KR102236212B1 - A double layer composite membrane for photothermal therapy of bone tissue and method for preparing the same - Google Patents

Abstract

The present invention relates to: a method for synthesizing hydroxyapatite in which a part of a magnesium (Mg) element is substituted and which contains a photothermal agent with a photothermal effect, for preparing a double-layered organic-inorganic composite membrane for photothermal treatment of osteosarcoma; a method for manufacturing a porous monolith support for a lower layer; a method for manufacturing a hydrogel layer using a 3D printing technique of gelatin/hyaluronic acid/hydroxyapatite of an upper layer; and the composite membrane. The present invention has excellent effects in preventing recurrence of osteosarcoma caused by bone tumor tissue that may remain after surgical removal of osteosarcoma, and inducing regeneration of normal bone tissue.

Description

A double layer composite membrane for photothermal therapy of bone tissue and method for preparing the same}

The present invention relates to a dual-layered composite membrane for bone tissue photothermal treatment and a method for manufacturing the same, and more particularly, a biodegradable polymer is formed into a porous monolith support by heat-induced phase separation, and gelatin/hyaluronic acid/hydroxyapatite is added to the support. After making a layer of hydration gel by 3D printing, the surface is coated with hydroxyapatite in which magnesium (Mg) element is partially substituted, which contains a photothermal treatment agent with photothermal effect. It can be used to prevent the recurrence of osteosarcoma caused by and induce the regeneration of normal bone tissue, to a composite membrane of a bilayer structure for photothermal treatment of bone tissue, and a method of manufacturing the same.

Osteosarcoma is a representative disease among primary malignant tumors that occur in bones. Normal bone tissue is destroyed by tumor cells, forms masses, and spreads to surrounding tissues (muscles, nerves, blood vessels, adjacent bones), and can occur in all bones. It usually occurs at the ends of long bones, such as around the knee joint and the upper part of the humerus in the shoulder area. Osteosarcoma is difficult to diagnose early and has a very low 5-year survival rate of about 20-30% of cases with metastases, and currently widely used tumor treatment methods include surgery, chemotherapy, and radiation therapy.

Osteosarcoma has been mostly removed by surgical operation, but since the boundary of the tumor is ambiguous, when the tumor is removed, normal tissue in addition to the actual tumor tissue is removed, resulting in large bone loss and a side effect of recurrence due to tumor cells that were not removed during surgery There is this. Therefore, it minimizes the problems of surgical surgery and anticancer drug treatment, which are representative osteosarcoma osteomatosis treatments, and promotes regeneration of bone defects and selectively treats residual tumor cells to enable complete osteosarcoma treatment without recurrence. Development is needed.

Photothermal therapy (PTT), one of the cancer treatment methods that has been receiving much attention in recent years, is relatively easy to perform and is very effective in local cancer treatment, so it can improve the quality of life of patients after treatment compared to conventional cancer treatment methods. There is an advantage that there is. When the body temperature rises, the cells of the normal tissue expand the blood vessels and can discharge heat, but the new blood vessels of the tumor tissue locally increase the temperature because the blood vessels do not expand and thus cannot discharge heat. This is the principle that blood clots are generated by the heat generated at this time, reducing blood flow, and thus the supply of nutrients is blocked, resulting in necrosis of cancer cells. Therefore, the photothermal treatment method converts near-infrared energy into heat by irradiating it with a photothermal treatment agent, and an elevated temperature of 42-50°C can rapidly kill cancer cells.

Most of the photothermal treatments used for photothermal treatment of osteosarcoma mainly use heptametin-based and cyanine-based dyes with low thermal stability and short half-life, and metal nanoparticles with a high possibility of remaining in the body.These photothermal treatments are caused by osteosarcoma surgery. Inability to treat bone defects. Therefore, it is necessary to develop a composite membrane in which a photothermal treatment agent capable of regenerating bone defects caused by surgery while simultaneously killing bone tumor cells that may remain after osteosarcoma removal surgery is introduced.

