KR102080628B1 - A manufacturing method of the porous biphasic calcium phosphate bone substitute materials having anti-inflammatory activity - Google Patents

Abstract

The present invention is a bicomponent calcium phosphate powder used in the preparation of a bicomponent calcium phosphate porous bone graft having excellent anti-inflammatory activity by using a three-dimensional printing technique cerium (Ce) or silicon (Si) having anti-inflammatory activity It is to provide a porous bone graft prepared by using a three-dimensional printing technique and a method for producing a porous bone graft prepared using these powders and water-soluble polymer binders and dispersants synthesized by the substitution of elements.

Description

Method for manufacturing anti-inflammatory active bicomponent calcium phosphate porous bone graft {A MANUFACTURING METHOD OF THE POROUS BIPHASIC CALCIUM PHOSPHATE BONE SUBSTITUTE MATERIALS HAVING ANTI-INFLAMMATORY ACTIVITY}

The present invention relates to a method for producing a bicomponent calcium phosphate-based porous bone graft slurry having excellent anti-inflammatory activity, and more particularly, a binary component in which some cerium (Ce) or silicon (Si) elements having inflammatory activity are partially substituted. The present invention relates to a method for producing a bone graft using a calcium phosphate-based powder, a water-soluble polymer binder and a dispersant, and to a bone graft prepared by a three-dimensional printing technique using the same.

Bone grafts are widely used to treat large and severe bone defects due to trauma or resection. Bone grafts used in such bone grafts can be broadly classified into autologous bone grafts, allogeneic bone grafts, xenograft grafts, and synthetic bone grafts. Biomaterials have been in the spotlight in that they can be manufactured in various forms that are advantageous in selecting materials and structurally producing bones.

Among the calcium phosphate-based materials mainly used as synthetic bone graft materials, hydroxyapatite having excellent biocompatibility and tricalcium phosphate showing biodegradability have been studied as biomaterials. However, hydroxyapatite has a disadvantage of slow biodegradation rate, and tricalcium phosphate has a problem of dissolving before bone recovery because of its weak strength and fast decomposition and absorption. In order to solve these problems, biphasic calcium phosphate (BCP), which is a mixture of hydroxyapatite and tricalcium phosphate, has been developed, and an appropriate combination of hydroxyapatite which is stable in vivo and tricalcium phosphate with excellent bioabsorption has been developed. It is known to exhibit a much better effect functionally by playing a role of complementing the strengths and weaknesses of each other, but there is a disadvantage that hydroxyapatite and tricalcium phosphate must be prepared and used, respectively.

In addition, in the case of these two-component calcium phosphate, there is a possibility of causing an inflammatory reaction if the powder form is used as it is, and in most cases, a mold of a specific shape is produced, and a mixture of calcium phosphate powder and a polymer is injected thereinto form Making bone grafts is a common method for making porous bone grafts. However, the bone graft prepared by this method has a disadvantage in that the shape and size are limited, and many molds are required to prepare bone grafts of various shapes and sizes.

Recently, three-dimensional printing technology has been used to manufacture porous bone grafts, and this technology enables the production of bone grafts having a three-dimensional structure with high precision and accuracy. These bone grafts can provide a uniform and consistent pore size, structure and thickness of each graft compared to conventional bone grafts, providing a surface area favorable for cell attachment and proliferation, as well as bones of various sizes and shapes. The advantage is that the preparation of the implant is possible. In order to manufacture bone graft using 3D printing technique, ceramic powder is mixed with polymer and printed and then used as a composite material mixed with polymer (see Patent Document 1). There is (refer patent document 2).

Although the above structure can be used as a bone graft material, infection may occur when the bone graft material is used in a bone defect site, and thus, a development of a bone graft material containing an antimicrobial agent has recently been attempted (see Patent Document 3). However, these antimicrobial agents are likely to be released at a rapid rate early, and there is a problem in that there is no method of introducing an antimicrobial agent in a bone graft composed only of ceramics.

