KR20090104475A - Manufacturing method for nanoparticles-biogradable polymer composites - Google Patents

Abstract

PURPOSE: A manufacturing method of nanoparticle-biodegradable polymer composite is provided, which forms new bone tissue in a short time by promoting breeding of the bone cell. CONSTITUTION: A manufacturing method of nanoparticle-biodegradable polymer composite comprises a process of preparing the biomaterial particulate slurry of nano-size; a process of preparing the bio-degradable high molecule solvent; and a process of removing the moisture by heating to the boiling point of the organic solvent with boiling point of higher than 120deg.C by gradually raising temperature while mixing and stirring biomaterial particulate slurry of nano-size and bio-degradable high molecule solvent.

Description

Manufacturing method for nanoparticles-biogradable polymer composites

The present invention relates to a method for producing an organic-inorganic complex of nano-sized biomaterials and biodegradable polymers, the presence or absence of nanoparticles and biodegradable polymers of biomaterials such as apatite hydroxide useful as a support for the treatment and regeneration of bone tissue The present invention relates to a method for preparing a group complex.

Tissue engineering supports for the treatment and regeneration of bone tissue should use materials that degrade in vivo in time to repair and repair damaged bone tissue. The support for bone tissue regeneration must satisfy three conditions as well as in vivo stability. First, it should be made of a material that helps the adhesion, proliferation, and differentiation of bone cells. Second, it should be made of a porous structure that facilitates the proliferation of bone cells and the formation of bone tissue. Interconnectivity should be good.

Hydroxyapatite, one of these biomaterials, is similar to human hard tissue components and has biocompatibility, adsorption capacity of heavy metals and bacteria. Hydroxyapatite having the same composition as the bioinorganic component has excellent human-friendliness, so that the implant in bone or skin can be easily integrated with surrounding biological tissues, and thus has been applied to a wide range of biomaterials.

On the other hand, biodegradable polymers, which are one of the biomaterials, are polymers that are decomposed and destroyed by simple hydrolysis or the action of enzymes, such as polypeptides, polysaccharides, polynucleotides, and polyesters produced by microorganisms. Are each broken down by unique enzymes. Most synthetic polymers do not degrade, but some aliphatic polyesters or polycarbonates degrade slowly by hydrolysis. Biodegradable polymers for medical use should not cause foreign body reactions in the living body, should be decomposed into harmless substances during decomposition, and have appropriate processing characteristics and strength. In this respect, the enzyme-degradable polymer has the advantage of high biocompatibility, but may also act as a disadvantage of inducing an immune response. In addition, the enzyme-degradable polymer is difficult to arbitrarily control the biodegradation rate and mechanical properties compared to the hydrolyzable polymer that is degraded by hydrolysis. On the other hand, hydrolyzable polymers have a low immune response in the body, easy control of mechanical properties and relatively simple structure changes depending on the use. In addition, most of these structures contain ester bonds. When the ester bonds are broken by water hydration in the body, the molecular weight decreases, and the low molecular weight polymers thus carried are transported through the blood and discharged out of the body in the form of urine. do. Therefore, synthetic polymers such as aliphatic polyesters are widely used in various applications.

However, biodegradable polymers are known to be hydrophobic and have poor adhesion and proliferation of cells. Thus, organic-inorganic complexes are prepared by incorporating bioactive materials similar to bone constituents such as hydroxyapatide. Many studies have been conducted to overcome. In addition, since bone is a composite composed mainly of nano-sized apatite and collagen in terms of materials, studies are being conducted to prepare nano-sized apatite and use it as a support material in biomimetic aspects.

However, the nano-sized biomaterials are inferior in dispersion stability in admixture with the polymer solution, so that the biomaterials are not evenly distributed in the nanostructures, which ultimately leads to osteoblast proliferation due to the inclusion of nano-sized biomaterials. There was a problem that did not take full advantage of the effect.

The present invention is to provide a method for producing a composite of biomaterial inorganic nanoparticles and biodegradable polymer resin in which nano-sized biomaterial particles are evenly distributed in the composite by solving the coagulation problem due to the residual amount of water.

In addition, an embodiment of the present invention is to provide a composite of biomaterial inorganic nanoparticles and a biodegradable polymer resin that can be nanoparticles of the biomaterial particles evenly distributed to express the osteoblast proliferation effect.

One embodiment of the present invention comprises the steps of preparing a nano-sized biomaterial particle slurry; Separately preparing a biodegradable polymer solution; And a nano-sized biomaterial particle slurry, a biodegradable polymer solution, and an organic solvent having a boiling point of 120 ° C. or higher, and gradually heated to a boiling point of an organic solvent having a boiling point of 120 ° C. or higher while gradually raising the temperature in a temperature range of 100 ° C. to 120 ° C. while stirring. It provides a method for producing a composite of a nano-size biomaterial and a biodegradable polymer comprising a step of removing moisture.

According to the invention a subsequent casting process; And a vacuum drying process.

In one embodiment according to the present invention, the nano-sized biomaterial particle slurry comprises the steps of preparing an aqueous solution of a phosphorus (P) source and a calcium (Ca) source; Mixing a calcium source aqueous solution with a phosphorus source aqueous solution to heat 1.5 to 2 hours at 80 to 100 ° C. under alkaline conditions, and reacting at room temperature for 24 to 48 hours to obtain a calcium phosphate precipitate; It may be obtained by washing the precipitate with distilled water to remove the residual amount of impurities to obtain a nano-sized calcium phosphate-based biomaterial particles containing substantially water.

In another embodiment of the present invention, the nano-sized biomaterial particle slurry is prepared by the steps of preparing an aqueous solution of a silicon (Si) source and a calcium (Ca) source; Calcium silicate-based precipitates by mixing an aqueous calcium source solution with an aqueous silicon source solution and heating at 80 to 100 ° C. under alkaline conditions for 1.5 to 2 hours and reacting at room temperature for 24 to 48 hours; And it may be obtained by washing the precipitate with distilled water to remove the residual amount of impurities to obtain a nano-sized calcium silicate-based biomaterial particles containing substantially water.

In the above embodiments, after obtaining the calcium phosphate-based biomaterial particles or calcium silicate-based particles substantially containing water, the particles are stored in an alcohol solution of 95-99% concentration for 10 to 15 days, but 2 to Once every three days by replacing the alcohol solution may further comprise the step of removing the moisture contained in the particles.

In an embodiment of the present invention, the boiling point is gradually heated in a temperature range of 100 ℃ to 120 ℃ while stirring the calcium phosphate-based biomaterial particles or calcium silicate-based particles substantially containing water in an organic solvent having a boiling point of 120 ℃ or more 120 The method may further include a step of removing water by heating to a boiling point of an organic solvent having a temperature of about 0 ° C. or more.

In one embodiment of the present invention, the calcium phosphate-based biomaterial particles or calcium silicate-based particles substantially containing water may be present in a dispersed state in an alcohol solution.

In one embodiment of the present invention, at least one of the aqueous solution of calcium source, the aqueous solution of phosphorus source and the aqueous solution of silicon source may further comprise a dispersant.