The membrane for bone-guided regeneration currently used for bone tissue regeneration is a method of inducing the growth of bone tissue while blocking the invasion of undifferentiated fibroblasts, which are soft tissue components, into the space where new bone is regenerated. To this end, various types of absorbent membranes having a multilayer structure have been used, and such a multilayered absorbent membrane for bone tissue regeneration is known as a patent (see Patent Document 1-3). However, in the case of a known multilayered absorbent membrane for bone tissue regeneration, a multilayered structure of collagen having a different degree of crosslinking or a multilayered structure using collagen and hydration gel can be effectively prevented from penetrating the soft tissue into the space inside the membrane. It is difficult to effectively promote the regeneration of bone tissue.

Therefore, nowhere in the patent literature is known about the manufacture of a double-layered composite membrane that can be used to induce regeneration of normal bone tissue while at the same time treating tumor cells that may remain after osteosarcoma removal surgery.

Republic of Korea Patent Publication No. 10-1684790 (December 02, 2016) Republic of Korea Patent Publication No. 10-1260757 (April 29, 2013) Republic of Korea Patent Publication No. 10-1964907 (March 27, 2019)

Therefore, the problem to be solved of the present invention is to create a porous monolith support by thermal-induced phase separation of a biodegradable polymer, and after making a hydration gel layer by a three-dimensional printing technique of gelatin/hyaluronic acid/hydroxyapatite on the support, the photothermal effect is achieved. By coating the surface of hydroxyapatite, which is partially substituted with magnesium (Mg), which contains photothermal therapeutic agents, it prevents recurrence of osteosarcoma due to bone tumor tissue that may remain after surgical removal of osteosarcoma, and effectively blocks penetration of soft tissues. It is to provide a composite membrane of a double-layer structure for photothermal treatment of bone tissue that can be used to effectively induce the regeneration of normal bone tissue and a method of manufacturing the same.

According to an aspect of the present invention, (a) preparing a polymer template solution by dissolving a water-soluble polymer in distilled water; (b) preparing a calcium ion precursor solution, a phosphate ion precursor solution, a magnesium ion precursor solution, and a photothermal treatment agent solution, respectively; (c) The photothermal treatment agent solution is first added to the polymer template solution, and the calcium ion precursor solution, the phosphate ion precursor solution, and the magnesium ion precursor solution are used to generate the hydroxide apatite. And magnesium (Mg) molar ratio {(Ca+Mg)/P} = C (where C is a predetermined ratio), stirred, centrifuged, and dried to partially replace magnesium containing photothermal therapy. Preparing hydroxyapatite powder (Mg-HA-IR); (d) preparing a slurry by mixing the magnesium-substituted hydroxide apatite powder containing the photothermal treatment agent obtained in step (c) with gelatin and distilled water; (e) heating the biodegradable polymer to a first predetermined temperature to prepare a homogeneous solution, and then putting the solution into a mold and cooling to a second predetermined temperature to separate and precipitate phases; (f) washing and drying the biodegradable polymer precipitated in step (e) to prepare a porous monolith; (g) preparing a solution for 3D printing by dispersing gelatin, hyaluronic acid, and hydroxyapatite in distilled water, and then printing the solution on the porous monolith prepared in step (f) to prepare a three-dimensional hydrogel layer; (g) The hydration gel layer was prepared with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide. And crosslinking using a mixture of NHS) to prepare a double-layered composite membrane; And (h) coating the surface of the double-layered composite membrane with hydroxyapatite partially substituted with magnesium containing a photothermal treatment agent. Can be.

The water-soluble polymer for the polymer template of step (a) is block copolymerized with at least one selected from the group consisting of hyaluronic acid, sodium alginate, beta-cyclodextrin, polyvinylpyrrolidone, polyvinyl alcohol, laminarin, pullulan, and fucoidan. Chain polyethylene glycol-polypropylene glycol-polyethylene glycol (PEG-PPG-PEG) may be included.

The calcium ion precursor of step (b) may include at least one selected from the group consisting of calcium nitrate, calcium chloride, calcium sulfate, and calcium hydroxide.

The phosphate ion precursor of step (b) is a first ammonium phosphate, a second ammonium phosphate, a first sodium phosphate, a second sodium phosphate, a third sodium phosphate, a first potassium phosphate, a second potassium phosphate, and a third potassium phosphate. It may include one or more selected from the group consisting of.

The magnesium ion precursor of step (b) may include at least one selected from the group consisting of magnesium sulfate, magnesium nitrate, magnesium chloride, magnesium hydroxide, and magnesium carbonate.