Therefore, in order to improve these problems, a method of preparing a bicomponent calcium phosphate powder containing a hydroxyapatite component and a tricalcium phosphate component, a method of preparing porous bone grafts of various shapes and sizes, and anti-inflammatory which can be sustained for a long time Development of a method to grant this is necessary.

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

The present invention has been made to solve the problems raised above, by using a bicomponent calcium phosphate-based powder and a water-soluble polymer binder and dispersant partially substituted with cerium (Ce) and silicon (Si) elements having anti-inflammatory activity It is to provide a porous bone graft prepared using a method for producing a bone graft and a three-dimensional printing technique.

The object of the present invention is

(a) dissolving a water-soluble polymer in distilled water to prepare a polymer template solution;

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

(c) the calcium ion precursor solution, the phosphate ion precursor solution, and the cerium ion precursor solution to produce a bicomponent calcium phosphate containing hydroxyapatite and tricalcium phosphate in a 9: 1 to 5: 5 molar ratio in the polymer template solution; (Ca + Ce) /P=1.585/1 to 1.653 / 1 in a molar ratio, followed by stirring, centrifugation and drying to prepare a bicomponent calcium phosphate powder in which cerium was partially substituted (Ce-BCP);

(d) the calcium ion precursor solution, the phosphate ion precursor solution, and the silicon ion precursor solution to produce a bicomponent calcium phosphate containing hydroxyapatite and tricalcium phosphate in a ratio of 9: 1 to 5: 5 in the polymer template solution. Ca / (P + Si) = 1.585 / 1 to 1.653 / 1 in a molar ratio, followed by stirring, centrifugation and drying to prepare a bicomponent calcium phosphate powder in which silicon was partially substituted (Si-BCP);

(e) mixing the dried cerium or silicon partially substituted bicomponent calcium phosphate powder obtained in the step with a polymer binder, a dispersant and distilled water to prepare a slurry, wherein the porous bone graft having an anti-inflammatory activity It is to provide a manufacturing method.

Another object of the present invention

The prepared slurry was printed using a three-dimensional printer, dried at 40 ° C. to 50 ° C., and then sintered with a polymer binder and a dispersant at 1,000˜1,200 ° C. for 1 to 3 hours. To provide a porous bone graft.

The present invention is a porous bone prepared by a three-dimensional printing technique after mixing a powder, a polymer binder and a dispersant synthesized by partially substituting cerium or silicon elements with a bicomponent calcium phosphate having excellent biocompatibility and controlled biodegradability and mechanical properties. Implants have an excellent effect on improving anti-inflammatory activity.

In addition, when used in dentistry and orthopedics for bone regeneration greatly improve the initial adhesion and proliferation of bone cells, and has an excellent effect of greatly improving the anti-inflammatory properties there is an advantage that can be applied to the field of dentistry and orthopedic.

1 is a bicomponent calcium phosphate (BCP) powder according to the present invention, the bicomponent calcium phosphate (Ce-BCP) powder partially substituted with cerium and the bicomponent calcium phosphate (Si-BCP) powder partially substituted with silicon This is a schematic diagram of a method for producing porous bone graft with anti-inflammatory activity using a method and a three-dimensional printer.
2 is 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-BCP) powder partially substituted with silicon Is a photograph taken at 50,000 magnification by scanning electron microscope.
3 is 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-BCP) powder partially substituted with silicon Digital and scanning electron microscopy images of porous bone grafts with anti-inflammatory activity prepared using
Figure 4 is a graph of the elemental analysis of the anti-inflammatory active porous bone graft prepared according to the present invention using energy dispersive X-ray spectroscopy.
Figure 5 is a graph of the surface analysis of the anti-inflammatory activity porous bone graft prepared according to the present invention using infrared spectroscopy.
6 is a graph showing the results of cell proliferation of the porous bone graft with anti-inflammatory activity prepared according to the present invention.
7 is a graph showing the results of measuring the inflammatory activity of the anti-inflammatory activity of the porous bone graft prepared according to the present invention.
FIG. 8 is a measurement of inflammatory activity according to the substitution rate of cerium or silicon of partially substituted calcium phosphate (Ce-BCP) and partially substituted calcium phosphate (Si-BCP) partially substituted according to the present invention. A graph showing the results.

