KR20190074437A - A manufacturing method of the porous biphasic calcium phosphate bone substitute materials having anti-inflammatory activity - Google Patents

Abstract

The present invention, in manufacturing a biphasic calcium phosphate-based porous bone graft material having excellent anti-inflammatory activity by using a three-dimensional printing technique, provides a method for manufacturing the porous bone graft material using a powder, water-soluble polymer binder and dispersant, and the porous bone graft material manufactured by using the three-dimensional printing technique, by synthesizing the biphasic calcium phosphate powder such that elements cerium (Ce) or silicon (Si) having anti-inflammatory activity are partially substituted.

Description

TECHNICAL FIELD [0001] The present invention relates to a porous bone graft material, and more particularly, to a porous bone graft material having an anti-

The present invention relates to a method for producing a calcium phosphate porous bone graft material slurry having excellent antiinflammatory activity, and more particularly, to a method for producing a porous calcium phosphate bone graft material slurry containing a cerium (Ce) or silicon (Si) The present invention relates to a method for manufacturing a bone graft material using a calcium phosphate-based powder, a water-soluble polymer binder and a dispersing agent, and a bone graft material prepared by the three-dimensional printing technique using the same.

Bone grafting is widely used to treat large and serious bone defects due to trauma or resection. Bone grafts used in these bone grafts can be classified into autografts, allografts, heterogeneous bone grafts, and synthetic bone grafts. Unlike other bone graft materials, synthetic graft grafts are easy to supply, It is in the spotlight that it can be manufactured in various forms favoring biocompatibility and structurally favorable bone formation.

Among the calcium phosphate-based materials mainly used as synthetic bone graft materials, hydroxyapatite having excellent biocompatibility and tricalcium phosphate having biodegradability have been studied as biomaterials. However, hydroxyapatite has a disadvantage in that the biodegradation rate is slow and tricalcium phosphate has a weak strength and is fast in decomposition and absorption, so that it is dissolved before the bone is recovered. To solve this problem, biphasic calcium phosphate (BCP), which is a mixture of hydroxyapatite and tricalcium phosphate, has been developed, and a suitable combination of hydroxyapatite stable in vivo and tricalcium phosphate excellent in bioabsorbability It is known that it has a far superior effect on function because it plays a role of complementing the advantages and disadvantages of each other. However, hydroxyapatite and tricalcium phosphate have to be prepared and used respectively.

In the case of these biphasic calcium phosphates, too, there is a possibility of causing an inflammatory reaction when the powder form is used as it is. Therefore, a mold of a specific shape is prepared, and a mixture of calcium phosphate powder and a polymer is injected into the mold. The production of bone graft materials is a general method for producing porous bone graft materials. However, the bone graft materials manufactured by this method have a limited shape and size, and there are disadvantages that many molds are required to manufacture bone graft materials of various shapes and sizes.

In recent years, three-dimensional printing technology has been used to produce porous bone graft materials, and it has become possible to manufacture a bone graft material having a three-dimensional structure with high precision and accuracy. These bone graft materials can provide a surface area favorable for adhesion and proliferation of cells because each layer can have a uniform and consistent pore size, structure and thickness as compared with bone graft materials manufactured by conventional methods, It is possible to produce a graft material. In order to manufacture a bone graft material using a three-dimensional printing technique, a ceramic powder is mixed with a polymer and printed and then used as a composite material mixed with a polymer (see Patent Document 1), or a method in which a polymer is sintered to use only a ceramic bone graft material (See Patent Document 2).

Although the above-described composition can be used as a bone graft material, when a bone graft material is used for a bone defect site, infection may occur. Recently, development of a bone graft material containing an antimicrobial agent has been attempted (see Patent Document 3). However, these antimicrobial agents are likely to be released at an early stage, and there is also a problem in that there is no way to introduce antimicrobial agents into bone graft materials composed of only ceramics.

In order to solve these problems, there have been proposed a method for producing a calcium phosphate powder having a hydroxyapatite component and a calcium phosphate component, a method for preparing porous bone grafts of various shapes and sizes, a long- Is required to be developed.

