KR20230070969A - Bone graft material derived from starfish, a marine-derived material, and method for manufacturing the same - Google Patents

Abstract

It relates to a bone graft material derived from a starfish, which is a material of marine origin, and a method for manufacturing the same. There is an effect of manufacturing a bone graft material with significantly improved porosity, and according to the bone graft material manufactured by the above method, cell proliferation, differentiation into osteoblasts, or bone regeneration or formation are remarkably increased to be useful as a bone graft material. There are possible effects.

Description

Bone graft material derived from starfish, a marine-derived material, and method for manufacturing the same

It relates to a bone graft material derived from starfish and a manufacturing method thereof.

Biomaterials for bone grafting depended on their characteristics of being inactive in vivo in the early stages of development, but there were many limitations in their use due to infection and inflammatory reactions in surrounding tissues after the procedure. Since then, with the rapid development of biomaterial technology using metals, ceramics, and polymers, we have designed and developed materials that are biocompatible rather than bioinert to provide various kinds of bioactive scaffolds for bone tissue regeneration depending on the site and purpose of use. has reached the development of These bioactive scaffolds for regenerating bone tissue have different physical properties depending on the implantation site, have no toxic reaction to surrounding tissues, and require relatively high mechanical properties compared to other artificial organs. These bioactive scaffolds for bone tissue regeneration are marketed and developed as various biomaterials depending on the characteristics of the raw materials and the purpose of use.

Natural hydroxyapatite is emerging as a biomaterial for bone tissue transplantation, but research on it is still insufficient (Korean Application No. 10-2019-7020453).

Therefore, in order to process the biomaterial for bone tissue transplantation and use it as a bone graft material, it is necessary to develop a naturally-derived material having higher cell proliferation and adhesion.

One aspect includes (a) separating bone fragments from starfish; (b) reacting by adding an acidic aqueous solution to the separated bone fragments; (c) performing a hydrothermal reaction on the bone fragments obtained in step (b); (d) performing a sintering process on the bone fragments on which the hydrothermalization reaction is completed; and (e) cooling the bone fragments that have undergone the sintering process.

Another aspect is to provide a starfish-derived bone graft material prepared by the above method.

Another aspect is to provide a bone graft material comprising a starfish-derived porous bone fragment.

Another aspect is to provide a pharmaceutical composition for preventing or treating bone disease comprising the bone graft material as an active ingredient.

One aspect includes (a) separating bone fragments from starfish; (b) reacting by adding an acidic aqueous solution to the separated bone fragments; (c) performing a hydrothermal reaction on the bone fragments obtained in step (b); (d) performing a sintering process on the bone fragments on which the hydrothermalization reaction is completed; and (e) cooling the bone fragments that have undergone the sintering process.

The term "bone graft" can mean a material that can be used for preservation of bone defects, stimulation of bone formation, joint union, immobilization of joints and prevention of dislocation, and can be used for bone grafting. there is. Accordingly, the bone graft material may be for treating bone defects. The term "bone graft material" may be used interchangeably with "bone filler", "bone substitute", "bone scaffold" and the like.

The bone graft material may be, for example, ethmoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic bone ( zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula ), humerus, radius, ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium, pubis, It can be applied to the femur, tibia, fibula, patella, calcaneus, tarsal, or metatarsal bones.

The term “porosity” can refer to the property of having many pores within. High porosity may mean that there are a large number of pores and that the diameter of the pores is large.

The starfish-derived bone graft material having porosity may be a starfish-derived bone graft material having improved porosity. That is, the starfish-derived bone graft material prepared by the above method may have significantly improved porosity compared to existing bone graft materials.

The method of manufacturing the starfish-derived bone graft material may include a step (step (a)) of separating a bone fragment from the starfish.

In the step (a), both physical and chemical methods may be used to separate the bone fragments from the starfish.

The starfish is Asterias amureasis, spider starfish (Ophiolocus japonicus), red starfish (Certonardoa semiregularis), star starfish (Asterina pectinifera), sun starfish (Solarstar borealis), rod starfish (Solarstar paxillatus), thorn maple starfish (Astropecten polyacantbus), and at least one starfish selected from the group consisting of Marthasterias glaciallis.

The method of manufacturing the starfish-derived bone graft material may include a step (step (b)) of reacting the separated bone fragment with an acidic aqueous solution.

The pH of the acidic aqueous solution in step (b) may be about 0.1 to 6. Specifically, the acidic aqueous solution may have a pH of about 0.1 to 6, about 0.1 to 5, about 0.1 to 4, about 0.1 to 3, about 1 to 6, about 2 to 6, or about 3 to 6.

The acidic aqueous solution is hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, It may be an aqueous solution of methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or p-toluenesulfonic acid.

The step (b) may further include adding an acidic aqueous solution to the separated bone fragments and then stirring them.

Step (b) may be adding an acidic aqueous solution to the separated bone fragments and reacting for about 10 to 50 hours. Preferably, the step (b) may be to react for about 12 to 48 hours by adding an acidic aqueous solution to the separated bone fragments. According to one embodiment, if the reaction is performed for a time exceeding about 50 hours in the step (b), the size of the hole becomes excessively large, and the shape of the bone fragment may not be maintained intact and may be fragmented, and less than about 10 hours When the above reaction is performed for a time of , the porosity can be significantly reduced, and thus, the effect of promoting the proliferation of stem cells of the bone graft material prepared by the method, the effect of promoting the differentiation of stem cells into osteoblasts, or bone regeneration or The formation-promoting effect may be significantly reduced.

Step (b) may be to react by treating with ultrasonic waves. Specifically, the ultrasonic treatment may include adding an acidic aqueous solution to the bone fragments separated in step (b) and then treating the bone fragments immersed in the acidic aqueous solution with ultrasonic waves.