Method for producing a porous bone graft having anti-inflammatory activity according to the present invention

(a) dissolving a water-soluble polymer in distilled water to prepare a polymer template solution;

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

(c) the calcium ion precursor solution, the phosphate ion precursor solution, and the cerium ion precursor solution to produce a bicomponent calcium phosphate containing hydroxyapatite and tricalcium phosphate in a 9: 1 to 5: 5 molar ratio in the polymer template solution; To (Ca + Ce) / P = 1.585 / 1 to 1.653 / 1 molar ratio was added, stirred, centrifuged and dried to prepare a bicomponent calcium phosphate powder substituted with cerium (Ce-BCP);

(d) the calcium ion precursor solution, the phosphate ion precursor solution, and the silicon ion precursor solution to produce a bicomponent calcium phosphate containing hydroxyapatite and tricalcium phosphate in a ratio of 9: 1 to 5: 5 in the polymer template solution. Ca / (P + Si) = 1.585 / 1 to 1.653 / 1 in a molar ratio, followed by stirring, centrifugation and drying to prepare a bicomponent calcium phosphate powder in which silicon was partially substituted (Si-BCP);

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

According to the present invention for achieving the above object (a) the water-soluble polymer for the polymer template is composed of sodium alginate, beta-cyclodextrin, polyvinylpyrrolidone, polyvinyl alcohol, hyaluronic acid, laminarin, pullulan and fucoidan It may include one or more selected from the group.

The calcium ion precursor in step (b) is not particularly limited as long as it is a material capable of providing calcium ions. Specifically, the calcium ion precursor may include one or more 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 phosphate ions, and specifically, the first ammonium phosphate, the second ammonium phosphate, the first sodium phosphate, the second sodium phosphate, the third sodium phosphate, and the first potassium phosphate It may include one or more selected from the group consisting of dipotassium potassium phosphate and tertiary potassium phosphate. The cerium ion precursor is not particularly limited as long as it is a material capable of providing trivalent cerium ions, but may specifically include one or more 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. Specifically, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate (TPOS), tetra It may include one or more selected from the group consisting of isopropyl orthosilicate (TiPOS) and tetrabutyl orthosilicate (TBOS).

In the step (c), the ratio of cerium may occupy 6 mol% to 18 mol% of the combined value of calcium and cerium constituting the cation, and this value is bicomponent calcium phosphate (Ce- BCP) is the same as the cerium element occupies 4.7% to 12.8% by weight of the total weight in the powder. When the Ca / P ratio is less than 1.585, the strength may be lowered. When the Ca / P ratio is larger than 1.653, the biocompatibility may be lowered.

In the step (d), the ratio of silicon may occupy 6 mol% to 18 mol% of the sum of the phosphate and the silicon constituting the anion, and this value is a bicomponent calcium phosphate (Si- BCP) powder is equivalent to the silicon element accounts for 1.3% to 4.8% by weight based on the total weight. When the Ca / P ratio is less than 1.585, the strength may be lowered. When the Ca / P ratio is larger than 1.653, the biocompatibility may be lowered.

In the step (e), the polymer binder may include at least one selected from polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol, and the dispersing agent is ammonium polyacrylate, polyacrylic acid-maleic acid copolymer ammonium salt, and polygamma glutamate ammonium salt. It may include one or more selected from the group consisting of. Especially in the case of a dispersant, a polymer having a large amount of ammonium carboxylic acid salt is suitable.

The bone graft slurry prepared in the present invention can be made into a bone graft 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 ℃ to 50 ℃ and sintered polymer binder and dispersant at 1,000 ~ 1,200 ℃ for 1 to 3 hours It is prepared through the step of.

When the sintering temperature is lower than 1,000 ° C, the polymer material may not be sufficiently removed to act as a foreign matter. When the sintering temperature is higher than 1,300 ° C, the sintering may occur, thereby reducing the pores.