The photothermal treatment agent of step (b) is selected from the group consisting of indocyanine green (ICG), which is a cyanine dye, and IR-780, IR-783, IR-808, and IR-825, which are heptamethine cyanine dyes. It may contain one or more.

In the step (c), the ratio of magnesium accounts for 10 mol% to 30 mol% from the sum of calcium and magnesium constituting the cation, and the magnesium element in the hydroxide apatite partially substituted with magnesium is 2.5% by weight to the total weight. It can occupy 7.6% by weight.

The content of the photothermal treatment agent in step (c) may account for 1.3% to 4.8% by weight of the total weight of the hydroxyapatite partially substituted with magnesium.

In the step (e), the biodegradable polymer may include at least one selected from polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly-gamma-glutamic acid type, and poly-gamma-glutamic acid salt type. I can.

The first temperature may be 70 to 90°C, the second temperature may be -5 to 15°C, and C = 1.67/1.

According to another aspect of the present invention, a composite membrane having a double layer structure for photothermal treatment of bone tissue prepared by the above manufacturing method may be provided.

According to the present invention, by 3D printing an organic-inorganic complex mixed solution on a porous monolith prepared using a biodegradable polymer, and then coating a partially substituted hydroxyapatite containing magnesium containing a photothermal agent, a dual-layered composite membrane is prepared. , It has an excellent effect in preventing recurrence of osteosarcoma due to bone tumor tissue that may remain after surgical removal of osteosarcoma, and effectively inducing the regeneration of normal bone tissue by effectively blocking the penetration of soft tissue.

1 is a diagram schematically illustrating a method of manufacturing a double-layered composite membrane for photothermal treatment of bone tissue according to the present invention.
2 is a digital photograph of a biodegradable polymer monolith and a double-layer composite membrane prepared according to the present invention.
3 is a photograph of a hydroxy-cyclic apatite powder partially substituted with magnesium containing a photothermal treatment agent prepared according to the present invention, observed with a scanning electron microscope.
4 is a graph showing a surface analysis of a partially substituted hydroxyapatite powder containing magnesium containing a photothermal treatment agent prepared according to the present invention using infrared spectroscopy.
5 is a graph showing an elemental analysis of a hydroxycyclic apatite powder partially substituted with magnesium containing a photothermal treatment agent prepared according to the present invention using an energy dispersive X-ray spectroscopy method.
6 is a graph showing changes in exothermic temperature and thermal stability according to infrared irradiation of magnesium-substituted hydroxyapatite powder containing a photothermal treatment agent and a photothermal treatment agent prepared according to the present invention.
7 is a photograph of a porous monolith using a biodegradable polymer prepared according to the present invention observed with a scanning electron microscope.
8 is a photograph of an organic-inorganic composite hydration gel having different hydroxyapatite content prepared according to the present invention observed with a scanning microscope.
9 is a photograph of an organic-inorganic composite hydration gel having a different internal structure manufactured according to the present invention observed with a scanning electron microscope.
10 is a photograph of a double-layer structure composite membrane for photothermal treatment of bone tissue prepared according to the present invention observed with a scanning electron microscope.
11 is a photograph and graph of measuring a change in surface temperature after irradiation with infrared energy by immersing the bilayer structured composite membrane for photothermal treatment of bone tissue prepared according to the present invention in a cell culture medium.
12 is a photograph and graph showing the cytotoxicity results of the bilayer structured composite membrane for photothermal treatment of bone tissue prepared according to the present invention.

In order to fully understand the present invention, operational advantages of the present invention, and objects achieved by the implementation of the present invention, reference should be made to the accompanying drawings illustrating preferred embodiments of the present invention and the contents described in the accompanying drawings.

Hereinafter, the present invention will be described in detail by describing a preferred embodiment of the present invention with reference to the accompanying drawings. However, in describing the present invention, a description of a function or configuration that is already known will be omitted in order to clarify the gist of the present invention.

Unless otherwise defined 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 including 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, several methods and materials have been described. It should not be understood as limiting the invention to specific methodologies, protocols and reagents, as those skilled in the art can use it in a variety of ways, depending on the context in which they are used.