The ultrasonic treatment in step (b) is performed simultaneously with adding an acidic aqueous solution to the separated bone fragments, after adding an acidic aqueous solution to the separated bone fragments, or after adding an acidic aqueous solution to the separated bone fragments. It may be performed in the step of stirring. The ultrasonic treatment may be performed by administering the separated bone fragment and an acidic aqueous solution into an ultrasonic reactor, and irradiating ultrasonic waves for about 5 minutes with an irradiation interval of about 20 minutes. The irradiation interval may be about 10 to 40 minutes, about 10 to 35 minutes, about 10 to 30 minutes, about 15 to 30 minutes, or about 15 to 25 minutes, and the irradiation time is about 1 to 15 minutes, about 1 to 25 minutes. 13 minutes, about 1 to 10 minutes, about 1 to 8 minutes, or about 3 to 8 minutes.

The ultrasonic treatment in step (b) may be about 10 to 70 kHz ultrasonic treatment. Specifically, the ultrasonic treatment in step (b) is about 15 to 70, about 20 to 70, about 25 to 70, about 30 to 70, about 35 to 70, about 40 to 70, about 10 to 65, about 15 to 65, about 20 to 65, about 25 to 65, about 30 to 65, about 35 to 65, about 40 to 65, about 10 to 60, about 15 to 60, about 20 to 60, about 25 to 60, about 30 to 60, about 35 to 60, or about 40 to 60 kHz. According to one embodiment, in step (b), when ultrasonic waves of more than about 70 kHz are processed, the size of the hole becomes excessively large, and the shape of the bone fragment may not be maintained intact and may be fragmented, and ultrasonic waves of less than about 10 kHz are treated In this case, the porosity can be significantly reduced, and as a result, the effect of promoting the proliferation of stem cells, the effect of promoting the differentiation of stem cells into osteoblasts, or the effect of promoting bone regeneration or formation of the bone graft material prepared by the above method is significantly reduced. can do.

The method of manufacturing the starfish-derived bone graft material may include a step (step (c)) of performing a hydrothermal reaction on the bone fragment obtained in step (b).

The term "hydrothermalization reaction" may refer to a synthesis reaction of a material in the presence of high-temperature water or high-temperature, high-pressure water.

Step (c) includes adding the bone fragments obtained in step (b) to an aqueous solution containing a compound capable of providing phosphoric acid; and performing a hydrothermal reaction on the bone fragments added to an aqueous solution containing a compound capable of providing phosphoric acid. Examples of the compound capable of providing the phosphoric acid may include (NH ₄ )H ₂ PO _{4 ,} H ₃ PO ₄ , Na ₃ PO ₄ , or Na ₂ HPO ₄ .

Step (c) may include performing a hydrothermal reaction by treating the bone fragment obtained in step (b) with microwaves.

The term "microwave" may refer to electromagnetic waves having a short wavelength and properties such as straightness, reflection, refraction, and interference similar to those of light. The microwave may be an electromagnetic wave having a frequency of about 1 to 300 GHz and a wavelength of about 1 mm to 1 m.

Specifically, in step (c), the bone fragments obtained in step (b) are treated with microwaves of about 300 MHz to 300 GHz in an amount of about 100 to 1500 W for about 0.5 minutes to 48 hours, resulting in a hydrothermal reaction. It may include the step of performing.

The hydrothermal reaction of step (c) may be performed at about 30 to 600 ° C for about 2 to 40 hours.

The hydrothermalization reaction is carried out at at least about 30 ° C, about 50 ° C, about 80 ° C, or about 100 ° C or more, for example, about 30 to 600 ° C, about 80 to 600 ° C, about 100 to 600 ° C, about 100 to about 100 ° C It may be a hydrothermal reaction at 500 ° C, about 100 to 400 ° C, or about 100 to 300 ° C for about 2 to 40 hours, about 2 to 30 hours, about 10 to 40 hours, or about 12 to 36 hours.

The method of manufacturing the starfish-derived bone graft material may include a step (step (d)) of performing a sintering process on the bone fragments for which the hydrothermalization reaction has been completed.

The term "sintering process" refers to one of heat treatment processing methods in which press-molded powders into an appropriate shape are heated to form a tightly adhered assembly. For example, the sintering process means a process of heating the bone fragments on which hydroxyapatite crystals are formed on the surface of the bone fragments after the hydrothermalization reaction has been completed to form a tightly adherent combination of various components constituting the bone fragments, including hydroxyapatite. can

The sintering process of step (d) includes raising the temperature to about 400 to 800° C. at a rate of about 1 to 20° C./min and maintaining the temperature for about 1 to 8 hours; and raising the temperature to about 600 to 1500 °C at a rate of about 1 to 20 °C/min and maintaining the temperature for about 1 to 8 hours.

The sintering process is performed after raising the temperature to about 400 to 700, about 500 to 700, or about 550 to 650 ° C at a rate of about 1 to 18, about 1 to 15, about 1 to 10, or about 3 to 8 ° C / min holding for about 3 to 8, about 4 to 8, or about 5 to 7 hours; and from about 600 to 1300, from about 700 to 1300, from about 700 to 1200, from about 800 to 1200, from about 800 to about 18, from about 1 to 15, from about 1 to 10, or from about 3 to 8 °C/min. It may include a step of raising the temperature to 1100, about 800 to 1000, or about 850 to 950 ° C and then maintaining it for about 3 to 8, about 4 to 8, or about 5 to 7 hours.

Another aspect provides a starfish-derived bone graft material prepared by the above method.