Patent Document 1: Korean Patent Application Publication No. 10-2016-0095481 (published on Aug. 11, 2016) Patent Document 2: Korean Patent Application Publication No. 10-2017-0120766 (published on November 01, 2017) Patent Document 3: Korean Patent Application Publication No. 10-2017-0099578 (published on September 01, 2017)

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a method for producing a calcium phosphate- A method for manufacturing a bone graft material, and a porous bone graft material manufactured using a three-dimensional printing technique.

SUMMARY OF THE INVENTION

(a) preparing a polymer template solution by dissolving a water-soluble polymer in distilled water;

(b) providing a calcium ion precursor solution, a phosphate ion precursor, a cerium ion precursor and a silicon ion precursor, respectively;

(c) adding the calcium ion precursor solution, phosphate ion precursor solution and cerium ion precursor solution (Ca + Ce) solution to the polymer template solution so as to produce a binary calcium phosphate mixed with hydroxyapatite and tricalcium phosphate in a ratio of 6: /P=1.585/1 to 1.653 / 1, followed by stirring, centrifuging and drying to prepare a calcium phosphate powder having a partial substitution of cerium (Ce-BCP);

(d) adding the calcium ion precursor solution, the phosphate ion precursor solution and the silicon ion precursor solution to the polymer template solution so that a biphasic calcium phosphate having a ratio of hydroxyapatite and tricalcium phosphate of 6: 4 is formed, Si) = 1.585 / 1 to 1.653 / 1, stirring the mixture, centrifuging and drying to prepare a calcium phosphate powder partially substituted with silicon (Si-BCP);

(e) mixing the dried calcium cerium or silicon-partially substituted calcium phosphate powder obtained in the above step with a polymer binder, a dispersing agent and distilled water to prepare a slurry. And a method for producing the same.

Another object of the present invention is to provide

Drying the prepared slurry at 40 ° C to 50 ° C using a three-dimensional printer, and sintering the polymer binder and the dispersing agent for 1 to 3 hours at 1,000 to 1,200 ° C. The present invention also provides a porous bone graft material.

The present invention relates to a method for producing a porous bone, which comprises mixing a powder obtained by partially replacing cerium or a silicon element with a biodegradable calcium phosphate having excellent biocompatibility and having controlled biodegradability and mechanical properties, a polymer binder and a dispersing agent, The implant has an excellent effect in improving anti-inflammatory activity.

In addition, when used in dentistry and orthopedic surgery for bone regeneration, early adhesion and proliferation of osteocytes are significantly improved, and there is an excellent effect of greatly improving anti-inflammatory properties, which is advantageous in dental and orthopedic fields.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the preparation of a bicomponent calcium phosphate (BCP) powder according to the present invention, a bicomponent calcium phosphate (Ce-BCP) powder partially substituted with cerium and a bicomponent calcium phosphate (Si- And a method of manufacturing a porous bone graft material having an anti-inflammatory activity using a three-dimensional printer.
FIG. 2 is a graph showing the results of a comparison between a binary calcium phosphate (BCP) powder prepared according to the present invention, a bismuth cobalt phosphate (Ce-BCP) powder partially substituted with cerium and a bicomponent calcium phosphate (Si- Was observed with a scanning electron microscope at a magnification of 50,000.
FIG. 3 is a graph showing the results of a comparison between a bicomponent calcium phosphate (BCP) powder prepared according to the present invention, a bicomponent calcium phosphate (Ce-BCP) powder partially substituted with cerium and a bicomponent calcium phosphate (Si- The results of this study were as follows: 1.
FIG. 4 is a graph showing the elemental analysis of the porous bone graft material having anti-inflammatory activity prepared according to the present invention by energy dispersive X-ray spectroscopy.
FIG. 5 is a graph showing the surface analysis of the porous bone graft material having anti-inflammatory activity prepared according to the present invention by infrared spectroscopy.
FIG. 6 is a graph showing the results of cell proliferation of the porous bone graft material having anti-inflammatory activity according to the present invention.
FIG. 7 is a graph showing the results of measuring the inflammatory activity of the porous bone graft material having anti-inflammatory activity prepared according to the present invention.
8 is a graph showing the results of measurement of the inflammatory activity according to the substitution ratio of cerium or silicon in the cerium-substituted bicarbonate calcium phosphate (Ce-BCP) and the partially substituted silicon bicarbonate (Si-BCP) Fig.