Unless defined otherwise herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art. Various scientific dictionaries that include the terms included herein are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the Examples or Experimental Examples of the present invention, some methods and materials have been described. Depending on the context used by those of ordinary skill in the art, they may be used in a variety of ways, and therefore should not be construed as limiting the invention to particular methodologies, protocols and reagents.

As used herein, the singular encompasses the plural objects unless the context clearly dictates otherwise. As used herein, "or" means "and / or" unless stated otherwise. Moreover, the terms “comprising” as well as other forms, such as “having”, “consisting of” and “consisting of” are not limiting.

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

Example  One

According to the invention Two-component  Calcium Phosphate Powder Manufacturing

In order to prepare the bicomponent calcium phosphate (BCP) powder of the present invention, calcium nitrate solution, which is a calcium ion precursor, was added to a polymer template solution in which sodium alginate, a water-soluble polymer, was dissolved in distilled water at 0.03% by weight, to be 0.1-0.3 M. After that, the pH was adjusted to 10 with 0.1N hydrochloric acid or ammonia water. Subsequently, the second ammonium phosphate solution, a 0.1-0.5 M phosphate ion precursor having a pH of 10, was stirred at 45 ° C. to a Ca / P = 1.602 / 1 mole ratio in a solution containing the polymer template and the calcium ion precursor. A small amount was added, and the reaction was continued for 24 hours after the addition was completed to prepare a bicomponent calcium phosphate powder. Here, Ca / P represents the molar ratio of calcium (Ca) to phosphorus (P).

Example  2

Partially substituted cerium according to the present invention Two-component  Calcium Phosphate Powder Manufacturing

In order to prepare a bicomponent calcium phosphate (Ce-BCP) powder partially substituted with cerium of the present invention, calcium nitrate solution and cerium ion, which are calcium ion precursors, are dissolved in a polymer template solution in which sodium alginate, a water-soluble polymer, is dissolved in distilled water at 0.03% by weight. After adding a cerium nitrate solution as a precursor to 0.1 ~ 0.3M to adjust the pH to 10 to 0.1N hydrochloric acid or ammonia water. Subsequently, a solution of the second ammonium phosphate, a 0.1-0.5 M phosphate ion precursor with a pH of 10, was added to the solution containing the polymer template, the calcium ion precursor, and the cerium ion precursor (Ca + Ce) / P = 1.602 / 1 mol A small amount was added with stirring at 45 ° C. to a ratio, and the reaction was continued for 24 hours to prepare a bicomponent calcium phosphate powder in which cerium was partially substituted. Herein, the two-component calcium phosphate substituted with cerium in Preparation Examples 1 to 3 by varying the amounts of the calcium ion precursor and the cerium ion precursor was prepared as shown in FIG. 1.

Production Example  One

Preparation of Bicomponent Calcium Phosphate Powders with Cerium Ion Substituted 6 Mole% of Calcium Ions

In the preparation method of the calcium ion precursor and the cerium ion precursor solution in Example 2, 94 mol% of the calcium ion precursor was used, and 6 mol% of the cerium ion precursor was used to prepare a mixed solution. A precursor was added to a Ca / P = 1.602 / 1 molar ratio to prepare a bicomponent calcium phosphate (Ce6-BCP) powder in which cerium ions substituted 6 mol% of calcium ions.

Production Example  2

Preparation of Binary Calcium Phosphate Powders with 18 Mole Percent Substituted Calcium Ions

In the preparation method of the calcium ion precursor and the cerium ion precursor solution of Example 2, a mixed solution was prepared using 82 mol% of calcium ion precursor and 18 mol% of cerium ion precursor, and phosphate ions. The precursor was added in a Ca / P = 1.602 / 1 molar ratio to prepare a bicomponent calcium phosphate (Ce18-BCP) powder in which cerium ions substituted 18 mol% of calcium ions.

Production Example  3

Preparation of Binary Calcium Phosphate Powders with Cerium Ions Substituted 30 Mole% of Calcium Ions

In the preparation method of the calcium ion precursor and the cerium ion precursor solution of Example 2, a mixed solution was prepared using 70 mol% of calcium ion precursor and 30 mol% of cerium ion precursor, and phosphate ions. The precursor was added in a Ca / P = 1.602 / 1 molar ratio to prepare a bicomponent calcium phosphate (Ce30-BCP) powder in which cerium ions replaced 30 mol% of calcium ions.