According to one embodiment, the starfish-derived bone graft material prepared by the above method has significantly improved porosity compared to artificially synthesized bone graft materials (or commercially available bone graft materials) and bone fragments simply isolated from starfish without using the above method. It can have, and because of this, it can exhibit unique chemical and structural properties. Specifically, the starfish-derived bone graft material prepared by the above method may have a remarkably improved porosity while containing microporosity and trace elements inherent in starfish bone fragments. For this reason, the starfish-derived bone graft material prepared by the above method has significantly improved cell proliferation promoting effect and osteoblast compared to artificially synthesized bone graft materials (or commercially available bone graft materials) and bone fragments simply isolated from starfish without using the above method. It may exhibit an effect of promoting differentiation into cells (or osteoblasts) or an effect of promoting bone regeneration or formation.

The starfish-derived bone graft material prepared by the above method may include hydroxyapatite and/or tricalcium phosphate.

The starfish-derived bone graft material prepared by the above method may have a porosity of about 50 to 80%. According to one embodiment, the bone graft material may have a porosity of about 60 to 80, about 65 to 80, about 70 to 80, about 75 to 80, or about 70 to 75%. According to one embodiment, when the above range is exceeded, the structure of the bone graft material may be collapsed, and when the above range is not reached, the porosity is too low, and it may be difficult for bone cells to penetrate and proliferate inside the bone graft material. As a result, the bone graft material prepared by the above method may significantly reduce the effect of promoting stem cell proliferation, the effect of promoting the differentiation of stem cells into osteoblasts, or the effect of promoting bone regeneration or formation.

The term "porosity" is the ratio of voids to the total volume of a solid, expressed as a percentage (%).

The pore diameter of the starfish-derived bone graft material prepared by the above method may be about 15 to 50 um. Specifically, the diameter of the pore is about 20 to 50, about 25 to 50, about 30 to 50, about 35 to 50, about 15 to 45, about 20 to 45, about 25 to 45, about 30 to 45, about 35 to 45, about 30 to 40, or about 35 to 40 um. According to one embodiment, when the above range is exceeded, the structure of the bone graft material may be collapsed, and when the above range is not reached, the porosity is too low, and it may be difficult for bone cells to penetrate and proliferate inside the bone graft material. As a result, the bone graft material prepared by the above method may significantly reduce the effect of promoting stem cell proliferation, the effect of promoting the differentiation of stem cells into osteoblasts, or the effect of promoting bone regeneration or formation.

The starfish-derived bone graft material prepared by the above method may be composed of a plurality of particles. The particle size of the starfish-derived bone graft material prepared by the above method may be about 0.1 to 3 mm. Therefore, the starfish-derived bone graft material prepared by the above method may exhibit a form that is easy to be transplanted to various tissue sites. For example, by filling the bone damage site with starfish-derived bone graft material prepared by the above method, the defect site can be repaired regardless of the shape and thickness of the bone damage site, and the bone graft material can be injected into the damaged site without a large incision. can be transplanted

According to one embodiment, the starfish-derived bone graft material prepared by the above method may exhibit an effect of promoting cell proliferation, cell adhesion, or differentiation into osteoblasts (or osteoblasts). In addition, according to one embodiment, the starfish-derived bone graft material prepared by the above method can be used in dentistry (dental implant), plastic surgery, or orthopedics, and has significantly improved bone regeneration or formation, bone conduction, or bone induction. It may be indicative of skill.

The term "osteogenic potential" refers to the function of inducing or promoting bone matrix formation and bone regeneration (osteoanagensis) by osteoblasts, including both cartilaginous bone formation, connective tissue bone formation, and preformal bone formation. to include

The term "bone conduction ability" refers to a function of inducing or promoting bone matrix formation of osteoblasts by attracting osteoblasts.

The term "bone inducing ability" refers to a function of inducing regeneration into a desired bone tissue by accessing only cells and substances useful for bone tissue regeneration to a defect.

Among the terms or elements mentioned in the starfish-derived bone graft material manufactured by the method, the same ones mentioned in the description of the manufacturing method are understood to be the same as those mentioned in the description of the manufacturing method above.

Another aspect provides a bone graft material comprising a starfish-derived porous bone fragment.

The term "spicule" refers to a fine structure having a function of a skeleton in the body of an invertebrate animal, and is also called an incus. Specifically, the bone fragment may refer to a small bone fragment in the body of an invertebrate animal.

The bone fragment or bone graft material may include hydroxyapatite and/or tricalcium phosphate.

The starfish-derived porous bone fragments may have significantly improved porosity compared to general starfish-derived bone fragments. In addition, since the bone graft material includes the porous bone fragment derived from the starfish, it may be a bone graft material having porosity.

The bone graft material may have a porosity of about 50 to 80%. According to one embodiment, the bone graft material may have a porosity of about 60 to 80, about 65 to 80, about 70 to 80, about 75 to 80, or about 70 to 75%. According to one embodiment, when the above range is exceeded, the structure of the bone graft material may be collapsed, and when the above range is not reached, the porosity is too low, and it may be difficult for bone cells to penetrate and proliferate inside the bone graft material. As a result, the effect of promoting stem cell proliferation, the effect of promoting the differentiation of stem cells into osteoblasts, or the effect of promoting bone regeneration or formation of the bone graft material may be significantly reduced.

The diameter of the pores of the bone graft material may be about 15 to 50 um. Specifically, the diameter of the pore is about 20 to 50, about 25 to 50, about 30 to 50, about 35 to 50, about 15 to 45, about 20 to 45, about 25 to 45, about 30 to 45, about 35 to 45, about 30 to 40, or about 35 to 40 um. According to one embodiment, when the above range is exceeded, the structure of the bone graft material may be collapsed, and when the above range is not reached, the porosity is too low, and it may be difficult for bone cells to penetrate and proliferate inside the bone graft material. As a result, the effect of promoting stem cell proliferation, the effect of promoting the differentiation of stem cells into osteoblasts, or the effect of promoting bone regeneration or formation of the bone graft material may be significantly reduced.