The method for preparing a porous bone graft material having anti-inflammatory activity according to the present invention comprises

(a) preparing a polymer template solution by dissolving a water-soluble polymer in distilled water;

(b) providing a calcium ion precursor solution, a phosphate ion precursor, a cerium ion precursor and a silicon ion precursor, respectively;

(c) adding the calcium ion precursor solution, phosphate ion precursor solution and cerium ion precursor solution (Ca + Ce) solution to the polymer template solution so as to produce a binary calcium phosphate mixed with hydroxyapatite and tricalcium phosphate in a ratio of 6: / P = 1.585 / 1 to 1.653 / 1, stirring the mixture, centrifuging and drying to prepare a calcium phosphate powder having a partial substitution of cerium (Ce-BCP);

(d) adding the calcium ion precursor solution, the phosphate ion precursor solution and the silicon ion precursor solution to the polymer template solution so that a biphasic calcium phosphate having a ratio of hydroxyapatite and tricalcium phosphate of 6: 4 is formed, Si) = 1.585 / 1 to 1.653 / 1, stirring the mixture, centrifuging and drying to prepare a calcium phosphate powder partially substituted with silicon (Si-BCP);

(e) mixing the dried calcium cerium or silicon-substituted calcium phosphate powder obtained in the above step with a polymer binder, a dispersant and distilled water to prepare a slurry.

To achieve the above object, according to the present invention, in the step (a), the water-soluble polymer for a polymer template comprises sodium alginate, beta-cyclodextrin, polyvinylpyrrolidone, polyvinyl alcohol, hyaluronic acid, laminarin, pullulan and fucoidan One or more selected from the group.

In the step (b), the calcium ion precursor is not particularly limited as long as it is a substance capable of providing calcium ions, but may specifically include at least one selected from the group consisting of calcium nitrate, calcium chloride, calcium sulfate and calcium hydroxide. The phosphate ion precursor is not particularly limited as long as it is a substance capable of providing a phosphate ion, but specifically includes ammonium phosphate, dibasic ammonium phosphate, sodium phosphate dibasic, sodium phosphate dibasic, sodium phosphate dibasic, , Dibasic potassium phosphate, and dibasic potassium phosphate. The cerium ion precursor is not particularly limited as long as it is a substance capable of providing trivalent cerium ions, and specifically, it may include at least one selected from the group consisting of cerium nitrate, cerium sulfate, cerium chloride, cerium acetate and cerium carbonate .

The silicon ion precursor according to the present invention is not particularly limited as long as it is a material capable of providing silicon ions, but specifically includes tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate (TPOS) (TiPOS), and tetrabutylososilicate (TBOS). [0035] The term " a "

In the step (c), the ratio of cerium may be from 6 mol% to 18 mol% based on the sum of calcium and cerium constituting the cation, BCP) powder occupies 4.7 wt% to 12.8 wt% based on the total weight of the cerium element. When the ratio of Ca / P is less than 1.585, the strength is lowered, and when it is more than 1.653, the biocompatibility may be deteriorated.

In the step (d), the proportion of silicon may account for 6 to 18 mol% of the combined amount of phosphate and silicon constituting the anion, BCP) powder occupies 1.3% by weight to 4.8% by weight based on the total weight of the silicon element. When the ratio of Ca / P is less than 1.585, the strength is lowered, and when it is more than 1.653, the biocompatibility may be deteriorated.

In the step (e), the polymer binder may include at least one selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone and polyethylene glycol, and the dispersing agent may be at least one selected from the group consisting of ammonium polyacrylate, ammonium polyacrylate- ≪ RTI ID = 0.0 > and / or < / RTI > In particular, in the case of a dispersing agent, a polymer having a large amount of carboxylic acid ammonium salt is suitable.

The bone graft material slurry prepared in the present invention can be produced as a bone graft material by a three-dimensional printing technique. Specifically, the slurry prepared according to the present invention is printed using a three-dimensional printer, the printed slurry is dried at 40 ° C to 50 ° C, sintered at 1,000 to 1,200 ° C for 1 to 3 hours to sinter the polymer binder and dispersant .