Example  3

Partially substituted silicone according to the present invention Two-component  Calcium Phosphate Powder Manufacturing

To prepare a bicomponent calcium phosphate (Si-BCP) powder in which the silicon of the present invention is partially substituted, first, a calcium nitrate solution, which is a calcium ion precursor, is added to a polymer template solution in which sodium alginate, a water-soluble polymer, is dissolved in distilled water at 0.03% by weight. After adjusting to 0.1 ~ 0.3M pH was adjusted to 10 to 0.1N hydrochloric acid or ammonia water. Subsequently, the second ammonium phosphate solution of 0.1-0.5 M phosphate ion precursor adjusted to pH 10 and the tetraethyl orthosilicate (TEOS) solution of 0.1-0.5 M silicon ion precursor adjusted to pH 2 were mixed with a polymer template. A small amount of silicon is substituted by adding a small amount of stirring at 45 ° C. to a Ca / (P + Si) = 1.602 / 1 molar ratio, and continuously reacting for 24 hours after the addition was completed. Powdered calcium phosphate powder was prepared. Here, the two-component calcium phosphate in which the silicon of Preparation Examples 4 to 6 was partially substituted by varying the amounts of the phosphate ion precursor and the silicide ion precursor was prepared as shown in FIG. 1.

Production Example  4

Silicon ions Phosphate  Ionic 6 mol%  Substituted Two-component  Preparation of Calcium Phosphate Powder

In the process of preparing the phosphate ion precursor and the silicon ion precursor solution in the manufacturing method of Example 3, a mixed solution was prepared using 94 mol% of phosphate ion precursor and 6 mol% of silicon ion precursor, and the solution was calcium A bicomponent calcium phosphate (Si6-BCP) powder was prepared in which the ion precursor solution was added at a Ca / (P + Si) = 1.602 / 1 molar ratio, in which 6 mol% of silicon ions substituted phosphate ions.

Production Example  5

Preparation of Bicomponent Calcium Phosphate Powders with Silicone Ions Substituted with 18 Mole Percent of Phosphate Ions

In the process of preparing the phosphate ion precursor and the silicon ion precursor solution in the preparation method of Example 3, a mixed solution was prepared by using 82 mol% of phosphate ion precursors and 18 mol% of silicon ion precursors, and the solution was calcium A bicomponent calcium phosphate (Si18-BCP) powder in which silicon ions substituted 18 mol% of phosphate ions was prepared by adding an ion precursor solution to a Ca / (P + Si) = 1.602 / 1 molar ratio.

Production Example  6

Preparation of Binary Calcium Phosphate Powders with Silicon Ions Substituted 30 Mole% of Phosphate Ions

In the process of preparing the phosphate ion precursor and the silicon ion precursor solution in the preparation method of Example 3, a mixed solution was prepared using 70 mol% of phosphate ion precursors and 30 mol% of silicon ion precursors. A bicomponent calcium phosphate (Si30-BCP) powder was prepared in which the ion precursor solution and Ca / (P + Si) = 1.602 / 1 molar ratio were added so that the silicon ions substituted the phosphate ions by 30 mol%.

Example  4

Porous bone partially substituted with cerium and silicon according to the present invention Implants  Produce

After mixing the powder obtained from Examples 1 to 3, polyvinyl alcohol as a polymer binder, polyammonium ammonium salt as a dispersant, and distilled water to prepare a slurry capable of three-dimensional printing, a scaffold was printed using a three-dimensional printer. . After the completion of printing, dried at 40 ℃ to remove a certain amount of moisture and then sintered at 1,000 ~ 1,200 ℃ for 2 hours to prepare a porous bone graft with anti-inflammatory activity, the production method is shown in Figure 3.