The bone graft material may be composed of a plurality of particles. The particle size of the bone graft material may be about 0.1 to 3 mm. Accordingly, the bone graft material may exhibit a form that is easy to be transplanted to various tissue sites. For example, by filling the bone damaged area with the bone graft material, the defective area can be repaired regardless of the shape and thickness of the bone damaged area, and the bone graft material can be injected into the damaged area without a large incision.

According to one embodiment, the bone graft material may exhibit an effect of promoting stem cell proliferation, an effect of promoting the differentiation of stem cells into osteoblasts, or an effect of promoting bone regeneration or formation. Specifically, the bone graft material may have a structure with significantly improved porosity compared to an artificially synthesized bone graft material (or a commercially available bone graft material) and a bone fragment simply isolated from a starfish without using the above method. Chemical and structural properties can be displayed. Specifically, the bone graft material may have significantly improved porosity while containing microporosity and trace elements inherent in starfish bone fragments. As a result, the bone graft material has a significantly improved stem cell proliferation promoting effect, stem cell transformation into osteoblasts, compared to artificially synthesized bone graft materials (or commercially available bone graft materials) and bone fragments simply isolated from starfish without using the above method. It may exhibit a differentiation promoting effect or a bone regeneration or formation promoting effect.

Stem cells whose proliferation or differentiation is promoted by the bone graft material may be cells having bone differentiation potential.

The term "cells with bone differentiation potential" refers to all cells that have the ability to differentiate into osteogenic cells (eg, osteoblasts (or osteoblasts), chondrocytes, osteoblasts, etc.).

Specifically, the stem cells whose proliferation or differentiation is promoted by the bone graft material may be at least one selected from the group consisting of mesenchymal stem cells, bone progenitor cells, osteoblasts, and chondrocytes, but is not limited thereto.

The term "osteocyte progenitor cells" can refer to cells that have the potential to become bone cells and are present in the periosteum and bone marrow.

The term “osteoblasts (or osteoblasts)” or “chondrocytes” is a concept that includes even progenitor cells, which are cells whose differentiation direction has already been determined into osteoblasts (or osteoblasts) or chondrocytes.

The bone graft material may further include cells, for example, somatic cells or stem cells.

The term “stem cell” may refer to undifferentiated cells having the ability to differentiate into other cells. The stem cells may be embryonic stem cells, adult stem cells, induced pluripotent stem cells, or mesenchymal stem cells. The mesenchymal stem cells may be isolated from humans of various tissues, various races, or various ages. For example, the mesenchymal stem cells may be mesenchymal stem cells derived from adipose tissue, placenta, umbilical cord blood, muscle tissue, corneal tissue, or bone marrow tissue. Also, for example, the mesenchymal stem cells may be adipose stem cells, bone marrow stem cells, cord blood stem cells, neural stem cells, placental stem cells, or cord blood stem cells.

The bone graft material may further include a binding agent. The binder may refer to a material that physically fixes, binds, or aggregates the particles or components constituting the bone graft material, thereby preventing the bone graft material from immediately leaving the bone defect when the bone graft material is applied to the bone defect. can Examples of binders include dental resin cement, glass ionomer cement, collagen based glues, zinc phosphate, or phosphate cement such as magnesium phosphate ( phosphate-based cements); or known biocompatible materials such as protein-based binders such as zinc carboxylate, fibrin glues, or mussel-derived adhesive proteins.

The bone graft material may further include additives. The additive may be a medically acceptable additional component added to prevent infection of a bone defect or to further improve the bone formation, bone conduction, or bone induction function of the bone graft material. Examples of additives include drugs such as antiviral agents, antibacterial agents, antibiotics, anticancer agents, or angiogenic drugs; polysaccharide aqueous solution; amino acid; peptide; vitamin; cofactors for protein synthesis; growth hormones such as somatotropin; endocrine tissue fragments; enzymes such as collagenase, peptidases, or oxidases; live cells such as cartilage fragments, chondrocytes, or bone marrow cells; immunosuppressants; fatty acid esters; or nucleic acids, but is not limited thereto, and those skilled in the art may select one or more suitable additives according to the bone defect site and the condition of the target to be applied.

The bone graft material may be formulated in the form of a powder, suspension, emulsion, ointment, or injection according to a conventional method and applied to a bone defect site or surrounding area.

The term "dosage" means an amount sufficient to induce or promote bone formation or regeneration by being applied to a target site in need thereof. The dose of the bone graft material may vary depending on the patient's weight, age, sex, health condition, and degree of bone defect, and may be appropriately selected by a person skilled in the art.

The bone graft material may further include, but is not limited to, a medium, a differentiation promoting factor or protein, and a gene for improving the performance of stem cells in order to promote proliferation and osteogenic differentiation of stem cells.

In addition, the bone graft material may additionally apply a drug to the bone graft material for rapid recovery of damaged bone tissue. As the drug, genes involved in tissue regeneration, genes improving the performance of stem cells, regeneration proteins, etc. may be used, but are not limited thereto.

Among the terms or elements mentioned in the bone graft material, the same as those mentioned in the description of the manufacturing method and the starfish-derived bone graft material manufactured by the method are the same as those mentioned above in the manufacturing method and the starfish-derived bone graft material manufactured by the method. It is understood as the same as mentioned in the description of.

Another aspect provides a pharmaceutical composition for preventing or treating bone disease comprising the bone graft material as an active ingredient.

The term "effective ingredient" means an appropriately effective amount of an ingredient that affects a beneficial or desirable clinical or biochemical outcome. Specifically, it may mean an effective amount of bone graft material.

The effective amount can be administered once or more and prevent disease, or, without limitation, alleviation of symptoms, reduction of disease extent, stabilization of disease state (i.e., not worsening), delay of disease progression, or It may be an appropriate amount for reduction in rate, or amelioration or palliation and alleviation (partial or total) of the disease state.

The term "prevention" refers to any action that prevents the occurrence of a disease in advance, suppresses the disease, or delays the progression of the disease.