When the sintering temperature is lower than 1,000 ° C., the polymer material can not be sufficiently removed and can act as a foreign substance. When the sintering temperature is higher than 1,300 ° C., the pores may be reduced due to over-sintering.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein have been found to be used in the examples or experimental examples of the present invention, some methods and materials have been described. Should not be construed as limiting the invention to the particular methodology, protocols and reagents, as they may be used in various ways in accordance with the context in which those skilled in the art use them.

As used herein, the singular forms include plural objects unless the context clearly dictates otherwise. As used herein, unless otherwise stated, " or " means " and / or ". Furthermore, it is to be understood that other forms, such as " having ", " consisting ", and " consisting "

The present invention will be described in more detail with reference to Examples. However, the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

Example  One

According to the invention Heterosexual  Manufacture of calcium phosphate powder

In order to prepare the binary calcium phosphate (BCP) powder of the present invention, sodium calcium alginate, which is a water-soluble polymer, is dissolved in distilled water at a concentration of 0.03% by weight, and then calcium nitrate solution as a calcium ion precursor is added thereto. And then adjusted to pH 10 with 0.1 N hydrochloric acid or aqueous ammonia. Subsequently, a dibasic ammonium phosphate solution of 0.1 to 0.5 M phosphate ion precursor adjusted to pH 10 was added to a solution containing a polymer template and a calcium ion precursor in a small amount while stirring at 45 캜 so that Ca / P was 1.602 / 1 And the mixture was continuously reacted for 24 hours after completion of the addition to prepare a calcium phosphate powder. Here, Ca / P represents the molar ratio of calcium (Ca) to phosphorus (P).

Example  2

When cerium according to the present invention is partially substituted Heterosexual  Manufacture of calcium phosphate powder

In order to prepare the calcium phosphate (Ce-BCP) powder in which the cerium is partially substituted according to the present invention, firstly, sodium alginate, which is a water-soluble polymer, is dissolved in distilled water in a concentration of 0.03% by weight, and a solution of calcium nitrate The precursor, cerium nitrate solution, was added to adjust the pH to 0.1-0.3 M and then adjusted to pH 10 with 0.1 N hydrochloric acid or aqueous ammonia. (Ca + Ce) / P = 1.602 / 1 was added to a solution containing a polymer template, a calcium ion precursor and a cerium ion precursor in a solution of a dibasic ammonium phosphate solution of 0.1 to 0.5 M phosphate ion precursor having a pH of 10 The mixture was stirred at 45 캜 for a few minutes while stirring. The mixture was continuously reacted for 24 hours after the completion of the addition to prepare a calcium phosphate powder partially substituted with cerium. Herein, the cerium-substituted binary calcium phosphate of Preparation Examples 1 to 3 was prepared in the following manner by varying the amounts of the calcium ion precursor and the cerium ion precursor.

Manufacturing example  One

Preparation of 6 mol% substituted calcium phosphate powder of cerium ion of calcium ion

In the preparation process of the calcium ion precursor and the cerium ion precursor solution in Example 2, a mixed solution was prepared by using 94 mol% of a calcium ion precursor and 6 mol% of a cerium ion precursor, The precursor was added so that Ca / P = 1.602 / 1 to prepare a binary calcium phosphate (Ce6-BCP) powder in which cerium ions were substituted with 6 mol% of calcium ions.

Manufacturing example  2

Preparation of calcium phosphate powder with 18 mol% substitution of cerium ion for calcium ion

In the preparation of the calcium ion precursor and the cerium ion precursor solution in Example 2, a mixed solution was prepared by using 82 mol% of a calcium ion precursor and 18 mol% of a cerium ion precursor, The precursor was added so that Ca / P = 1.602 / 1 to prepare a binary calcium phosphate (Ce18-BCP) powder in which cerium ions were replaced by 18 mol% of calcium ions.