In embodiments of the present invention, as an organic solvent having a boiling point of 120 ° C. or more, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), toluene or diglyme may be used.

According to embodiments of the present invention may further comprise the step of subsequently vacuum drying to obtain the biomaterial particles.

According to the embodiment of the present invention, the calcium phosphate-based particles may be apatite hydroxide particles or tricalcium phosphate particles.

Another embodiment of the present invention provides a composite of a nanoscale biomaterial and a biodegradable polymer obtained by the above-described methods.

In an embodiment of the present invention, the nano-sized biomaterial may be calcium phosphate particles or calcium silicate particles.

In an embodiment of the present invention, the biodegradable polymer may be at least one selected from poly (ε-caprolactone), poly (lactic acid), poly (glycolic acid) and copolymers of D, L-lactic acid and glycolic acid.

According to the present invention, in the preparation of organic-inorganic composites by mixing nano-sized biomaterials and biodegradable polymers, the nano-sized biomaterials are prevented from agglomerating, thereby being uniformly dispersed in the composites while maintaining the original particle size of the biomaterials. Its surface area is much larger than that of microparticles, which is advantageous for early attachment to bone cells. Also, it has superior protein absorption and osteoblast adhesion and can interact with extracellular matrix. In addition, it is possible to fully express the inherent effects of nanoparticles in forming new bone tissue in a short time by matching the material components of bone and promoting adhesion and proliferation of bone cells compared to microparticles.

The present invention will be described in more detail as follows.

According to the present invention, a method for preparing a composite of a nanoscale biomaterial and a biodegradable polymer (hereinafter referred to as an organic / inorganic composite) includes preparing a slurry of nanoscale biomaterial particles; Separately preparing a biodegradable polymer solution; And a nano-sized biomaterial particle slurry, a biodegradable polymer solution, and an organic solvent having a boiling point of 120 ° C. or higher, and gradually heated to a boiling point of an organic solvent having a boiling point of 120 ° C. or higher while gradually raising the temperature in a temperature range of 100 ° C. to 120 ° C. while stirring. And removing the moisture.

In preparing the biodegradable polymer solution, chloroform, dioxin or dimethyl formaldehyde may be used as the solvent, but the present invention is not limited thereto because it is to be selected as a solvent according to the type, viscosity, molecular weight, etc. of the biodegradable polymer. For example, if the biodegradable polymer is poly (ε-caprolactone) (PCL), the preferred solvent is chloroform, and if it is a copolymer of D, L-lactic acid and glycolic acid (PLGA), dimethyl formaldehyde and tetrahydrofuran Mixed solvents may be preferred. In the present invention, the biodegradable polymer is not particularly limited, and may be, for example, poly (ε-caprolactone), poly (lactic acid), poly (glycolic acid), and a copolymer of D, L-lactic acid and glycolic acid. In particular, poly (ε-caprolactone), which is inexpensive and easy to purchase, may be preferable because it has a lower decomposition rate than other biodegradable polymers, and serves as a support until the damaged bone is healed.

 As nano-sized biomaterials, apatite hydroxide nanoparticles known by the applicant may be used, and calcium silicates obtained from calcium oxide and silica known as bioactive ceramics as well as biomaterials obtained from other methods, silk apatite, etc. Any of the biological materials can be used as long as they have a nano size.

One example of the nano-sized biomaterial used in the preparation of the organic-inorganic composite of the present invention according to the embodiment of the present invention is calcium phosphate-based biomaterial particles, and one example of the preparation method is shown in Scheme 1 below.

The calcium source used in one embodiment of the present invention is calcium nitrate (Ca (NO) ₃ ) ₂ 4H ₂ O), the phosphorus source is ammonium phosphate ((NH ₄ ) ₃ PO ₄ )), but other water-soluble Phosphorus Various calcium sources and phosphorus sources can of course be used.

In this case, the stoichiometric ratio of Ca / P in the reaction of calcium source and phosphorus source is irrelevant to the production of nanoparticles, and it is possible to manufacture various calcium phosphate-based nanoparticles under the control of stoichiometric ratio. It will be known to those who have.

10Ca (NO ₃ ) ₂ + 6 (NH ₄ ) ₃ PO ₄ + 8NH ₄ OH → Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ + 20NH ₄ NO ₃ + 6H ₂ O

As shown in Scheme 1, when the calcium source and the phosphorus source are reacted under alkaline conditions, impurities and a large amount of water are generated, and the obtained apatite hydroxide is also poor in stability by moisture.

This entails a physical removal process to remove the water generated during the reaction, but it contains a residual amount of water in the particles, which was not sufficiently removed through the physical removal process.

Therefore, after the particles are obtained in the nano-size, the aggregation between the particles occurs. In one preferred embodiment of the present invention, the calcium phosphate-based particles obtained as nano-sized particles containing water in this manner have a boiling point of 120 ° C. or more. The nano-sized calcium phosphate-based particles from which water is completely removed are further subjected to a step of removing water by heating and stirring to the boiling point of the organic solvent while gradually raising the temperature (hereinafter referred to as the present invention water removing step). You can get it.

In the water removal process of the present invention, the nano-sized calcium phosphate-based particles substantially containing water are specifically stirred while gradually raising the temperature from 100 ° C to 120 ° C in a solvent having a boiling point of 120 ° C or higher. In the description above and below it will be understood that " substantially water-containing " is at a level of at least 30%.

According to the water removal process of the present invention, when the temperature is gradually raised to a temperature range of 100 ° C. to 120 ° C., the water is completely evaporated, and the solvent having a boiling point of 120 ° C. or higher is volatilized thereafter to efficiently and easily remove water. Can be. In this case, by heating above the boiling point can be removed most of the temperature range.

The remaining amount of the solvent can be easily removed by performing vacuum drying, thereby obtaining the desired nano-sized calcium phosphate-based biomaterial particles.

It can also be used for a subsequent step without removing the solvent. For example, even if a small amount of solvent is present, it does not affect the aggregation of particles, and in the future, in the preparation of organic-inorganic composites, there are aspects of miscibility with organic solvents in dispersing and using nano-sized calcium phosphate particles in organic solvents. Does not work against, but rather disperses well.

Solvents having a boiling point of 120 ° C. or higher may include dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), toluene or diglyme, but are not limited thereto. In consideration of the fact that the boiling point of the water is 100 ℃ can be selected from a variety of solvents having a boiling point higher than that.

Stirring in an organic solvent having a boiling point of 120 ° C. or higher may be preferably performed for 1 to 1.5 hours in terms of preventing damage to the nanoparticles.

In the present invention, the method for producing the nano-sized calcium phosphate particles substantially containing water is not particularly limited, but the precipitation method, the hydrolysis method, and the sol-reaction of the water-soluble calcium source and the water-soluble phosphorus source are reacted in an aqueous solution. In the case of calcium phosphate-based particles obtained by a wet method for producing a liquid medium, such as a gel method, the particles are obtained as substantially water-containing particles.