Experimental Example  One

Morphological Analysis of Porous Bone Graft Formed from Bicomponent Calcium Phosphate Substituted with Cerium and Silicon Partially

4 is an example using bicomponent calcium phosphate synthesized according to Examples 1 to 3, bicomponent calcium phosphate partially substituted with cerium and bicomponent calcium phosphate powder partially substituted with silicon, a polymer binder and a dispersant Digital and kettle microscopy (SEM, S-4300, Hitachi) images of anti-inflammatory active bone grafts prepared by the three-dimensional printing method according to Figure 4 was confirmed to have a porosity.

Experimental Example  2

Elemental Analysis of Porous Bone Grafts Prepared with Bicomponent Calcium Phosphate Substituted Partially with Cerium and Silicon

5 is an example using bicomponent calcium phosphate synthesized according to Examples 1 to 3, bicomponent calcium phosphate partially substituted with cerium and bicomponent calcium phosphate powder partially substituted with silicon, a polymer binder and a dispersant Porous bone graft with anti-inflammatory activity prepared by the three-dimensional printing method according to 4 was subjected to elemental analysis using energy dispersive X-ray spectroscopy (EDX, Horiba, Ltd., 0-20 keV).

As shown in FIG. 5, a peak attributable to oxygen (O) was found at 0.52 keV and a peak attributable to phosphorus (P) at 2.02 keV, and attributable to a calcium (Ca) at 3.72keV. Peaks appeared. In addition, the peak attributable to the silicon (Si) element was found to be 1.76 keV by the substitution of silicon, and the peak attributable to the cerium (Ce) element was represented at 4.86 keV by the substitution of cerium. It was confirmed that the preparation of porous bone graft using calcium phosphate was successful.

Experimental Example  3

Partially substituted cerium and silicon of the present invention Two-component  Porous bone made of calcium phosphate Of implant  Surface Absorbance Analysis

6 is an example using bicomponent calcium phosphate synthesized according to Examples 1 to 3, bicomponent calcium phosphate partially substituted with cerium and bicomponent calcium phosphate powder partially substituted with silicon, a polymer binder and a dispersant Total Absorption Measurement of Surface Absorption of Anti-Inflammatory Porous Bone Grafts Prepared by Three-Dimensional Printing Method According to Fig. 4 Analysis in the range of 400 ~ 4000 cm ^-1 using Fourier transform infrared spectroscopy It was.

As shown in FIG. 6, peaks attributable to phosphate ions (PO ₄ ³⁻ ) were found at 1093, 1025 and 989 cm ⁻¹ in all samples, and peaks attributable to Ce—O depending on the substitution of cerium were 445 cm ^−1. The peak attributable to Si-O-Si was found at 880 cm ⁻¹ depending on the substitution of silicon.

Experimental Example  4

Partially substituted cerium and silicon of the present invention Two-component  Porous bone made of calcium phosphate Of implant  Compressive strength evaluation

Binary calcium phosphate (manufacturing group 1) synthesized according to Examples 1 to 3, bicomponent calcium phosphate (manufacturing group 2) partially substituted with cerium and bicomponent calcium phosphate (manufacturing group 3) partially substituted with silicon As a powder and a control, hydroxyapatite (control group 1) having Ca / P = 1.67 and binary calcium phosphate (control group 2) powder having Ca / P = 1.551 were three-dimensionally printed according to Example 4 using a polymer binder and a dispersant. The compressive strength of the anti-inflammatory active bone graft prepared by the method was measured using a universal testing machine (UTM, University Testing Mschine, Test One, TO-101), and the results are shown in [Table 1].

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 group 1 1.67 5.1 MPa Control 2 1.551 2.1 MPa

Referring to Table 1, it was confirmed that the compressive strength of the porous bone graft prepared in the example and the bone graft prepared from the hydroxyapatite was high in the case of the control group 2 having a high compressive strength but a Ca / P ratio of less than 1.585. It was.