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

Example 1

Preparation of hydroxyapatite powder partially substituted with magnesium containing photothermal therapy according to the present invention

In order to prepare a partially substituted hydroxyapatite (Mg-HA-IR) powder containing the photothermal agent of the present invention, a water-soluble polymer, polyethylene glycol-polypropylene glycol-polyethylene glycol copolymer 2% by weight and hyaluronic acid 0.2% by weight In a polymer template solution prepared by dissolving in distilled water, 3% by weight of the photothermal treatment agent IR-780 was dissolved based on the weight of the water-soluble polymer and the ion precursor to prepare a mixed solution of the water-soluble polymer and the photothermal treatment agent. To this mixed solution, a calcium nitrate solution as a calcium ion precursor and a magnesium nitrate solution as a magnesium ion precursor were added to adjust the concentration to 0.1 to 0.3M, and then adjusted with 0.1N hydrochloric acid or aqueous ammonia so that the pH was 10. Subsequently, a 0.1~0.5M phosphate ion precursor, a second ammonium phosphate solution, whose pH is adjusted to 10, is added to a solution containing a polymer template, a photothermal agent, and a calcium ion and magnesium ion precursor (Ca+Mg)/P = 1.67/ A small amount was added while stirring at 45° C. so as to be 1, and the reaction was continued for 24 hours after the addition was completed to prepare a hydroxyapatite powder in which magnesium containing a photothermal treatment was partially substituted. Here, (Ca+Mg)/P represents the molar ratio of calcium (Ca) and magnesium (Mg) to phosphorus (P).

Example 2

Porous monolith production according to the present invention

A solution was prepared by dissolving 12% by weight polylactic acid in a 1,4-dioxane and 2-butanol 1:1 mixed solvent while heating at 70 to 90°C, for example, 80°C for 2 hours. At this time, it is difficult to prepare a uniform solution because the biodegradable polymer is not completely dissolved below 70°C, and there is a possibility of thermal decomposition of the biodegradable polymer above 90°C. After putting the solution obtained in the above step into a Teflon mold capable of forming a disc shape, cooling it at -5 to 15°C, for example, 0°C for 24 hours to proceed with a phase separation reaction, followed by immersion in methanol as a non-solvent to 1,4- A porous monolith was prepared by removing dioxane and 2-butanol and simultaneously forming pores. At this time, there is a possibility that phase separation does not occur completely because the sedimentation rate of the biodegradable polymer is too fast below -5°C, and there is a possibility that the precipitation of the biodegradable polymer does not occur above 15°C.

Example 3

Fabrication of organic-inorganic composite hydration gel layer using 3D printing technique according to the present invention

6% by weight of gelatin and 0.3% by weight of hyaluronic acid were dissolved in distilled water, and hydroxyapatite nanoparticles were dispersed to prepare a 3D printing solution. The solution obtained in the above step was put in a 3D printer, and the hydration gel layer was printed on the upper layer using the porous monolith prepared in Example 2 as a support for the lower layer, and then immersed in an ethanol solution in which 3 wt% EDC and 3 wt% NHS were dissolved for 12 hours. After crosslinking, washing with distilled water and freeze-drying were performed to prepare a double-layered composite membrane as shown in FIG. 1.

Manufacturing Example 1

Preparation of organic-inorganic composite hydration gel layer containing 10% by weight of hydroxyapatite nanoparticles

In the process of preparing the solution for 3D printing in the manufacturing method of Example 3, 10% by weight of hydroxyapatite nanoparticles were used based on the weight of the polymer, and using this solution, the upper hydrogel layer was crossed so that the horizontal and vertical strands cross 90 degrees. By printing, a double-layered composite membrane (GEHA10) was prepared.

Manufacturing Example 2

Preparation of organic-inorganic composite hydration gel layer containing 20% by weight of hydroxyapatite nanoparticles

In the process of preparing the solution for 3D printing in the manufacturing method of Example 3, 20% by weight of hydroxyapatite nanoparticles based on the weight of the polymer was used, and using this solution, the upper hydrogel layer was crossed so that the horizontal and vertical strands cross 90 degrees. By printing, a double-layered composite membrane (GEHA20) was prepared.

Manufacturing Example 3

Preparation of organic-inorganic composite hydration gel layer containing 40% by weight of hydroxyapatite nanoparticles

In the process of preparing the solution for 3D printing in the manufacturing method of Example 3, 40% by weight of hydroxyapatite nanoparticles based on the weight of the polymer was used, and using this solution, the upper hydrogel layer was crossed so that the horizontal and vertical strands cross 90 degrees. By printing, a double-layered composite membrane (GEHA40) was prepared.