As an example, one embodiment according to the present invention shows an example of particle production by precipitation in which a calcium source and a water-soluble phosphorus source are reacted in an aqueous solution, specifically, a phosphorus (P) source and a calcium (Ca) source. Preparing an aqueous solution; Mixing a calcium source aqueous solution with a phosphorus source aqueous solution to heat 1.5 hours to 2 hours at 80 to 100 ° C. under alkaline conditions, and reacting at room temperature for 24 to 48 hours to obtain a calcium phosphate precipitate; And the precipitate may be washed with distilled water to remove the residual amount of impurities to obtain a nano-sized calcium phosphate-based biomaterial particles containing substantially water.

In the preparation of the aqueous solution of calcium source and the aqueous solution of phosphorus source, a dispersant may be further included as necessary in order to increase dispersibility. Examples of the dispersant include polyethylene and the like. The content of the dispersant may be in an amount of about 20 to 30% by weight based on the volume of the aqueous solution.

By this precipitation method, nano-sized calcium phosphate-based biomaterial particles substantially containing water may be obtained, and then subjected to a primary water removal process before going through the water removal process of the present invention. The method may be a method for storing the nano-sized calcium phosphate-based biomaterial particles substantially containing water obtained by the above method in an alcohol solution for a certain period of time. At this time, the alcohol is preferably 95 to 99% concentration of the water removal efficiency, preferably stored for at least 10 to 15 days, it is more efficient to replace the alcohol solution every two to three days. Thus, after storing in the alcohol, it can be applied to the post-process as it is in the slurry state dispersed in the alcohol solution, may be more advantageous in terms of dispersion stability of the particles when used in a dispersed state in the alcohol solution.

In addition, after the primary water removal process stored in alcohol can be used separately in the form of particles by removing the alcohol of course.

Another example of the biomaterial nanoparticles of the present invention may be a calcium silicate-based biomaterial obtained from calcium oxide and silica, and an example of a method of preparing the same is similar to the method of obtaining the calcium phosphate-based biomaterial particles.

Specific examples of the method for producing a calcium silicate-based biomaterial include preparing an aqueous solution of a silicon (Si) source and a calcium (Ca) source;

Mixing a calcium source aqueous solution with a silicon source aqueous solution to heat 1.5 to 2 hours at 80 to 100 ° C. under alkaline conditions and stirring at room temperature for 24 to 48 hours to obtain a calcium silicate-based precipitate; And removing the residual amount of impurities by washing the precipitate with distilled water to obtain a nano-sized calcium silicate-based biomaterial particle substantially including water, and subsequently including a water removal process as described above. What is obtained may be more advantageous in terms of dispersion stability.

According to the present invention, the organic-inorganic composite is prepared by complexing nano-sized biomaterial particles and biodegradable polymer solution with or without a separate water removal process, and removing water during the manufacturing process of nano-sized biomaterial particles. Even though it passes through the organic-inorganic composites, it is exposed to moisture due to environmental factors or process factors, which hinders the dispersion stability of nano-sized biomaterial particles.

Thus, in the organic-inorganic composite production method according to the present invention, in mixing a nano-sized biomaterial particle slurry and a biodegradable polymer solution, a certain amount of a solvent having a boiling point of 120 ℃ or more is added separately. Herein, 'constant amount' may be, for example, about 100 to 200 parts by volume of an organic solvent based on 100 parts by weight of a composite.

Then, the process is performed to remove the residual amount of organic solvent by stirring while gradually raising the temperature in a temperature range of 100 ° C. to 120 ° C. and then heating the boiling point of the organic solvent having a boiling point of 120 ° C. or more.

According to this process, if the mixed solution of nano-sized biomaterial and biodegradable polymer solution containing an organic solvent having a boiling point of 120 ° C. or more is gradually heated to a temperature range of 100 ° C. to 120 ° C., the water contained in the solution is completely evaporated. With or after this, a solvent having a boiling point of 120 ° C. or more may be volatilized to remove moisture efficiently and easily. In this case, by heating above the boiling point can be removed most of the temperature range.

In this case, the solvent having a boiling point of 120 ° C. or more may be the same as or different from an organic solvent that can be used in the water removal process of the nano-sized biomaterial, and specific examples thereof include dimethyl formamide (DMF) and dimethyl sulfoxide. (DMSO), toluene or diglyme, but the present invention is not limited thereto, and may be selected from various solvents having a boiling point higher than that of water at a boiling point of 100 ° C. for removing residual moisture. Of course it can.

Stirring in an organic solvent having a boiling point of 120 ° C. or higher may be preferably performed for 1 to 1.5 hours in terms of preventing damage to the nanoparticles.

As such, in the preparation of the organic-inorganic composite, the nano-size of the desired inorganic-inorganic composite is finally improved as it is possible to improve the dispersion stability without causing the aggregation of the nano-sized biomaterial in the mixed solution. The biomaterial may be evenly dispersed.

The preparation of the organic-inorganic composite according to the present invention is then subjected to a casting process as in the conventional organic-inorganic nanocomposite production, and then to a process of removing the solvent contained in the solution through a drying process such as vacuum drying.

The casting process is not particularly limited and may be referred to as a manufacturing process as a unit capable of functioning as a support, and may be performed at room temperature.

Hereinafter, examples of the present invention will be described in more detail, but the scope of the present invention is not limited to these examples.

Preparation Example 1

Calcium nitrate (Ca (NO ₃ ) ₂ 4H ₂ O, made by Junsei Chemical Co., Ltd., Japan) and ammonium phosphate ((NH ₄ ) ₂ HPO ₄ , made by Junsei Chemical Co., Ltd., Japan) Apatite was prepared by the following method with a ratio of / P of 1.67.

First, the phosphorus source and the calcium source were sufficiently dissolved in distilled water at about 80 ° C. to prepare respective aqueous solutions, and the calcium source solution was slowly added to the phosphorus source solution and mixed well to obtain a nano-sized calcium phosphate precipitate.

At this time, the pH of the solution was maintained by adding ammonium hydroxide to 11 or more, and the mixture was heated at about 80 ° C. for 2 hours and then sufficiently synthesized at room temperature for 24 hours or more.

In order to remove the remaining impurities, washed with distilled water three times or more to obtain apatite hydroxide particles (hereinafter referred to as initial particles) having a particle size of 50 to 100 nm.

25 g of the obtained apatite hydroxide particles were placed in 100 ml of dimethylformamide (boiling point of 165 ° C.) and stirred for 5 hours using a magnetic bar while gradually raising the temperature to 100 to 120 ° C. to remove water (hereinafter referred to as a water removal process). box).

Then, the resultant was dried in vacuo to remove the residual amount of the organic solvent.

On the other hand, the particle size was observed using a transmission electron microscopy (Transmation electron microscopy, TEM, JEOL, JEM-2100F, Japan), the results are as shown in FIG. It can be seen from the results of FIG. 1 that nanoparticles having a width of about 20-40 nm and a length of 50-100 nm were obtained. In FIG. 1, a solid line shows 100 nm.