Experimental Example  5

Evaluation of Cell Proliferative Capacity of Porous Bone Grafts Prepared with Bicomponent Calcium Phosphate Substituted with Cerium and Silicon Partially

7 is a bicomponent calcium phosphate synthesized according to Examples 1 to 3, bicomponent calcium phosphate partially substituted with cerium and bicomponent calcium phosphate and hydroxyapatite (control 1) powder partially substituted with silicone and a polymer binder And the results of evaluating the cell proliferation of the anti-inflammatory activity porous bone graft prepared by the three-dimensional printing method according to Example 4 using a dispersant. Osteoblastic progenitor cells (MC3T3-E1, ATCC) were used as cells, and α-MEM with fetal calf serum (FBS) and 1% penicillin-streptomycin was used as cell culture medium. Porous bone grafts were sterilized using autoclave, sterilized with 75%, 50% and 25% ethanol, and washed twice with phosphate buffered saline (PBS) for 48-well cell culture plates. Put in. The osteoblastic progenitor cells were dispensed into the bone graft at a concentration of 2 × 10 ⁴ cells / well, and cultured for 4 hours, 1, 3, 5, and 7 days at 37 ° C. and 5% CO ₂ , respectively to attach and proliferate the cells. The ability was evaluated using the MTT method and the results are shown in FIG.

The initial attachment and cell proliferation of osteoblastic progenitor cells from FIG. 7 occurred most rapidly in bone grafts made of bicomponent calcium phosphate partially substituted with cerium, and cells rather than bone grafts made of bicomponent calcium phosphate partially substituted with silicon. It was confirmed that the initial adhesion and proliferation of the fast progress, and the slowest cell proliferation rate in the bone graft made of hydroxyapatite. This is the result of more active proliferation behavior of the cells because the substituted cerium and silicon stimulate the osteogenic progenitor cells to improve proliferative capacity, and also show some anti-inflammatory and antimicrobial properties.

Experimental Example  6

Anti-inflammatory Evaluation of Porous Bone Grafts Prepared with Bicomponent Calcium Phosphate Partially Substituted with Cerium and Silicon

8 is a bicomponent calcium phosphate synthesized according to Examples 1 to 3, bicomponent calcium phosphate partially substituted with cerium and bicomponent calcium phosphate powder partially substituted with silicon, a polymer binder, and a dispersant. According to 4, the anti-inflammatory activity of the porous bone graft prepared by the three-dimensional printing method was evaluated. The anti-inflammatory properties of the porous bone graft were pulsated using RAW264.7 cells, which are mouse-derived phagocytic cells induced by inflammatory reactions with lipopolysaccharide (LPS). RAW264.7 cells, a Murine macrophage cell line, were adjusted to 1 × 10 ⁵ cells / well using DMEM medium containing penicillin (100 IU / mL) and streptomycin (100 μg / mL) and 10% FBS. , 48-well cell culture plate containing the porous bone graft of the present invention prepared according to the above method, and incubated for 48 hours at 5% CO ₂ and 37 ℃. Inflammatory factors, interleukin-6 (IL-6) and interleukin-1β (IL-1β), generated at this time were dispensed on the plate to which these protein antibodies were attached, and then measured by ELISA kit (R & D Systems).

From FIG. 8, the production of IL-6 and IL-1β was most suppressed in the porous bone graft prepared using cerium-substituted bicomponent calcium phosphate, and also prepared using silicon-substituted bicomponent calcium phosphate. One porous bone graft also showed superior anti-inflammatory activity compared to the bicomponent bone graft.

Based on these results, to determine the anti-inflammatory activity according to the substitution rate of cerium or silicon, three cerium-substituted porous bone grafts and three silicon-substituted substitutions were obtained by varying the amount of cerium and silicon used as in Preparation Examples 1 to 6. Porous bone grafts were prepared to measure the amount of IL-6 produced and are shown in FIG. 9.

9, it was confirmed that the anti-inflammatory properties of the cerium in the case of the substitution of up to 18 mol% of calcium with the increase of the concentration of elemental cerium, but when the cerium is substituted with 30 mol% of calcium, the amount of IL-6 is increased and It was confirmed that the inflammatory fall. In addition, in the case of silicon, as in the case of cerium, when the phosphate is substituted up to 18 mol% of silicon, the anti-inflammatory property is improved by increasing the concentration of elemental silicon, but when the silicon is substituted by 30 mol% of phosphate, IL-6 It was found that the amount of produced and the anti-inflammatory was less.