Manufacturing Example 4

Fabrication of organic-inorganic complex hydration gel layer intersecting in zigzag

In the manufacturing method of Preparation Example 3, a double-layered composite membrane (GEHA40ZZ) was prepared by printing the upper hydration gel layer so that the horizontal and vertical strands intersect in a zigzag form.

Manufacturing Example 5

Fabrication of organic-inorganic complex hydration gel layer crossing 45 degrees

In the manufacturing method of Preparation Example 3, a double-layered composite membrane (GEHA4045) was prepared by printing the upper hydration gel layer so that the horizontal and vertical strands intersect at 45 degrees.

Example 4

Preparation of a composite membrane of a double layer structure for photothermal treatment of bone tissue according to the present invention

Bone tissue photothermal treatment by dispersing the surface of the double-layered composite membrane (GEHA40) obtained in Preparation Example 3 above in an aqueous solution in which 2% by weight of gelatin was dissolved in magnesium containing a photothermal agent having the same weight as the polymer and partially substituted hydroxide apatite, followed by coating and washing. A composite membrane of a two-layer structure (GEHA40IR) was prepared.

Experimental Example 1

Analysis of the form of hydroxyapatite powder in which magnesium containing a photothermal agent is partially substituted and the content of the photothermal agent according to the present invention

FIG. 3 shows a scanning electron microscope (SEM, MIRA3, TESCAN) image of a partially substituted magnesium hydroxide apatite powder containing a photodynamic therapeutic agent synthesized according to Example 1. FIG. It was confirmed that the powder formed nanoparticles, but showed some secondary agglomeration. The content of the photothermal treatment agent contained in the nanoparticles was analyzed using an ultraviolet-visible spectrophotometer (U-2900, Hitachi) and found to be 1.4% by weight of the final product.

Experimental Example 2

Surface absorbance analysis of hydroxyapatite powder partially substituted with magnesium containing photothermal treatment according to the present invention

4 is a total reflection measurement of the surface absorbance of a partially substituted hydroxyapatite powder containing magnesium containing a photothermal treatment agent prepared according to Example 1 using Fourier transform infrared spectroscopy (ATR-FTIR, ALPHA, Bruker optics) from 400 to 4000. It was analyzed in the range of ^{cm -1.}

^{As shown in FIG. 4, peaks due to phosphate ions (PO4 3-} ) appeared at 1093, 1022, 972, 569 and 558 cm ^-1 in all samples regardless of the presence or absence of all photothermal therapeutic agents, and use of a water-soluble polymer template The peak attributable to a hydroxyl group was confirmed at ^{3296 cm -1 by.}

Experimental Example 3

Elemental analysis of hydroxyapatite powder partially substituted with magnesium containing photothermal therapy according to the present invention

FIG. 5 is an elemental analysis of magnesium-substituted hydroxide apatite powder containing a photothermal treatment agent prepared according to Example 1 using an energy dispersive X-ray spectroscopy (EDX, Horiba, Ltd., 0-20 keV). .

As shown in FIG. 5, in all samples regardless of the presence or absence of the photothermal treatment agent, a peak due to phosphorus (P) element appeared at 2.10 keV, and a peak due to calcium (Ca) element appeared at 3.66 keV, and magnesium (Mg ) A peak due to the element appeared at 1.28 keV, confirming that the preparation of a partially substituted magnesium hydroxide apatite powder containing a photothermal agent was successfully performed.

Experimental Example 4

Measurement of surface temperature change and thermal stability of hydroxyapatite powder partially substituted with magnesium containing photothermal therapy according to the present invention

Figure 6 is a thermal imaging camera (FLIR, E53) repeatedly using infrared energy after irradiation of infrared energy to a partially substituted hydroxyapatite powder containing magnesium containing the photothermal treatment agent prepared according to Example 1 above. It was measured as.