In addition, X-ray diffraction analysis (XRD, Geigerflex, Figaku, Japan) to analyze the production of apatite hydroxide production was shown in Figure 2 the results. In FIG. 2, n-HA is a particle obtained by the example, and m-HA is a commercially available micro-sized apatite obtained from Sigma-Aldrich, and nanoparticles obtained by the present invention from the results of FIG. 2. It can be seen that the particles of size match well with the commercially available microsized apatite hydroxide in terms of peak position and intensity. However, nanosized particles have significantly lower peak intensities than microsized particles, indicating that nanosized particles are less crystallized than microsized particles.

Preparation Example 2

In Preparation Example 1, the initial particles were obtained, and then 25 g of the initial particles were stored for 10 days in 200 ml of 95% ethyl alcohol. However, once every two days, the old ethyl alcohol was discarded and replaced with the same amount of new ethyl alcohol.

Then, the ethyl alcohol solution was removed from the ethyl alcohol solution in which the initial particles were dispersed, 100 ml of dimethyl formamide solution was added, and water was removed by stirring for 5 hours using a magnetic bar while gradually raising the temperature to 100 to 120 ° C. .

The final particle size was 20-40 nm wide and 50-100 nm long.

Preparation Example 3

Tricalcium phosphate particles were prepared by adjusting the reaction ratio between the calcium source and the phosphorus source in Preparation Example 1 such that the stoichiometric ratio of Ca / P was 1.5. All other series of steps are the same as in Example 1.

The final particle size was 15-35 nm wide and 40-80 nm long.

Preparation Example 4

Tricalcium phosphate particles were prepared by preparing the reaction ratio of the calcium source and the phosphorus source in Preparation Example 2 such that the stoichiometric ratio of Ca / P was 1.5. All other series of steps are the same as in Production Example 2.

The final particle size was 15-35 nm wide and 40-80 nm long.

Preparation Example 5

Calcium nitrate (Ca (NO ₃ ) ₂ 4H ₂ O, made by Junsei Chemical Co., Ltd., Japan) and sodium silicate ((Na ₂ SiO ₃ · 9H ₂ O), made by Junsei Chemical Co., Ltd., Japan Calcium silicate was prepared by the following method using Ca / Si as 1).

First, the silicon source and the calcium source were sufficiently dissolved in distilled water at about 80 ° C. to prepare respective aqueous solutions, and the calcium source solution was slowly added to the silicon source solution and mixed well to obtain a nano-sized calcium silicate precipitate.

At this time, the pH of the solution was maintained by adding ammonium hydroxide to 11 or more, and the mixture was heated at about 80 ° C. for 2 hours and then sufficiently synthesized at room temperature for 24 hours or more.

In order to remove the remaining impurities, calcium silicate particles having a particle size of 200 to 300 nm (hereinafter referred to as initial particles) were obtained by washing three times or more with distilled water.

25 g of the calcium silicate particles thus obtained were placed in 300 ml of dimethylformamide, and the water was removed by stirring for 5 hours using a magnetic bar while gradually increasing the temperature to 100 to 120 ° C (hereinafter, referred to as a water removal process).

Then, the resultant was dried in vacuo to remove the residual amount of the organic solvent.

On the other hand, the particle size was observed using a transmission electron microscopy (Transmation electron microscopy, TEM, JEOL, JEM-2100F, Japan), the results are as shown in FIG. From the results of FIG. 10, it can be seen that nano-sized particles having a width of about 50 nm and a length of 300 nm were obtained.

In addition, it was analyzed by X-ray diffraction analysis (XRD, Geigerflex, Figaku, Japan) for the phase composition and crystallographic analysis of calcium silicate and the results are shown in FIG. In Figure 11 (a) is a nano-sized calcium silicate particles obtained by this example, (b) is a micro-sized calcium silicate, the nano-sized particles obtained by the present invention from the results of FIG. It can be seen that the calcium silicates of size match well in peak position and intensity. However, nanosized particles have significantly lower peak intensities than microsized particles, indicating that nanosized particles are less crystallized than microsized particles.

Preparation Example 6

In Preparation Example 5, the initial particles were obtained, and then 25 g of the initial particles were stored in 95% concentration of ethyl alcohol for 10 days. However, once every two days, the old ethyl alcohol was discarded and replaced with the same amount of new ethyl alcohol.

Then, the ethyl alcohol solution was removed from the ethyl alcohol solution in which the initial particles were dispersed, 100 ml of dimethyl formamide solution was added, and water was removed by stirring for 5 hours using a magnetic bar while gradually raising the temperature to 100 to 120 ° C. .

The final particle size was 50-100 nm wide and 200-300 nm long.

Examples 1-4

Using the calcium phosphate-based biomaterials obtained in Preparation Examples 1 to 4, an organic-inorganic composite was prepared by a conventional solvent casting method together with a biodegradable polymer. Specifically, the organic-inorganic composite three-dimensional support was manufactured using the apparatus shown in FIG. 3 by the following method.

First, a biodegradable polymer solution (hereinafter, PCL solution) was prepared by dissolving poly (ε-caprolactone) pellet (Sigma-Aldrich, St. Louis, USA; Mw = 65,000) in 20% (w / v) in chloroform. It was.

On the other hand, each biomaterial particles obtained according to Production Examples 1 to 4 (hereinafter, Production Example 1-2 particles are referred to as n-HA, Production Example 3-4 particles referred to as n-TCP) 25g (particle usage) Was dispersed in DMF to prepare n-HA / DMF slurry or n-TCP / DMF slurry.

An n-HA / DMF slurry or n-TCP / DMF slurry was added to the PCL solution described above and stirred continuously for 2 hours to obtain a homogeneous dispersion. The mixture was heated at 80 ° C. for 2 hours to volatilize chloroform to obtain an n-HA / PCL / DMF mixture or an n-TCP / PCL / DMF mixture, of which the nanoparticle content was 40% by weight. The temperature was then gradually raised to 100-120 ° C. with constant stirring and maintained for 2 hours. The mixture was then cast in a Teflon mold (100 × 100 × 3 mm 3) and dried in air for 24 hours to evaporate DMF. The remaining solvent was removed by vacuum drying for 48 hours to obtain n-HA / PCL complex or n-TCP complex. The size of the three-dimensional support is 10 × 10 × 5 mm 3.

Examples 5-6

Using the calcium silicate-based biomaterials obtained according to Preparation Examples 5 to 6, an organic-inorganic composite was prepared by a conventional solvent casting method together with a biodegradable polymer. Specifically, the organic-inorganic composite three-dimensional support was manufactured using the apparatus shown in FIG. 3 by the following method.

First, a biodegradable polymer solution (hereinafter, PCL solution) was prepared by dissolving poly (ε-caprolactone) pellet (Sigma-Aldrich, St. Louis, USA; Mw = 80,000) in 20% (w / v) in chloroform. It was.

On the other hand, 25 g (particle usage) of each biomaterial particle (hereinafter referred to as n-CS) obtained according to Production Examples 5 to 6 were dispersed in DMF to prepare an n-CS / DMF slurry.