Manufacturing example  3

Preparation of calcium phosphate powder with 30 mol% substitution of cerium ion for calcium ion

In the preparation of the calcium ion precursor and the cerium ion precursor solution in Example 2, a mixed solution was prepared by using 70 mol% of a calcium ion precursor and 30 mol% of a cerium ion precursor in the course of preparing a solution of a calcium ion precursor and a cerium ion precursor, The precursor was added so that Ca / P = 1.602 / 1 to prepare a binary calcium phosphate (Ce30-BCP) powder in which cerium ions were substituted with calcium ions by 30 mol%.

Example  3

When the silicon according to the invention is partially substituted Heterosexual  Manufacture of calcium phosphate powder

In order to prepare the silicon-bicarbonate (Si-BCP) powder in which the silicon of the present invention is partially substituted, first, a calcium nitrate solution as a calcium ion precursor is added to a polymer template solution in which sodium alginate as a water-soluble polymer is dissolved in distilled water at 0.03 wt% 0.1 to 0.3 M and then adjusted to pH 10 with 0.1 N hydrochloric acid or aqueous ammonia. Subsequently, a solution of tetraethylorthosilicate (TEOS), a 0.1 to 0.5M phosphate ion precursor, a dibasic ammonium phosphate solution and a 0.1 to 0.5M silicon ion precursor, pH adjusted to 2, was added to the polymer template (P + Si) = 1.602 / 1 was added to the solution containing the calcium ion precursor at 45 ° C while stirring at a constant rate, and the reaction was continued for 24 hours after the completion of the addition, Calcium powder was prepared. Herein, the binary calcium phosphate of Preparation Examples 4 to 6 in which the amounts of the phosphate ion precursor and the silicate ion precursor were substituted was partially prepared as shown in FIG. 1.

Manufacturing example  4

Silicon ions Phosphate  Ionic 6 mol%  Substituted Heterosexual  Preparation of calcium phosphate powder

In the preparation process of the phosphate ion precursor and the silicon ion precursor solution in the preparation method of Example 3, a mixed solution was prepared by using 94 mol% of a phosphate ion precursor and 6 mol% of a silicon ion precursor, Ionic precursor solution and Ca / (P + Si) = 1.602 / 1 to prepare a binary calcium phosphate (Si6-BCP) powder in which the phosphate ion was replaced by 6 mole% of the silicon ion.

Manufacturing example  5

Preparation of a calcium phosphate powder in which the silicon ion was substituted with 18 mol% of phosphate ion

In the preparation process of the phosphate ion precursor and the silicon ion precursor solution in Example 3, a mixed solution was prepared by using 82 mol% of a phosphate ion precursor and 18 mol% of a silicon ion precursor, Ion precursor solution and Ca / (P + Si) = 1.602 / 1 to prepare a binary calcium phosphate (Si18-BCP) powder in which the phosphate ion was replaced by 18 mole% of the silicon ion.

Manufacturing example  6

Production of a calcium phosphate powder in which a silicon ion is substituted with 30 mol% of a phosphate ion

In the preparation process of the phosphate ion precursor and the silicon ion precursor solution in the preparation method of Example 3, a mixed solution was prepared by using 70 mol% of phosphate ion precursor and 30 mol% of a silicon ion precursor, Ionic precursor solution and Ca / (P + Si) = 1.602 / 1 to prepare a binary calcium phosphate (Si30-BCP) powder in which the phosphate ion was replaced by 30 mole% of the silicon ion.

Example  4

The porous cores in which cerium and silicon are partially substituted according to the present invention Implantable material  Produce

A slurry capable of three-dimensional printing was prepared by mixing the powder obtained in Examples 1 to 3 with polyvinyl alcohol as a polymer binder, an ammonium salt of polyacrylic acid as a dispersant, and distilled water, and printing the scaffold using a three-dimensional printer . After completion of printing, drying was performed at 40 ° C to remove a certain amount of water, and sintering was performed at 1,000 to 1,200 ° C for 2 hours to prepare a porous bone graft having anti-inflammatory activity.

Experimental Example  One

Porous bone graft material morphology analysis using bicomponent calcium phosphate with partially substituted cerium and silicon

FIG. 4 is a graph showing the results of a comparison between the results of Example 1 and Example 3 using the binary calcium phosphate, bicarbonate of partially substituted cerium, and calcium phosphate powder partially substituted with silicone and a polymeric binder and dispersant, (SEM, S-4300, Hitachi) images of porous bone graft materials with antiinflammatory activity, which were prepared by 3D printing according to the method of Sambrook et al. (2004), were confirmed to have porosity.