The term "treatment" refers to both therapeutic treatment and prophylactic or prophylactic measures. In addition, it means any action that improves or beneficially changes the symptoms of a disease.

The bone disease is osteomalacia, rickets, osteopenia, metabolic bone disease such as calcium dysregulation, osteolysis caused by bone metastases of cancer cells or wear debrid of artificial joints, endocrine diseases, or secondary diseases caused by drugs. It may be at least one selected from the group consisting of secondary bone loss, Paget disease, bone defect, alveolar bone defect due to periodontitis, osteonecrosis, and fibrous osteitis, but is not limited thereto.

The pharmaceutical composition may further include suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions. Usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil; and the like.

Among the terms or elements mentioned in the composition, the same as those mentioned in the description of the manufacturing method, the starfish-derived bone graft material produced by the method, and the bone graft material, are the same as those mentioned in the above manufacturing method and starfish manufactured by the method. It is understood as mentioned in the derived bone graft material, and in the description of said bone graft material.

Another aspect provides a method for preventing or treating bone disease comprising administering the bone graft material or a pharmaceutical composition for preventing or treating bone disease containing the bone graft material as an active ingredient to a subject.

The administration method of the bone graft material or the pharmaceutical composition containing the same as an active ingredient is not particularly limited, and may be, for example, injection into a bone damaged or diseased area or instillation during surgery of the area. For example, by filling the bone damaged area with the bone graft material, the defective area can be repaired regardless of the shape and thickness of the bone damaged area, and the bone graft material can be injected into the damaged area without a large incision.

The terms "administering", "introducing", and "implanting" are used interchangeably and result in at least partial localization of the bone graft material according to one embodiment or a pharmaceutical composition comprising the same as an active ingredient to a desired site. It can mean placement into an object by a method or path. The bone graft material according to one embodiment or a pharmaceutical composition containing the bone graft material as an active ingredient may be administered to a desired location in a living subject by any suitable route.

The subject may be a human or a non-human mammal. More specifically, the subject means a subject in need of prevention or treatment of a disease, and may mean mammals such as humans, non-human primates, mice, dogs, cats, horses, and cows.

Among the terms or elements mentioned in the treatment method, the same as those mentioned in the description of the manufacturing method, the starfish-derived bone graft material produced by the method, the bone graft material, and the composition, are the same as those mentioned in the above manufacturing method and the method. It is understood as mentioned in the description of the starfish-derived bone graft material prepared by, the bone graft material, and the composition.

According to the method for manufacturing a bone graft material derived from starfish according to one aspect, there is an effect of manufacturing a bone graft material with remarkably improved porosity while containing micropores and trace elements inherent in starfish bone fragments as they are. According to the bone graft material prepared by, cell proliferation, differentiation into osteoblasts, or bone regeneration or formation are significantly increased, so that it can be usefully used as a bone graft material.

1 is a result of confirming the structure of a starfish-derived bone fragment according to one embodiment with a scanning electron microscope.
2 is a result of confirming the change of the starfish-derived bone graft material subjected to the porosity improvement process according to one embodiment with a scanning electron microscope (A: before the porosity improvement process; B: after the porosity improvement process).
3 is a result of component analysis (A) and a result of confirming structural characteristics (B) of a starfish-derived bone graft material subjected to a porosity improvement process according to an embodiment.
4 is a graph showing the cell proliferation promoting effect of the starfish-derived bone graft material subjected to the porosity improvement process according to one embodiment.
5 is a graph showing the bone differentiation promoting effect of the starfish-derived bone graft material subjected to the porosity improvement process according to one embodiment.
6 is a graph (A) and an image (B) showing the bone formation promoting effect of the starfish-derived bone graft material subjected to a porosity improvement process according to one embodiment.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

[Comparative example]

Comparative Example 1: General bone graft material

As a comparative example, stoichiometrically synthesized HAp particles (pure HAp, particle size in the range of 200-500 μm) were used.

Comparative Example 2: Starfish-derived bone fragments

Starfish-derived bone fragments were prepared as follows. Amur starfish ( Asterias amurensis ) species collected from the west coast were used. Amur starfish individuals were cut to about 5x5 cm ² and immersed in about 10% NaOCl solution, followed by stirring for about 24 hours. After the first washing, the bone fragments were separated by stirring in about 10% NaOCl solution for about 12 hours and washing three or more times with a sufficient amount of distilled water. The separated bone fragments were dried in an oven at about 60°C for about 24 hours. Then, bone fragments dried in a state in which no additional porosity enhancement process was performed were used as comparative examples.

[Example]

Examples 1 to 12: Preparation of starfish-derived bone graft materials with improved porosity

A starfish-derived bone graft material having improved porosity was prepared as follows.

(1) Separation of bone fragments from starfish

Using the same method as in Comparative Example 2, bone fragments were separated from starfish to obtain starfish-derived bone fragments.

(2) treatment of acidic aqueous solution and ultrasonic

About 5 g of starfish-derived bone fragments were immersed in about 100 mL of an aqueous solution of about 0.2 pH HCl. After mixing for about 30 minutes at a speed of about 60 rpm using a propeller stirrer, the mixture was reacted at about 25° C. for 6, 12, 24, 48, 72, or 96 hours as shown in Table 1 below.

At the same time, as shown in Table 2 below, ultrasonic waves of about 20, 40, 60, 80, or 100 kHz are applied for about 5 minutes every about 20 minutes using an ultrasonic reactor to promote the reaction in the treatment reaction of the acidic aqueous solution. process was carried out.

After the acidic aqueous solution and ultrasonic treatment were completed, the bone fragment samples were washed with distilled water, continuously immersion washed for 3 days, and then dried in an oven at about 60 ° C for about 24 hours.