The n-CS / DMF slurry was added to the PCL solution described above and stirred continuously for 2 hours to obtain a homogeneous dispersion. The mixture was heated at 80 ° C. for 2 hours to volatilize chloroform to give an n-CS / PCL / DMF mixture, of which the content of n-CS was 40% by weight. The temperature was then gradually raised to 100-120 ° C. with constant stirring and maintained for 2 hours. The mixture was then cast in a Teflon mold (100 × 100 × 3 mm 3) and dried in air for 24 hours to evaporate DMF. The remaining solvent was removed by vacuum drying for 48 hours to obtain n-CS / PCL complex.

The size of the three-dimensional support is 10 × 10 × 3 mm 3.

Experimental Example 1. Evaluation of n-Ha / PCL complex and n-TCP / PCL complex

Characteristics of organic-inorganic complex

(1) Structural analysis of the support: mercury intrusion porosimetry (Auto-pore IV 9500, Micro-meritics Instrument Corporation, for porosity analysis of n-HA / PCL complex, n-TCP / PCL complex and n-CS / PCL complex) USA, n = 3).

And the scanning electron microscope (SEM, Hitachi LTd, S-4300SE, JAPAN) was used to analyze the structure of the surface and the cross section.

(2) mechanical properties of the support

Compression modulus of n-HA / PCL complex, n-TCP / PCL complex and n-CS / PCL complex was measured at room temperature using a Micro-load system (Micro-load system, R & B). The moving speed was set to 0.5 mm / min here. The surface area was measured by converting the extrusion load into compressive stress. Compression modulus was determined from the initial porous response region. Five samples were tested for each group and the mean and standard deviation were calculated for statistical analysis.

(3) wettability evaluation

The wettability of the organic-inorganic composite was determined by measuring the contact angle of water droplets on the surface using a contact angle measuring device (SEO 300A, Surface and Electro-Optics). Using a micro-syringe (Perfecktim, Popper & Sons, Japan), 10 [mu] l droplets were dropped in three places on each sample and the contact angle was measured after 300 seconds.

Cell culture on organic-inorganic complex

(1) osteoblast proliferation

In order to evaluate the osteoblast proliferation capacity of each of the prepared organic-inorganic complex scaffolds, MG-63 osteoblast-like cells (American Type Culture Collection, No. CRL-1427, USA) were used in DMEM (Dulbeccos's Modified Eagle Medium, Gibco, USA). The cells were cultured in medium containing FBS (fetal bovin serum) and 1% penicillin / streptomycin (P / S Hyclone, USA). The supports were preliminarily infiltrated in the culture medium for at least 48 hours, left in well plates and sterilized using ultraviolet light. Osteoblast adhesion and proliferation were assessed using methyl thiazolyl tetrazolium (MTT) assay. The MG-63 cells described above were seeded directly on the support at a concentration of 5 × 10 ⁴ cells / ml. The support seeded with the cells was incubated with 5% (v / v) CO ₂ in a 37 ° C. thermostat. Cell adhesion was observed 4 hours after sowing and cell proliferation was assessed over 1, 4 and 7 days. Briefly, complex-cell constructs were left in MTT containing culture medium and incubated at 37 ° C. for 4 hours in a humid atmosphere. Dimethyl sulfoxide was added to each well to completely dissolve the MTT. The optical density (OD) of each well was analyzed at 590 nm wavelength (reference wavelength 620 nm) in a microplate reader (Elisa, Synergy HT, Bio-Tek instruments, USA). Six species of each scaffold were tested during each incubation period and each test result was subjected to triple assay. The results were collected in optical density units.

(2) cell morphology

MG-63 osteoblastic cells were used for the biocompatibility evaluation of the support. About 50 μl of culture medium containing 5 × 10 4 cells was seeded onto the porous support in a 24-well culture plate. Cells were attached to the support for 4 hours, after which 1 mL of fresh culture medium was added to each well and cells were incubated for 1, 4 or 7 days in a humid atmosphere (37 ° C., 5% CO ₂ ). Cell morphology on the complex support was observed using SEM (Hitachi, S-4300SE, Japan). After another incubation time, the support-cell constructs were washed twice with PBS solution and fixed with 4% formalin (pH 7.4) in PBS for 20 minutes. This was washed twice with PBS solution and dehydrated at each concentration for 3 minutes with varying ethanol concentrations (50, 60, 70, 80, 90 and 100% v / v). Each species was air dried overnight in a desiccator. The dried species were adhered onto copper specimen stubs and deposited coated with gold for SEM observation.

Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA). All results are expressed as mean ± standard deviation. The statistical significance level was determined as p <0.05. Commercially available SPSS software (ver. 11.0, Standard Software Package, USA) was used for Fisher's LSD method.

result

(1) pore structure of the composite

The photograph of the three-dimensional composite obtained from Example 1 is attached to FIG. 4. From the results of FIG. 4, it can be seen that a support having goodly internally bonded and uniformly dispersed pores was obtained.

In addition, the SEM image is shown in Figure 5, Figure 5 (a) is a photograph observed in the plane and (b) is an image observed in the cross section. From this result, it can be seen that the pore size of the composite is 400-500㎛ in the vertical direction, 300㎛ in the horizontal direction, has a three-dimensional internally bonded pores. The SEM image is attached to FIG. 6 in relation to the surface microstructure shape, indicating that the composite has a smooth surface. The smooth surface would be the result of fine and good distribution of nano-sized biomaterials (n-HA) in the PCL matrix. It can be seen that the nanoparticles are exposed while maintaining their unique size and shape on the surface of the composite.

In tissue engineering, the scaffold is used as a temporary carrier for transplanted cells. Thus, the scaffold must provide a suitable space for the cells to grow and form new tissue. Cell growth and new tissue production may vary depending on the porosity, pore size and material of the support. Internally linked pore networks are also essential for cell internal growth, vascularization and nutrient diffusion.

According to the present invention it was possible to successfully produce a porous composite support. It can be seen that the organic-inorganic complex support is applicable to the field of bone tissue engineering.

(2) mechanical properties and hydrophilicity

The compressive modulus of the organic-inorganic composite support was 3.2 ± 0.1 Mpa and the porosity was 72.2 ± 0.3% (significance level p <0.01). These results can be said to be the result of good dispersion of nano-sized biomaterial particles. Therefore, it can be seen that the organic-inorganic composite support according to the present invention can be considered as a candidate for bone substitute.

On the other hand, the initial water contact angle of the organic-inorganic composite is 30 ㅀ, it can be seen that the excellent water absorption ability. This is an excellent hydrophilicity, and this result seems to be due to the high surface area of nanosize particles exposed to the composite surface. Wei et al. Reported that having hydrophilic surfaces rather than hydrophobic surfaces is better for cell attachment, differentiation and proliferation (Jianhua Wei, Masao Yoshinari.Adhesion of mouse fibroblasts on hexamethydisiloxane surfaces with wide range of wettability.J Biomed Mater Res Part B 2007; 81B; 66-75). Yang et al. Also reported that the hydrophilicity of material supplements is essential for in vitro osteoblast adhesion in the absorption of fibronectin (Yang M, Zhu S. Studies on bone marrow stromal cells affinity of poly (3-hydrocybutyrate-). Thus, the hydrophilic surface of the organic-inorganic complex according to the present invention can promote cell adhesion and proliferation.