As shown in FIG. 6, in the case of IR780 not encapsulated with hydroxyapatite partially substituted with magnesium, the temperature rises to 90°C by infrared irradiation, but the temperature rise partially decreases due to repeated infrared irradiation, so the thermal stability of IR780 is good. It was confirmed that it was not. However, when magnesium is partially substituted with hydroxyapatite, the temperature rises to about 105°C by infrared irradiation, and the temperature of the photothermal treatment agent is kept almost constant even by repeated infrared irradiation, indicating that the thermal stability is very excellent. have.

Experimental Example 5

Analysis of the morphology of the porous monolith according to the present invention

FIG. 7 shows a scanning electron microscope (SEM, MIRA3, TESCAN) image of the surface of the monolith prepared according to Example 2, and it was confirmed that the monolith had a good overall porosity.

Experimental Example 6

Form analysis of organic-inorganic complex hydration gel layer using 3D printing technique according to the present invention

FIG. 8 shows a scanning electron microscope (SEM, MIRA3, TESCAN) image of the surface of a double-layered composite membrane containing hydroxyapatite in the organic-inorganic composite hydration gel layer prepared according to Preparation Examples 1 to 3 above. It was confirmed that the hydration gel layer had a good overall porosity, and that an uneven structure was generated by the introduction of hydroxyapatite on the surface. 9 is a schematic diagram using Solidworks and an analysis photograph using an electron microscope (SEM, MIRA3, TESCAN) of a double-layered composite membrane having different internal structures manufactured according to Preparation Examples 3 to 5 above.

Experimental Example 7

Analysis of the morphology of the double-layered composite membrane for bone tissue treatment according to the present invention

10 is a scanning electron microscope (SEM, MIRA3, TESCAN) image of the surface of a double-layered composite membrane for treating osteosarcoma coated on the surface of a magnesium-substituted hydroxyapatite containing a photothermal treatment agent prepared according to Example 4 above. As shown, it was confirmed that hydroxyapatite was coated on the surface.

Experimental Example 8

Measurement of the surface temperature change of the double-layered composite membrane for bone tissue treatment according to the present invention

11 is a thermal imaging camera (FLIR, E53) showing the change in surface temperature according to the irradiation time after irradiating infrared energy in a wet state by immersing the double-layered composite membrane for bone tissue treatment prepared according to Example 4 in a cell culture medium. ) Was measured using.

As shown in FIG. 11, it was confirmed that the surface temperature of the composite membrane coated with hydroxyapatite partially substituted with magnesium containing a photothermal treatment agent was increased to 45 degrees or more upon irradiation with infrared rays.

Experimental Example 9

Cytotoxicity evaluation of the double-layered composite membrane for bone tissue treatment according to the present invention

12 shows the results of evaluating the biocompatibility of the bilayer composite membrane for bone tissue treatment prepared according to Example 4 through a cytotoxicity test. Mouse fibroblast L-929 cells were purchased from ATCC and used for cytotoxicity. The cells were 37° C. in RPMI 1640 medium, which is a basic medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Basic culture was carried out under% CO _{2 conditions.}

In order to confirm the cytotoxicity of the double-layered composite membrane according to the present invention, the membrane was first sterilized with a sterilizer and then sterilized for 2 minutes with 75%, 50%, and 25% ethanol under aseptic conditions, respectively. After disinfection, the membrane was put into 100 mL of RPMI 1640 medium and eluted at 37° C. for 3 days to prepare an elution medium.

The basic medium of L-929 cells cultured for 24 hours in the basic culture conditions was replaced with each elution medium prepared in the above step, and cytotoxicity was confirmed by MTT assay after 1 day incubation. Cells cultured in basic culture medium were used for the negative control group and cells cultured in sterile distilled water for the positive control group.

As shown in FIG. 12, a high cell viability of 93% was exhibited in the elution medium of the double-layered composite membrane. Through this, it was confirmed that the dual-layered composite membrane for treatment of bone tissue of the present invention has no cytotoxicity and excellent biocompatibility.

Although the present embodiment has been described in detail with reference to the drawings above, the scope of the present embodiment is not limited to the above-described drawings and description.

As described above, the present invention is not limited to the described embodiments, and it is apparent to those of ordinary skill in the art that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, it should be said that such modifications or variations belong to the scope of the claims of the present invention.