(3) hydrothermal reaction

The bone fragment sample was cut into small pieces with a grinder and filtered through a sieve to separate and collect bone fragments of a certain size, and a hydrothermal reaction was performed. The bone fragments were immersed in (NH ₄ )H ₂ PO ₄ at a concentration of about 2 M and reacted at about 200° C. for about 24 hours in a hydrothermal vessel. Thereafter, the bone fragments were sufficiently washed with distilled water and dried in an oven at about 60° C. for about 24 hours.

(4) sintering reaction

After the hydrothermalization reaction was completed, the bone fragment sample was heated at a rate of about 5 ° C per minute to reach about 600 ° C, heated for about 6 hours, and then heated again at about 5 ° C per minute to reach about 900 ° C, followed by about 6 heated for an hour.

Thereafter, natural cooling was performed to room temperature to finally prepare starfish-derived bone graft materials having improved porosity of Examples 1 to 12.

Comparative Example 2 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Acidic aqueous solution reaction time (h) 0 6 12 24 48 72 96 Ultrasound intensity (kHz) - 40 40 40 40 40 40 hydrothermalization ○ ○ ○ ○ ○ ○ ○ sintering ○ ○ ○ ○ ○ ○ ○

Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Acidic aqueous solution reaction time (h) 48 48 48 48 48 48 Ultrasound intensity (kHz) - 20 40 60 80 100 hydrothermalization ○ ○ ○ ○ ○ ○ sintering ○ ○ ○ ○ ○ ○

[Experimental Example]

Experimental Example 1: Confirmation of structure of starfish-derived bone graft material

In this experimental example, the following experiments were conducted to confirm the structural characteristics of the starfish-derived bone graft material.

Specifically, the starfish-derived bone fragments of Comparative Example 2 were observed under vacuum using a scanning electron microscope.

As a result, as shown in FIG. 1, it was confirmed that the starfish-derived bone fragment is a porous material and thus has the potential to be used as a material for a bone graft material.

Experimental Example 2: Confirmation of porosity improvement of starfish-derived bone graft material by additional process

(1) Check whether porosity is improved according to the acidic aqueous solution treatment reaction time

In this experimental example, the starfish-derived bone fragments of Comparative Example 2 were subjected to an additional process of treatment with an acidic aqueous solution for various times, and at the same time treated with ultrasonic waves at about 40 kHz to react, followed by a hydrothermal reaction and a sintering reaction. In order to confirm whether the porosity of the starfish-derived bone graft materials of Examples 1 to 6 in Table 1 was improved, an experiment was conducted to measure the porosity and average pore diameter.

Specifically, with respect to Comparative Example 2 and Examples 1 to 6, they were observed under vacuum using a scanning electron microscope. As for the porosity characteristics, the average pore diameter and porosity were measured using an automated mercury porosimeter by the mercury tube method. Values are expressed as mean ± standard deviation (SD), statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's using GraphPad Prism version 5.3 (GraphPad Software, SanDiego, CA, USA). It was performed using a post-hoc test. P<0.05 was considered statistically significant.

As a result, as shown in Table 3 below, Examples 1 to 6 showed superior porosity and larger average pore diameter than Comparative Example 2, and an example in which an additional process of acidic aqueous solution treatment reaction was performed for about 48 hours. It was confirmed that the bone graft material of No. 4 exhibited the most excellent porosity and the largest average pore diameter. In particular, when the process was performed for about 72 hours or more, the size of the hole increased too much, and the shape of the bone fragments could not be maintained intact and fragmented. did

Comparative Example 2 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Acidic aqueous solution reaction time (h) 0 6 12 24 48 72 96 Porosity (%) 50.23 55.36 67.28 70.91 73.42 - - Porous average diameter (um) 17.1 18.3 25.1 29.0 39.3 - -

In addition, as shown in FIG. 2, it was found that the size and number of holes in FIG. 2B showing Example 4 were significantly increased compared to FIG. 2A showing Comparative Example 2.

These results indicate that the bone graft materials of Examples 1 to 4 subjected to the additional porosity improvement step of the acidic aqueous solution treatment reaction were significantly improved in porosity compared to Comparative Example 2, making them more suitable as bone graft materials. In particular, the bone graft material of Example 4, which was subjected to an additional process of treatment with an acidic aqueous solution for about 48 hours, showed the most excellent porosity, which means that it was most suitable as a bone graft material.

(2) Check whether the porosity is improved according to the ultrasonic intensity

In this experimental example, the starfish-derived bone fragments of Comparative Example 2 were subjected to an additional process of treatment with an acidic aqueous solution for about 48 hours, and at the same time treated with ultrasonic waves of various kHz to react, followed by a hydrothermal reaction and a sintering reaction. In order to confirm that the porosity of the starfish-derived bone graft materials of Examples 7 to 12 in Table 2 was improved, an experiment was conducted to measure the porosity and average pore diameter.

Specifically, with respect to Comparative Example 2 and Examples 7 to 12, they were observed under vacuum using a scanning electron microscope. As for the porosity characteristics, the average pore diameter and porosity were measured using an automated mercury porosimeter by the mercury tube method. Values are expressed as mean ± standard deviation (SD), statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's using GraphPad Prism version 5.3 (GraphPad Software, SanDiego, CA, USA). It was performed using a post-hoc test. P<0.05 was considered statistically significant.

As a result, as shown in Table 4 below, Examples 8 to 12 prepared by ultrasonic treatment showed superior porosity and larger average pore diameter than Comparative Examples 2 and 7 prepared without ultrasonic treatment, and about It was confirmed that the bone graft materials of Examples 9 and 10 prepared by treating with ultrasonic waves at an intensity of 40 to 60 kHz exhibited the highest porosity and the largest average pore diameter. In particular, when ultrasound was treated at an intensity of about 80 kHz or higher, the size of the hole increased too much, and the shape of the bone fragment was not maintained intact and fragmented. confirmed that.