(3) cell adhesion and proliferation

Adhesion and proliferation of MG-63 osteoblast-like cells are shown in FIG. 8 by evaluation using MTT assay. According to the results of Figure 8 it can be seen that the adhesion of osteoblasts on the organic-inorganic complex support. In addition, when comparing the OD value of 1, 4 and 7 days, it can be seen that the osteoblasts proliferate faster with the passage of time.

The adhesion and proliferation of MG-63 cells on the organic-inorganic complex support also appears to be related to the surface structure of the support. Because nanoscale biomaterials have a small size and a large specific surface area, they have special surface characteristics that promote cell adhesion, and the hydrophilic surfaces of nanoscale particles also promote cell adhesion and proliferation.

(4) cell morphology

Figure 9 shows the SEM microstructure of the morphological structure of MG-63 cells on the organic-inorganic complex support cultured for 7 days. According to Figure 9 it can be seen that the cells are evenly distributed on the surface of the support and a confluent layer in intimate contact with the surface is formed. The dispersed cells are also kept in physical contact with each other. Cell density is also high. It can be seen that the cells cultured on the organic-inorganic complex support form a dense cell layer covering the surface.

Bioactivity and biocompatibility of biomaterials are closely related to cell contact and proliferation characteristics. The MG-63 cells grow better and proliferate on the organic-inorganic complex according to the present invention and also have a morphological feature with an intimate cell matrix.

Generally, biodegradable polymers are hydrophobic, and likewise, PCL is also hydrophobic, but the addition of hyperhydrophilic apatite improves cell adhesion and proliferation. n-HA / PCL has good wettability for cell adhesion because of the large specific surface area of n-HA particles. Cell proliferation is thus also more suitable.

Experimental Example 2 Evaluation of n-CS / PCL Complex

Mechanical properties, biodegradability and hydrophilicity

(1) Mechanical strength of the prepared calcium silicate / PCL organic-inorganic composite (hereinafter n-CPC) specimen (standard: 10 × 10 × 10 kPa) at room temperature using compression test at constant feed rate (2mm / min) Measured. The jigs were specially designed to provide uniform loads on the composite specimens and were tested using Bionix 858.20 (MTS Systems Corp., Eden Prairie, USA). Compressive strength and elastic modulus were calculated within the linear range of the stress-tensile curve. The test tested four of five species and averaged the results.

(2) The weight loss in test species of 10 × 10 × 10 mm 3 specification during degradation in phosphate buffered saline (PBS; pH 7.4) was measured using the change in dry weight after incubation for the specified test period. During each test period, the test species were removed, washed with distilled water and dried in a vacuum oven for one week. All values shown are the average of all three species. The percentage of weight loss is calculated by the following equation.

Weight loss rate (%) = 100 × (W ₀ -W _t ) / W ₀

Wherein W ₀ is the weight at the start and W _t is the dry weight at that time t.

(3) The surface wettability of standard 10 × 10 × 10 mm 3 was determined by measuring the contact angle of water droplets on the surface using a contact angle measuring device (SEO 300A, Surface and Electro-Optics, Korea). Using a micro-syringe (Perfecktim, Popper & Sons, Japan), 10 [mu] l droplets were dropped in three places on each sample and the contact angle was measured after 30 seconds. Each result is expressed as four means ± standard deviations for five contact angles for each test.

Specimen deposition and ion concentration measurement in SBF

In vitro bioactivity of n-CPC was assessed by testing for the formation of apatite on these surfaces in SBP.

SBF has an ion concentration similar to that found in human blood. SBF was prepared by dissolving sample grade NaCl, NaHCO ₃ , KCl, K ₂ HPO ₄ · 3H ₂ O, MgCl ₂ · 6H ₂ O, CaCl ₂ and Na ₂ SO ₄ in ion-exchanged distilled water.

Table 1 below shows the ion concentration in SBF solution and the ion concentration in human blood, where the solution was buffered to pH 7.4 with tris (hydroxymethyl) aminomethane ((CH ₂ OH) ₂ CNH ₂ ) and 1M HCl ( At 37 ° C).

Ion concentration (mM) Na ⁺ K ⁺ Mg ^{2 +} Ca ^{2 +} Cl ^- HCO ^{3 -} HPO ₄ ^{2 -} SO ₄ ^{2 -} SBF 142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5 blood 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5

Cut to specimen size 10 × 10 × 10 mm 3, grind with diamond paste, wash with distilled water, dry under vacuum at room temperature, and stir or refresh SBF solution for 1, 3, 5, 7 and 14 days at 37 ° C. It was deposited in 30 ml SBF without. After immersion, each specimen was removed from the SBF solution, washed lightly with distilled water and dried at room temperature. The morphology and composition of the apatite formed on the surface of the composite was monitored by energy-dispersive X-ray spectroscopy (EDS) under Hitachi S-4300SE field emission-scanning electron microscope (FE-SEM). At each test point, the ion concentrations of calcium, phosphorus and silicon in the SBF solution were measured using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES).

Cell adhesion, growth and proliferation

To examine cell adhesion and proliferation, n-CPC dense specimens of standard 10 × 10 × 10 mm 3 were sonicated in ethanol and sterilized using ultraviolet light. For cell adhesion testing, MG-63 osteoblastic cells (American Type Culture Collection, No. CRL-1427, USA) were added to DMEM (Dulbeccos's Modified Eagle Medium, Gibco, USA) with 10% fetal bovin serum (FBS). Cultivation was done in medium containing% penicillin / streptomycin (P / S Hyclone, USA). The supports were preliminarily infiltrated in the culture medium for at least 48 hours, left in well plates and sterilized using ultraviolet light. Osteoblast adhesion and proliferation were assessed using methyl thiazolyl tetrazolium (MTT) assay. The MG-63 cells described above were seeded directly on the support at a concentration of 2 × 10 ³ cells / ml. The support seeded with the cells was incubated with 5% (v / v) CO ₂ in a 37 ° C. thermostat. Cell adhesion was observed 4 hours after sowing and cell proliferation was assessed over 1, 4 and 7 days. Briefly, complex-cell constructs were left in MTT containing culture medium and incubated at 37 ° C. for 4 hours in a humid atmosphere. Dimethyl sulfoxide was added to each well to completely dissolve the MTT. The optical density (OD) of each well was analyzed at 590 nm wavelength (reference wavelength 620 nm) in a microplate reader (Elisa, Synergy HT, Bio-Tek instruments, USA). Six species of each scaffold were tested during each incubation period and each test result was subjected to triple assay. The results were collected in optical density units.