Experimental Example  2

Elemental Analysis of Porous Bone Implants Made from Barium Barium Phosphate Partially Substituted with Cerium and Silicon

FIG. 5 is a graph showing the results of a comparison between the results of Example 1 and Example 3 using the binary calcium phosphate, bicarbonate of partially substituted cerium, and calcium phosphate powder partially substituted with silicon and a polymeric binder and a dispersant, 4 was analyzed by energy dispersive X-ray spectroscopy (EDX, Horiba, Ltd., 0-20 keV) for porous bone grafts with antiinflammatory activity.

As shown in FIG. 5, peaks attributable to oxygen (O) elements were observed at 0.52 keV in all samples, peaks attributable to phosphorus (P) at 2.02 keV, and peaks attributable to phosphorus (P) The peak appeared. The peak due to the silicon (Si) element appeared at 1.76 keV due to the substitution of silicon and the peak due to the cerium (Ce) element appeared at 4.86 keV due to the substitution of cerium, It was confirmed that the preparation of porous bone graft material using calcium phosphate monobasic was successfully performed.

Experimental Example  3

In the present invention, cerium and silicon are partially substituted Heterosexual  Porous cores made of calcium phosphate Graft  Surface absorbance analysis

FIG. 6 is a graph showing the results of a comparison between the results of Example 1 and Example 3 using the binary calcium phosphate, the partially substituted bicarbonate of cerium, and the calcium phosphate powder partially substituted with silicone and the polymeric binder and dispersant, (ATR-FTIR, ALPHA, Bruker optics) in the range of 400 ~ 4000 cm ^-1 for the total absorbance of porous bone graft materials with antiinflammatory activity, which were prepared by 3D printing according to the method of Fourier Transform Infrared Spectroscopy Respectively.

As shown in FIG. 6, peaks attributed to phosphate ions (PO ₄ ^3- ) were observed at 1093, 1025 and 989 cm ^-1 in all the samples, and peaks due to Ce-O were found at 445 cm ^-1 And a peak due to Si-O-Si was confirmed at 880 cm ^-1 according to the substitution of silicon.

Experimental Example  4

In the present invention, cerium and silicon are partially substituted Heterosexual  Porous cores made of calcium phosphate Graft  Evaluation of compressive strength

(Manufactured in Group 1), bicomponent calcium phosphate (manufactured in Group 2) partially substituted with cerium and a bicomponent calcium phosphate (manufactured in Group 3) in which silicon was partially substituted were synthesized according to Examples 1 to 3, (Control 1) and Ca / P = 1.551 (calcium phosphate) (control 2) powder with Ca / P = 1.67 as a control and the polymer binder and a dispersant were subjected to three-dimensional printing Table 1 shows the results of measurement of the compressive strength of porous bone graft materials with anti-inflammatory activity using a universal testing machine (UTM, University Testing Mschine, Test One, TO-101).

Ca / P ratio Compressive strength Manufacturing group 1 1.602 4.3 MPa Manufacturing group 2 1.602 4.7 MPa Manufacturing group 3 1.602 4.6 MPa Control 1 1.67 5.1 MPa Control group 2 1.551 2.1 MPa

As shown in Table 1, the compressive strength of the porous bone graft material prepared in Examples and the bone graft material made of hydroxyapatite was high but the compressive strength was significantly reduced in the control 2 with a Ca / P ratio of less than 1.585 Respectively.