Claims (11)

(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 solution, a magnesium ion precursor solution, and a photothermal treatment agent solution, respectively;
(c) The photothermal treatment agent solution is first added to the polymer template solution, and the calcium ion precursor solution, the phosphate ion precursor solution, and the magnesium ion precursor solution are used to generate the hydroxide apatite. And magnesium (Mg) molar ratio {(Ca+Mg)/P} = C (where C is a predetermined ratio), stirred, centrifuged, and dried to partially replace magnesium containing photothermal therapy. Preparing hydroxyapatite powder (Mg-HA-IR);
(d) preparing a slurry by mixing the magnesium-substituted hydroxide apatite powder containing the photothermal treatment agent obtained in step (c) with gelatin and distilled water;
(e) heating the biodegradable polymer to a first predetermined temperature to prepare a homogeneous solution, and then putting the solution into a mold and cooling to a second predetermined temperature to separate and precipitate phases;
(f) washing and drying the biodegradable polymer precipitated in step (e) to prepare a porous monolith;
(g) preparing a solution for 3D printing by dispersing gelatin, hyaluronic acid, and hydroxyapatite in distilled water, and then printing the solution on the porous monolith prepared in step (f) to prepare a three-dimensional hydrogel layer;
(g) The hydration gel layer was prepared with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide. And crosslinking using a mixture of NHS) to prepare a double-layered composite membrane; And
(h) coating the surface of the double-layered composite membrane with hydroxyapatite partially substituted with magnesium containing a photothermal treatment agent. The method of claim 1,
The water-soluble polymer for the polymer template of step (a) is block copolymerized with at least one selected from the group consisting of hyaluronic acid, sodium alginate, beta-cyclodextrin, polyvinylpyrrolidone, polyvinyl alcohol, laminarin, pullulan, and fucoidan. Chain polyethylene glycol-polypropylene glycol-polyethylene glycol (PEG-PPG-PEG), characterized in that it comprises a composite membrane of a bilayer structure for photothermal treatment of bone tissue. The method of claim 1,
The calcium ion precursor of step (b) comprises at least one selected from the group consisting of calcium nitrate, calcium chloride, calcium sulfate, and calcium hydroxide. The method of claim 1,
The phosphate ion precursor of step (b) is a first ammonium phosphate, a second ammonium phosphate, a first sodium phosphate, a second sodium phosphate, a third sodium phosphate, a first potassium phosphate, a second potassium phosphate, and a third potassium phosphate. A method for producing a composite membrane of a bilayer structure for photothermal treatment of bone tissue, characterized in that it comprises at least one selected from the group consisting of. The method of claim 1,
The magnesium ion precursor of step (b) comprises at least one selected from the group consisting of magnesium sulfate, magnesium nitrate, magnesium chloride, magnesium hydroxide, and magnesium carbonate. Manufacturing method. The method of claim 1,
The photothermal treatment agent of step (b) is selected from the group consisting of indocyanine green (ICG), which is a cyanine dye, and IR-780, IR-783, IR-808, and IR-825, which are heptamethine cyanine dyes. A method for producing a composite membrane of a double-layer structure for photothermal treatment of bone tissue, characterized in that it comprises one or more. The method of claim 1,
In the step (c), the ratio of magnesium accounts for 10 mol% to 30 mol% from the sum of calcium and magnesium constituting the cation, and in the hydroxide apatite partially substituted with magnesium, the magnesium element is from 2.5 wt% to the total weight. A method of manufacturing a composite membrane having a double layer structure for photothermal treatment of bone tissue, characterized in that it occupies 7.6% by weight. The method of claim 1,
In the step (c), the content of the photothermal treatment agent accounts for 1.3% to 4.8% by weight of the total weight of the hydroxyapatite partially substituted with magnesium. The method of claim 1,
In the step (e), the biodegradable polymer comprises at least one selected from polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly-gamma-glutamic acid type, and poly-gamma-glutamic acid salt type. Characterized in that, the method for producing a composite membrane of a double-layer structure for photothermal treatment of bone tissue. The method of claim 1,
The first temperature is 70 ~ 90 ℃,
The second temperature is -5 ~ 15 ℃,
C = 1.67/1, characterized in that, the method for producing a composite membrane of a double-layer structure for photothermal treatment of bone tissue. A composite membrane of a double-layer structure for photothermal treatment of bone tissue, characterized in that produced by the manufacturing method according to any one of claims 1 to 10.

Images