Alkaline phosphatase activity

To assess alkaline phosphate (ALP) activity, 5 × 10 ⁴ hMSCs (human mesenchymal stem cells, Cambrex Bio Science Walkersville, Inc., Walkersvill, USA) were placed on n-CPC dense specimens of standard 10 × 10 × 10 mm 3. Seeds and ALP activity were measured at 1, 4, and 7 routines. Before seeding the cells, the supports were soaked in 75% ethanol solution for 60 minutes and lysed with PBS; Subsequent to this process, the cell lysis buffer containing 0.1% Triton X-100 was added to the specimen and frozen to -20 ° C. Frozen specimens were thawed at 37 ° C. for 5 minutes and ALP activity was measured according to ALP kit 104. 50 ml of sample were mixed with p-nitrophenol (1 mg / ml) in 1 M diethanolamine buffer containing 0.5 nM MgCl 2 (pH 9.8) and incubated on a bench shaker at 37 ° C. for 15 minutes. The reaction was stopped by addition to 25 mL 3N NaOH per 100 mL reaction mixture. Cell number was determined by measuring DNA content using Quant-iT (tm) Pico-Green dsDNA reagent kit (Molecular Probes, Engene, USA). Sample DNA was quantified by measuring fluorescence with Synergy HT Multi-Detection Microplate Reader (BioTek, VT, USA) (excitation wavelength 480 nm, emission wavelength 520 nm). Enzyme activity was quantified by measuring absorbance at 405 nm, and ALP activity was calculated from standard curves after normalizing to total DNA content. The results were expressed as nanocols of p-nitrophenol produced per minute per nanogram of DNA.

Cell morphology

MG-63 osteoblastic cells were used for the biocompatibility evaluation of the support. About 50 μl of culture medium containing 5 × 10 ⁴ cells was seeded onto the porous support in a 24-well culture plate. Cells were attached to the support for 4 hours, after which 1 mL of fresh culture medium was added to each well and cells were incubated for 1, 4 or 7 days in a humid atmosphere (37 ° C., 5% CO ₂ ). Cell morphology on the complex support was observed using SEM (Hitachi, S-4300SE, Japan). After another incubation time, the support-cell constructs were washed twice with PBS solution and fixed with 4% formalin (pH 7.4) in PBS for 20 minutes. This was washed twice with PBS solution and dehydrated at each concentration for 3 minutes with varying ethanol concentrations (50, 60, 70, 80, 90 and 100% v / v). Each species was air dried overnight in a desiccator. The dried species were adhered onto copper specimen stubs and deposited coated with gold for SEM observation.

Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA). All results are expressed as mean ± standard deviation. The statistical significance level was determined as p <0.05. Commercially available SPSS software (ver. 11.0, Standard Software Package, USA) was used for Fisher's LSD method.

result

(1) mechanical properties, hydrophilicity and degradability

Table 2 shows the compressive strength and modulus of elasticity of n-CPC according to n-CS content.

Compressive strength (MPa) Elastic Modulus (MPa) PCL 37 ± 2 191 ± 8 CS (20%) 43 ± 1 345 ± 21 CS (40%) 54 ± 1.5 537 ± 24 Cs (60%) 37 ± 3 246 ± 12

From the results in Table 2, the mechanical strength was the best when the CS content is 40%, the weight change in PBS at different incubation time for n-CPC containing 40% CS is shown in Figure 12 It was.

12, it can be seen that after 12 weeks of constant temperature, the weight loss rate of n-CPC is 30%. In the case of PCL, the weight loss rate at the end point (12 weeks) of the degradability test from the initial weight is about 2.1%.

Meanwhile, Table 3 shows the results of measuring the contact angle of the surface of the composite specimen.

n-CPC contact angle (°) PCL 77.5 ± 0.8 Contains 20% CS 59.2 ± 1.2 Contains 40% CS 26.7 ± 0.9 Contains 60% CS 0

It can be seen from the results of Table 3 that the contact angle becomes significantly smaller as the content of n-CS increases, indicating that the hydrophilicity is increased.

(2) Apatite Production in SBF

In vitro bioactivity of n-CPC was investigated by depositing samples in SBF. Figure 13 shows the surface properties of n-CPC before deposition on SBF.

14 shows a SEM image of n-CPC after 14 days of deposition in SBF. This image shows a photograph of the surface after deposition in SBF for 14 days with different magnifications. The results in FIG. 14 show that the apatite layer is typically in spherical granules. As the magnification increases, it can be seen that a large number of flake apatite crystals are present on the n-CPC. The flake apatite crystals have a length of about 100-200 nm and a width of about 20-60 nm. It can be seen that the apatite layer on the n-CPC flakes is also densely packed. It can also be seen that the apatite crystalline layer on n-CPC is compact and homogeneous.

EDS spectra were tested for apatite layers formed on the composite surface after 14 days of deposition in SBF. This is illustrated in FIG. 15, and as a result, both calcium and phosphorus peaks were observed. The ratio of Ca / P to n-CPC was 1.63. This is similar to hydroxyapatite with a Ca / P ratio of 1.67.

(3) Ion concentration change in SBF

Changes in elemental concentrations of calcium, phosphorus and silicon were observed in SBF after specimens were deposited in SBF for various periods of time. The result is shown in FIG.

According to the results of FIG. 16, in all specimens, calcium and silicon concentrations were increased up to 3 days, and then increased at a slower rate until 14 days thereafter. In addition, in contrast to the increase in the Ca and Si concentration it can be seen that the P ion concentration gradually decreases during the deposition period.

(4) cell adhesion

MTT analysis is used to analyze the relative number of cells attached to various materials, because the optical density absorbance value can be used as an indicator of the relative cell number on the complex. Cell adhesion was assessed using MTT analysis of MG63 cells cultured on neat PCL and n-CPC, respectively. The control is a tissue culture plate. The results are shown in FIG.

According to the results of FIG. 17, it can be seen that the absorbance is significantly increased with time after the cell attachment. These results show that the n-CPC specimens according to the invention are useful for attaching cells on these surfaces and promoting cell growth.

(5) cell growth and proliferation

Proliferation of MG63 osteoblastic cells cultured on n-CPC was also assessed using the MTT assay for the same reasons as described above. The results are shown in FIG.

According to the results of FIG. 18, the OD value is also significantly increased as the incubation time passes. These results show that n-CPC may promote cell growth and cell proliferation.

In addition, the results of morphology of MG63 cells cultured on n-CPC using Phase Contrast Microscopy are shown in FIG. 19. In the micrograph of FIG. 19, (a) shows the results observed at 1 day after the culture, (b) 4 days after the culture, and (c) 7 days after the culture. After 4 days incubation, the cells grow and show expansion. After 7 days, the MG63 population shows a clear increase and it can be seen that cell proliferation is normal and fused into monolayer cells. These results show that the complex according to the present invention has no negative effect on cell morphology, proliferation and is also excellent in biocompatibility.

(6) cell differentiation

Cell differentiation was assessed by ALP activity in hMSCs in 1, 4 and 7 day cultures. The result is shown in FIG.

According to the results of FIG. 20, ALP was shown to be low at 1 and 4 days, but ALP activity increased substantially with the culturing period. Cells on n-CPC showed the highest levels of ALP activity at 7 days, indicating that hMSCs were most degraded at 7 days.

(7) cell morphology

21 shows a SEM photograph to show the morphological characteristics of hMSCS cultured on n-CPC. In Fig. 21, (a) is the result of 4 days of culture and (b) is the result of 7 days.

