic collector.

Figure 7 (a) is a CLSM image (red-surviving cells, blue-nucleus) showing cell growth on the third day of culture in the porous microsphere of Example 4, (b) is a porous microsphere prepared in Example 4 It is a graph showing the results of analyzing the proliferation rate of bone marrow stem cells on the surface by MTS method.

Referring to Figure 7 (a), the fluorescence image constructed along the z-axis shows that there are living cells, the living cells are clustered on the surface of the microsphere, the active cytoskeletal process as shown in red ( It can be seen that it moves between the pore channels during the cytoskeletal process.

As shown in (b) of FIG. 7, it can be seen that more cells are crowded in the porous microsphere of Example 4 than the solid microsphere of Comparative Example 1 after the culture period. In addition, it can be seen that the number of cells cultured continuously increases with increasing incubation time. This is due to the large pores of the microspheres helped the movement of the cells and the internal space provided good culture conditions for the rBMSCs. From this, it can be seen that it is more preferable to design microspheres with pores therein.

In addition, the surface modification with calcium phosphate-based crystals, macromolecular morphology and bone-living body of the present invention, since the polymer material in the form of a film, nanofibers or porous scaffold promotes osteogenic development of osteogenic precursors and stem cells Surface-modified microspheres with bone-bioactive are thought to be useful for cell transplantation and tissue engineering in difficult tissue regeneration applications.

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 or cell carrier for tissue engineering for regeneration of bone and periodontal tissue.

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

2 (a) to 2 (f) are scanning electron micrographs in which the microsphere surfaces of Comparative Example 1 and Examples 1 to 5 are enlarged.

Figure 3 shows the size distribution and optical image of the porous microspheres prepared in Example 4 of the present invention.

Figure 4 (a) is a scanning electron micrograph taken after 6 hours treatment with 2N NaOH in Example 4, (b) is a scanning electron microscope picture taken after immersion in SBF solution for 5 days, (c) is SBF Scanning electron micrograph taken at high magnification of the inside of the microspheres after immersion in solution, (d) is a scanning electron micrograph taken at high magnification outside the microspheres after immersion in SBF solution.

5 is a photograph of the dispersion state (a) and the dispersion state (b) after surface modification in the microspheres of the microspheres of Example 4 before surface modification.

Figure 6 shows the EDS spectrum of the calcium phosphate-based crystals formed on the surface of the nanofiber film prepared in Example 4 of the present invention.

Figure 7 (a) is a CLSM image (red-surviving cells, blue-nucleus) showing cell growth on the third day of culture in the porous microsphere of Example 4, (b) is a porous microsphere prepared in Example 4 It is a graph showing the results of analyzing the proliferation rate of bone marrow stem cells on the surface by MTS method.

Claims (12)

In porous microspheres with pores formed on and inside a spherical body, The microsphere comprises a polyester-based polymer, the surface and the pore surface of the microsphere is coated with calcium phosphate-based crystals surface modified porous microspheres. The method of claim 1, wherein the polyester-based polymer Polycaprolactone, polylactic acid, polyglycolic acid, polylactide-co-glycolide, polyvalerolactone, polyhydroxy butyrate, polyhydroxy valerate, copolymers thereof and blends thereof One type of porous microspheres. The method of claim 1, wherein the calcium phosphate-based crystals A porous microsphere, characterized in that it is one selected from the group consisting of hydroxyapatite, oxyapatite, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate, calcium metaphosphate and mixtures thereof. The method of claim 1, wherein the average particle diameter of the microspheres Porous microspheres, characterized in that 100 to 1000 ㎛. According to claim 1, wherein the diameter of the micropores is Porous microspheres, characterized in that 1 to 100 ㎛. S1) polyester polymer; Organic solvents; And dispersing a polymer solution including a pore-forming agent in an aqueous phase containing a hydrophilic solvent and a surfactant to prepare an O / W emulsion. S2) removing the organic solvent from the O / W emulsion to obtain a microsphere S3) subliming the pore-forming agent to form pores in the microsphere body and inside; S4) activating the surface by treating the microspheres formed pores with an alkaline solution; And S5) immersing the surface activated microsphere in a mixed solution of calcium ions and phosphate ions Method for producing a porous microsphere comprising a. The method of claim 6, wherein the pore-forming agent Camphor, camphor, naphthalene, menthol, thymol, coumarin, vanillin, salicyamide, 2-amiminopyridine, t -butanol, trichloro- t -butanol, imidazole, dimethylsulfone, urea and 2-amidopyridine Production method characterized in that the one selected from the group consisting of. The method of claim 6, wherein the alkaline solution is A process for producing sodium hydroxide, potassium hydroxide or hydrogen peroxide. The method of claim 6, wherein the award of step S1) The method of claim 1, wherein the pore-forming agent is cooled below the melting point. The method of claim 6, wherein the mixed solution The concentration of calcium ions is 1 mM to 10 mM, the concentration of phosphate ions is 0.5 mM to 5 mM, pH is a manufacturing method characterized in that the buffer solution of 7.35 to 7.45. The method of claim 10, wherein the mixed solution Biomaterials comprising 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- Method for producing a similar solution. The method of claim 6, wherein step S5) Process for 3 to 28 days at 36.5 to 37.5 ℃ characterized in that the production method.

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