From this result, the range of substitution rate of anti-inflammatory cerium and silicon is 6-18 mol%, and this value is 4.7% by weight to 12.8% by weight of the cerium element in the bicomponent calcium phosphate (Ce-BCP) partially substituted with cerium. It is the same as occupying the weight%, and the silicon element is equivalent to the 1.0 weight% to 3.1 weight% of the total weight in the partially substituted calcium phosphate (Si-BCP).

Claims (11)

(a) dissolving at least one water-soluble polymer selected from sodium alginate, beta-cyclodextrin, polyvinylpyrrolidone, polyvinyl alcohol, hyaluronic acid, laminarin, pullulan and fucoidan to prepare a polymer template solution;
(b) at least one calcium ion precursor solution selected from calcium nitrate, calcium chloride, calcium sulfate and calcium hydroxide,
At least one phosphate ion precursor, nitric acid, selected from ammonium monophosphate, diammonium phosphate, sodium monophosphate, sodium diphosphate, trisodium phosphate, potassium monophosphate, potassium diphosphate and potassium triphosphate One or more cerium ion precursors selected from cerium, cerium sulfate, cerium chloride, cerium acetate and cerium carbonate and tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate (TPOS), tetraisopropyl Preparing at least one silicon ion precursor selected from the group consisting of orthosilicate (TiPOS) and tetrabutyl orthosilicate (TBOS);
(c) the calcium ion precursor solution, the phosphate ion precursor solution, and the cerium ion precursor solution to produce a bicomponent calcium phosphate containing hydroxyapatite and tricalcium phosphate in a 9: 1 to 5: 5 molar ratio in the polymer template solution; To (Ca + Ce) / P = 1.585 / 1 to 1.653 / 1 molar ratio, followed by stirring, centrifugation and drying to prepare a bicomponent calcium phosphate powder in which cerium was partially substituted, but the proportion of cerium constitutes a cation. Preparing a bicomponent calcium phosphate powder comprising 6 mol% to 18 mol% of the combined value of calcium and cerium (Ce-BCP);
(d) the calcium ion precursor solution, the phosphate ion precursor solution, and the silicon ion precursor solution to produce a bicomponent calcium phosphate containing hydroxyapatite and tricalcium phosphate in a 9: 1 to 5: 5 molar ratio in the polymer template solution Ca / (P + Si) = 1.585 / 1 to 1.653 / 1 in a molar ratio, followed by stirring, centrifugation and drying to prepare a bicomponent calcium phosphate powder in which silicon was partially substituted (Si-BCP);
(e) anti-inflammatory active binary component, comprising the step of mixing the dried cerium or silicon partially substituted bicomponent calcium phosphate powder obtained in the step with a polymeric binder, a dispersant and distilled water to form a slurry. Method for producing a calcium phosphate porous bone graft material.

delete delete delete delete delete delete The method of claim 1,
The method of manufacturing an anti-inflammatory active bicomponent calcium phosphate-based porous bone graft material, wherein the ratio of silicon in step (d) accounts for 6 mol% to 18 mol% of the sum of phosphate and silicon constituting an anion.
The method of claim 1,
The polymer binder in step (e) comprises at least one selected from polyvinyl alcohol, polyvinylpyrrolidone and polyethylene glycol, a method for producing an anti-inflammatory active bicomponent calcium phosphate-based porous bone graft.
The method of claim 1,
The dispersing agent in step (e) comprises at least one member selected from the group consisting of ammonium polyacrylic acid salt, polyacrylic acid-maleic acid copolymer ammonium salt and polygamma glutamic acid ammonium salt, anti-inflammatory active bicomponent calcium phosphate-based porous bone graft Way.
Claim 1, 8, 9 and 10 using a slurry prepared by any one of the steps of printing using a three-dimensional printer and the printed slurry after drying at 40 ℃ 1,000 ~ 1,200 An anti-inflammatory active bicomponent calcium phosphate-based porous bone graft material, comprising the step of sintering the polymeric binder and the dispersant at 2 ° C. for 2 hours.

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