Experimental Example  5

Evaluation of cell proliferation ability of porous bone graft made of calcium phosphate with partially substituted silicone and cerium

FIG. 7 is a graph showing the results of the comparison between the two types of calcium phosphate, bicarbonate calcium phosphate, calcium phosphate and hydroxyapatite (control 1) partially substituted with cerium, And the dispersibility of the porous bone graft material having the anti-inflammatory activity prepared by the three-dimensional printing method according to Example 4 above. As the cells, osteogenic precursor cells (MC3T3-E1, ATCC) were used and α-MEM supplemented with fetal bovine serum (FBS) and 1% penicillin-streptomycin was used as a cell culture medium. Porous bone graft materials were sterilized by autoclaving using high-temperature autoclave, and then sterilized with 75%, 50%, 25% ethanol and washed twice with phosphate-buffered saline (PBS) to obtain 48-well cell culture plates . The osteogenic precursor cells to 2 × 10 ⁴ in a concentration of cells / well dispensed in a bone grafting material and each incubated in 37 ℃, 5% CO ₂ environment for 4 hours, 1, 3, 5 and 7 days, adhesion and proliferation of cells The ability was evaluated using the MTT method and the results are shown in FIG.

7, the early attachment of osteogenic precursor cells and cell proliferation were the fastest in the bone graft made of calcium phosphate with a partial substitution of cerium, and the bone graft material made of calcium phosphate And that the proliferation rate of hydroxyapatite bone graft material was the slowest. This results from the fact that the proliferation behavior of the cells is enhanced because the substituted cerium and silicon stimulate the osteogenic precursor cells to improve the proliferation power and show some anti-inflammatory and antimicrobial properties.

Experimental Example  6

The present invention relates to the evaluation of the anti-inflammatory properties of porous bone grafts made of bicarbonate calcium phosphate with partially substituted cerium and silicon

FIG. 8 is a graph showing the results obtained by using the two-component calcium phosphate, bicomponent calcium phosphate partially substituted with cerium, and calcium phosphate powder partially substituted with silicon and a polymeric binder and dispersant synthesized according to Examples 1 to 3, 4 shows the results of evaluating the anti-inflammatory activity of the porous bone graft materials prepared by the three-dimensional printing method. The anti - inflammatory properties of porous bone graft materials were evaluated by RAW264.7 cells, a mouse - derived macrophage induced by inflammatory response with lipopolysaccharide (LPS). Murine macrophage cell line RAW264.7 cells were adjusted to 1 × 10 ⁵ cells / well using DMEM medium containing penicillin (100 IU / ml) and streptomycin (100 μg / ml) and 10% FBS , A 48-well cell culture plate containing the inventive porous bone graft material prepared according to the above method, and pre-cultured at 5% CO ₂ and 37 ° C for 48 hours. Interleukin-6 (IL-6) and interleukin-1β (IL-1β), which are inflammatory factors, were measured on ELISA kit (R & D Systems).

8, the production of IL-6 and IL-1β was most inhibited in the porous bone graft material prepared by using the cerium-partially substituted biphosphate calcium phosphate, One porous bone graft material showed better anti - inflammatory activity than the bone graft material.

Based on these results, in order to investigate the anti-inflammatory activity according to the substitution ratio of cerium or silicon, the substitution ratio of cerium and silicon was changed from three cerium-substituted porous bone graft materials to three silicone substitution materials Porous bone graft material was prepared and the amount of IL-6 produced was measured and shown in FIG.

From FIG. 9, it was confirmed that when cerium was substituted up to 18 mol% of calcium, the anti-inflammatory property was improved by increasing the concentration of cerium element. However, when cerium was replaced by 30 mol% of calcium, And the inflammation was found to be poor. In addition, in the case of silicon, as in the case of cerium, it was confirmed that the anti-inflammatory property was improved by increasing the concentration of the silicon element when the phosphate was substituted by up to 18 mol% of silicon. However, when the silicon was replaced by 30 mol% And the anti-inflammatory properties were decreased.

From this result, it can be seen that the substitution ratio range in which cerium and silicon have antiinflammatory properties is 6 to 18 mol%, and this value is in the range of 4.7 to 12.8% by weight based on the total weight of the cerium element in the calcium phosphate (Ce-BCP) (Si-BCP) in which silicon is partially substituted, and the silicon element occupies 1.0 wt% to 3.1 wt% with respect to the total weight.