From the results in FIG. 21, it can be seen that after 4 days, the cells are excellently expanded and extended and exist intimately attached to the complex surface.

After 7 days, it can be seen that the cells on the complex surface stretch well and form a fusion layer intimately bound to the surface and the stretched cells remain in physical contact with each other.

1 is a transmission electron micrograph of the nano-sized apatite hydroxide particles obtained according to the present invention.

2 is a graph comparing X-ray diffraction analysis results of nano-sized apatite hydroxide particles obtained according to the present invention with commercially-sized microsized apatite hydroxide particles.

3 shows an improved R.P. Photo (a) of the system and a photo showing the manufacturing method (b) of the composite support using the same.

Figure 4 is a photograph of the three-dimensional composite support produced by the experimental apparatus of FIG.

Figure 5 is a SEM photograph of the n-HA / PCL complex support, (a) is a photograph observed in the plane, (b) is a photograph observed in the cross section.

6 is a SEM photograph of the surface microstructure morphology of n-HA / PCL (solid line = 250 nm).

7 shows the results of surface hydrophilicity evaluation of n-HA / PCL.

8 is a graph of MTT analysis of n-HA / PCL support.

Figure 9 is a SEM photograph 7 days after MG63 cell culture on the n-HA / PCL support, (a) and (b) is a different magnification.

10 is a transmission electron micrograph of the nano-sized calcium silicate particles obtained according to the present invention.

11 is a graph comparing the results of X-ray diffraction analysis of nanosized calcium silicate particles obtained according to the present invention with commercially available microsized apatite hydroxide particles.

12 is a graph showing the weight change of n-CPC (containing 40% calcium silicate) in PBS at different incubation times.

FIG. 13 is a scanning electron micrograph of the surface of n-CPC before deposition on SBF. FIG.

14 is a scanning electron micrograph at different magnifications for n-CPC specimens deposited in SBF for 14 days.

FIG. 15 shows EDS spectra of n-CPC surfaces after immersion in SBF for 14 days.

16 is a graph of concentration change of Ca, Si, and P after n-CPCdmlk deposition in SBF at different times (◆: Ca, ■: Ca, Δ: P).

17 is a result graph showing the initial attachment of MG63 cells on n-CPC.

18 is a graph showing the proliferation of MG63 cells cultured on n-CPC for 1, 4 and 7 days.

Figure 19 is a Phase Contrast Microscopy photograph of MG63 cells cultured on n-CPC during different culture periods ((a) culture 1 day, (b) culture 4 days, (c) culture 7 days).

20 is a graph of ALP activity of hMSCs cultured on n-CPC for 1, 4 and 7 days.

FIG. 21 is an SEM image of hMSCs seeded on n-CPC surface at Days 4 (a) and 7 (b).

Claims (15)

Preparing a nano-sized biomaterial particle slurry; Separately preparing a biodegradable polymer solution; And A nano-sized biomaterial particle slurry, a biodegradable polymer solution, and an organic solvent having a boiling point of 120 ° C. or more are mixed, and gradually heated to a boiling point of an organic solvent having a boiling point of 120 ° C. or more while gradually heating in a temperature range of 100 ° C. to 120 ° C. with stirring. A method for producing a composite of a nano-sized biomaterial and a biodegradable polymer comprising a step of removing moisture. The process of claim 1 further comprising: a casting process; And a vacuum drying process further comprising a nanoscale biomaterial and a biodegradable polymer composite manufacturing method. The method of claim 1, wherein the nano-sized biomaterial particle slurry comprises the steps of preparing an aqueous solution of a phosphorus (P) source and a calcium (Ca) source; Mixing a calcium source aqueous solution with a phosphorus source aqueous solution to heat 1.5 to 2 hours at 80 to 100 ° C. under alkaline conditions, and reacting at room temperature for 24 to 48 hours to obtain a calcium phosphate precipitate; A composite of a nano-sized biomaterial and a biodegradable polymer, characterized in that obtained by washing the precipitate with distilled water to remove residual impurities to obtain a nano-sized calcium phosphate-based biomaterial particles containing substantially water. Manufacturing method. The method of claim 1, wherein the nano-sized biomaterial particle slurry comprises the steps of preparing an aqueous solution of a silicon (Si) source and a calcium (Ca) source; Mixing a calcium source aqueous solution with a silicon source aqueous solution and stirring at 80 to 100 ° C. for 1.5 to 2 hours under alkaline conditions to obtain a calcium silicate-based precipitate; And obtaining a nano-sized calcium silicate-based biomaterial particle substantially containing water by washing the precipitate with distilled water to remove residual impurities. Composite manufacturing method. The method according to claim 3 or 4, wherein the calcium phosphate-based biomaterial particles or calcium silicate-based particles containing substantially water are obtained, and then the particles are stored in an alcohol solution of 95-99% concentration for 10 to 15 days, Method of manufacturing a composite of a nano-sized biomaterial and a biodegradable polymer, characterized in that it further comprises the step of removing the water contained in the particles by replacing the alcohol solution every two to three days. The method according to claim 3 or 4, wherein the calcium phosphate-based biomaterial particles or calcium silicate-based particles substantially containing water are gradually heated in a temperature range of 100 ° C to 120 ° C while stirring in an organic solvent having a boiling point of 120 ° C or higher. A method of manufacturing a composite of a nano-sized biomaterial and a biodegradable polymer further comprising the step of removing moisture by heating to a boiling point of an organic solvent having a boiling point of 120 ° C. or higher. The nano-sized biomaterial and biodegradable polymer according to claim 3 or 4, wherein the calcium phosphate-based biomaterial particles or calcium silicate-based particles substantially containing water are dispersed in an alcohol solution. Method for producing a complex with. The method of claim 3 or 4, wherein at least one of the aqueous solution of calcium source, the aqueous solution of phosphorus source and the aqueous solution of silicon source further comprises a dispersing agent. The nanoscale biomaterial and biodegradation according to claim 1, wherein dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), toluene or diglyme are used as the organic solvent having a boiling point of 120 ° C or higher. Method for producing a composite with a sex polymer. The nanoscale biomaterial and biodegradation according to claim 6, wherein dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), toluene or diglyme are used as the organic solvent having a boiling point of 120 ° C or higher. Method for producing a composite with a sex polymer. 8. The method of claim 6, further comprising a step of subsequently vacuum drying. The method of claim 3, wherein the calcium phosphate-based particles are apatite hydroxide particles or tricalcium phosphate particles. A composite of a nanoscale biomaterial and a biodegradable polymer obtained according to the method of claim 1. 15. The composite of nano-sized biomaterial and biodegradable polymer according to claim 13, wherein the nano-sized biomaterial is calcium phosphate particles or calcium silicate particles. The method according to claim 13 or 14, wherein the biodegradable polymer is at least one selected from poly (ε-caprolactone), poly (lactic acid), poly (glycolic acid) and copolymers of D, L-lactic acid and glycolic acid. A composite of nanoscale biomaterials and biodegradable polymers.

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