Comparative Example 2 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Ultrasound intensity (kHz) - - 20 40 60 80 100 Porosity (%) 50.23 55.19 58.23 73.43 79.62 ND ND Porous average diameter (um) 17.1 18.69 20.61 39.51 42.1 ND ND

These results indicate that the bone graft materials of Examples 8 to 10 prepared by performing the acidic aqueous solution treatment reaction and the additional porosity improvement process of ultrasonic treatment were compared to those of Comparative Example 2 and Compared to Example 7 prepared by performing only the acidic aqueous solution treatment reaction without ultrasonic treatment, the porosity is significantly improved, which means that it is more suitable as a bone graft material. and 10 showed the most excellent porosity, meaning that it was most suitable as a bone graft material.

Experimental Example 3: Confirmation of chemical and structural properties of starfish-derived bone graft material with improved porosity

In this experimental example, the following experiments were conducted to confirm the chemical and structural properties of the starfish-derived bone graft material with improved porosity.

Specifically, the experiment was performed by selecting the bone graft material of Example 4 having the most excellent porosity in Experimental Example 2. In order to analyze the composition of the bone graft material of Example 4, X-ray diffraction analysis (XRD; D8, Bruker AXS, Karlsruhe, Germany) using CuKα radiation was performed, and the scan speed was about 0.02°/min (2θ), about 50 It was analyzed under the condition of about 30 mÅ in the range of kV, about 10-80 °. The ionic composition of the samples was evaluated by X-ray fluorescence spectroscopy (XRF, Bruker). Values are expressed as mean ± standard deviation (SD), statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's using GraphPad Prism version 5.3 (GraphPad Software, SanDiego, CA, USA). It was performed using a post-hoc test. P<0.05 was considered statistically significant.

The microstructure and surface morphology of the bone graft material of Example 4 were observed under vacuum using a scanning electron microscope (SEM; EM-30, COXEM, Daejeon, Korea). The pore size and porosity of the bone graft material of Example 4 were measured using an automated mercury porosimeter by the mercury tube method.

As a result, as shown in FIG. 3, as a result of XRD analysis, the bone graft material of Example 4 has hydroxyapatite as a main component, and hydroxyapatite crystals are evenly formed on the surface of the graft material even after manufacturing is completed, and a specific porous form is obtained. confirmed to be maintained.

This means that the starfish-derived bone graft material with improved porosity can be an excellent bone graft material due to its chemical and structural specificity.

Experimental Example 4: Confirmation of cell proliferation promotion of starfish-derived bone graft material with improved porosity

In this experimental example, starfish-derived with improved porosity In order to confirm the effect of promoting cell proliferation of the bone graft material, the cell growth rate was confirmed. The process of the cell growth rate confirmation experiment is as follows. As a comparative example, artificially synthesized bone graft materials (Comparative Example 1) and starfish-derived bone fragments not subjected to the porosity improvement process (Comparative Example 2) were used.

Specifically, the cytocompatibility of the bone graft material was evaluated using human-derived mesenchymal stem cells (hMSC). Cells were cultured in a-MEM (Gibco-BRL, Gaitherburg, MD) containing about 10% fetal bovine serum (FBS) under conditions of about 5% CO ₂ and about 37°C, and cells subcultured for up to 6 stages were obtained. used in this experiment. Cell proliferation was evaluated by a mitochondrial activity-based assay using CellTiter96 ^® Aqueous One solution (MTS assay, Invitrogen, Carlsbad, CA, USA). hMSCs were seeded and cultured in Comparative Example and Example 4 at a concentration of about 1x10 ⁴ cells/well for 1, 3, 6, 9, and 12 days. At a predetermined time point, about 50 μL of CellTiter96 ^® reagent solution was mixed with about 250 μL of normal medium and added to each well. After about 4 hours of cell culture, the supernatant was collected and absorbance was measured at about 490 nm using an ELISA plate reader (SpectraMAX M3; Molecular Devices, Sunnyvale, CA). Values are expressed as mean ± standard deviation (SD), statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's using GraphPad Prism version 5.3 (GraphPad Software, SanDiego, CA, USA). It was performed using a post-hoc test. P<0.05 was considered statistically significant.

As a result, as shown in FIG. 4, the absorbance of starfish-derived Comparative Example 2 and Example 4 was significantly higher than that of artificially synthesized Comparative Example 1. In particular, the absorbance of Example 4, which is a starfish-derived bone graft material with improved porosity, was the highest.

This means that the cell proliferation promoting effect of the starfish-derived bone graft material is higher than that of commercially available bone graft materials, which means that it has excellent performance as a bone graft material. means

Experimental Example 5: Confirmation of promotion of differentiation of starfish-derived bone graft material with improved porosity into osteoblasts

In this experimental example, starfish-derived with improved porosity In order to confirm the differentiation promoting effect of the bone graft material into osteoblasts, the expression levels of ALP, Runx2, and osteocalcin (OC), which are bone differentiation related genes, were analyzed through quantitative real-time polymerase chain reaction (RT-PCR). The measurement process of bone differentiation marker gene expression level is as follows. As a comparative example, an artificially synthesized bone graft material (Comparative Example 1) and starfish-derived bone fragments not subjected to a porosity improvement process (Comparative Example 2) were used.