Claims (11)

(a) preparing a polymer template solution by dissolving a water-soluble polymer in distilled water;
(b) providing a calcium ion precursor solution, a phosphate ion precursor, a cerium ion precursor and a silicon ion precursor, respectively;
(c) adding the calcium ion precursor solution, phosphate ion precursor solution and cerium ion precursor solution (Ca + Ce) solution to the polymer template solution so as to produce a binary calcium phosphate mixed with hydroxyapatite and tricalcium phosphate in a ratio of 6: / P = 1.585 / 1 to 1.653 / 1, stirring the mixture, centrifuging and drying to prepare a calcium phosphate powder having a partial substitution of cerium (Ce-BCP);
(d) adding the calcium ion precursor solution, the phosphate ion precursor solution and the silicon ion precursor solution to (Ca + Ce) solution so that the polymer template solution is mixed with 6: 4 hydroxyapatite and tricalcium phosphate, /P=1.585/1 to 1.653 / 1, followed by stirring, centrifugation and drying to prepare a calcium phosphate powder having a partially substituted silicone (Si-BCP);
(e) mixing the dried calcium cerium or silicon-partially substituted calcium phosphate powder obtained in the above step with a polymer binder, a dispersing agent and distilled water to form a slurry. A method for manufacturing a calcium phosphate-based porous bone graft material.
The method according to claim 1,
Wherein the water-soluble polymer for a polymer template in step a) comprises at least one selected from the group consisting of sodium alginate, beta-cyclodextrin, polyvinylpyrrolidone, polyvinyl alcohol, hyaluronic acid, laminarin, pullulan and fucoidan, A method for producing an antiinflammatory active diabody calcium phosphate based porous bone graft material.
The method according to claim 1,
Wherein the calcium ion precursor in step b) comprises at least one member selected from the group consisting of calcium nitrate, calcium chloride, calcium sulfate, and calcium hydroxide.
The method according to claim 1,
Wherein the phosphate ion precursor in step b) is selected from the group consisting of ammonium phosphate diphosphate, ammonium diphosphate, sodium phosphate dibasic, sodium phosphate dibasic, sodium dibasic, potassium dibasic, potassium dibasic and potassium dibasic. Wherein the porous bone graft material comprises at least one selected from the group consisting of calcium phosphate and calcium phosphate.
The method according to claim 1,
Wherein the cerium ion precursor in step b) comprises at least one selected from the group consisting of cerium nitrate, cerium sulfate, cerium chloride, cerium cerium and cerium carbonate, a method for producing an anti-inflammatory active type calcium phosphate porous bone graft material .
The method according to claim 1,
The silicon ion precursor in step b) may be selected from the group consisting of tetramethylorthosilicate (TMOS), tetraethylorthosilicate (TEOS), tetrapropylososilicate (TPOS), tetraisopropylorthosilicate (TiPOS) and tetrabutylorthosilicate Wherein the porous calcium phosphate-based porous bone graft material comprises at least one selected from the group consisting of calcium phosphate, calcium phosphate, and calcium phosphate.
The method according to claim 1,
In the step (c), the ratio of cerium accounts for 6 to 18 mol% of the sum of calcium and cerium constituting the cation, and in the case of calcium diacid phosphate partially substituted with cerium, the cerium element is 4.7 wt% % To 12.8% by weight of an anti-inflammatory active biocompatible calcium phosphate-based porous bone graft material.
The method according to claim 1,
In the step (d), the proportion of silicon accounts for from 6 mol% to 18 mol% in the sum of the phosphate and silicon constituting the anion, and in the case of the partially substituted silicon dioxide, the silicon element is 1.3 wt% To 4.8% by weight based on the weight of the porous calcium phosphate-based bone graft material.
The method according to claim 1,
Wherein the polymeric binder comprises at least one selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, and polyethylene glycol in the step (e).
The method according to claim 1,
In the step (e), the dispersing agent comprises at least one member selected from the group consisting of ammonium polyacrylate, ammonium polyacrylate-maleic acid copolymer ammonium salt and polygamma glutamate ammonium salt. Way.
A method for producing a color photographic material, comprising the steps of: printing using a three-dimensional printer using the slurry prepared according to any one of claims 1 to 11; drying the printed slurry at 40 DEG C and polymer binder and dispersing agent at 1,000 to 1,200 DEG C for 2 hours Sintering the porous calcium phosphate-based biocompatible porous bone graft material.

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