Specifically, for differentiation into osteoblasts, stimulants (about 0.1 mM dexamethasone, about 0.1 M b-glycerophosphte), About 50 ug/mL ascorbic acid) was added during cell culture. The normal growth medium and the medium containing the osteogenic stimulant were replaced every 2 days during the experiment. Total mRNA was isolated from the cells after 7 days of culture in a medium containing an osteogenic stimulant, and cDNA was transcribed with reverse transcriptase (Invitrogen) and oligo (dT) primers. cDNA was amplified with TaqMan Universal PCR Master mix (Applied Biosystem) and primers for ALP (Hs01029144_m1), Runx2, and OC (Hs01587814_g1) and TaqMan probe sets. All TaqMan PCRs were performed via the StepOne Plus RPCR system (Applied Biosystems, Foster City, CA, USA), and the 18S rRNA gene was co-amplified as an internal standard. Values are expressed as mean ± standard deviation (SD), statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's using GraphPad Prism version 5.3 (GraphPad Software, SanDiego, CA, USA). It was performed using a post-hoc test. P<0.05 was considered statistically significant.

As a result, as shown in FIG. 5 , the expression levels of bone differentiation-related marker genes of starfish-derived Comparative Example 2 and Example 4 were significantly higher than those of artificially synthesized Comparative Example 1. In particular, the expression level of marker genes related to bone differentiation in Example 4, which is a starfish-derived bone graft material with improved porosity, was the highest.

This means that the ability to promote bone differentiation of the starfish-derived bone graft material is higher than that of commercially available bone graft materials, which means that it has excellent performance as a bone graft material. means

Experimental Example 6: Confirmation of promotion of bone regeneration or formation of starfish-derived bone graft material with improved porosity

In this experimental example, starfish-derived with improved porosity Bone regeneration rate analysis and MT special staining were performed to confirm the effect of the bone graft material on promoting bone regeneration or formation. The experimental procedure is as follows. As a comparative example, an artificially synthesized bone graft material (Comparative Example 1) and starfish-derived bone fragments not subjected to a porosity improvement process (Comparative Example 2) were used.

Specifically, adult males (age > 3 months) using intramuscular doses of ketamine (ketamine, approximately 35 mg/kg; Yuhan Corporation, Seoul) and xylazine (approximately 5 mg/kg; Bayer Korea, Seoul) ) 4 New Zealand white rabbits (approximately 2.5–3.0 kg) were anesthetized. And local anesthesia was performed using about 2% lidocaine solution. After tissue resection, three isolated circular skull defects were made using a trephine with an outer diameter of about 6 mm. Each of the three defects was filled with about 40 mg of the bone graft material of Comparative Example 1, the bone fragment of Comparative Example 2, or the bone graft material of Example 4, respectively.

After transplanting Comparative Examples 1, 2, or Example 4 into the defect, the healing process was observed for 8 weeks, and after 8 weeks, the New Zealand white rabbit was euthanized, and the Comparative Examples 1, 2, or Example 4 were transplanted Bone tissue specimens were prepared by excising the defective defect from the rabbit bone together with the surrounding bone tissue. Before further analysis, the isolated bone tissue specimens were placed in a fixative (phosphate buffered 4% paraformaldehyde solution, pH 7.2) at about 4°C for 7 days. The newly formed bone was pathologically evaluated by micro-computed tomography (Micro-CT) (Sky-Scan 1172TM, Skyscan, Kontich, Belgium) under the following conditions.

Bone tissue specimens were analyzed using X-rays set at a voltage of about 60 kV and a current of about 167 μA using an about 0.5 mm aluminum filter. Based on the Micro-CT results, a qualitative image of the defect was observed with a 3D reconstruction image based on 3D software (CTVol, Skyscan), and the ratio of bone volume (bone volume, BV) was calculated as follows:

<Equation 1>

The ratio of bone volume (bone formation rate; BV, bone volume, %) = (new bone volume) - (volume of residual graft material) / total defect volume.

In addition, histological analysis was performed on bone tissue specimens. Bone tissue specimens were decalcified with about 8% formic acid/about 8% HCl and then dehydrated with graded aqueous alcohol solutions (about 70-100%). Finally, bone tissue specimens were embedded in paraffin. Sections of about 5 μm were cut using a rotary microtone (Microm, Walldorf, Germany). Five sections from the central part of each sample were stained with hematoxylin-eosin (HE) and Goldner's masson trichromand (MT). Then, samples were randomly selected and new bone formation was observed under a microscope (DMR, Leica, Nussloch, Germany). Values are expressed as mean ± standard deviation (SD), statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's using GraphPad Prism version 5.3 (GraphPad Software, SanDiego, CA, USA). It was performed using a post-hoc test. P<0.05 was considered statistically significant.

As a result, as shown in FIG. 6A, the bone graft material of Comparative Example 1 showed an average bone formation rate of about 23%. On the other hand, when the bone fragments of Comparative Example 2 were transplanted, the bone formation rate was about 35% on average, and the bone graft material of Example 4 showed the highest bone formation rate of about 43% on average.

In addition, as shown in FIG. 6B, it was observed that the bone graft material of Comparative Example 1 had no internal porosity and thus no new bone was formed inward, whereas the bone fragment of Comparative Example 2 formed new bone in the space due to the porous microstructure. It was confirmed that the new bone formation pattern inside the graft material was further increased in the bone graft material of Example 4.

This means that the bone graft material derived from starfish is an excellent bone graft material because it exhibits a significant new bone formation promoting effect compared to existing synthetic bone graft materials. In particular, new bone formation is significantly increased in the starfish-derived bone graft material with improved porosity compared to the original starfish-derived bone fragment, which means that the starfish-derived bone graft material with improved porosity is a superior bone graft material.

Claims (17)

(a) separating bone fragments from starfish;
(b) reacting by adding an acidic aqueous solution to the separated bone fragments;
(c) performing a hydrothermal reaction on the bone fragments obtained in step (b);
(d) performing a sintering process on the bone fragments on which the hydrothermalization reaction is completed; and
(e) a method of manufacturing a starfish-derived bone graft material having porosity comprising the step of cooling the bone fragments that have undergone the sintering process. The method according to claim 1, wherein the starfish is Asterias amureasis, spider starfish (Ophiolocus japonicus), red starfish (Certonardoa semiregularis), star starfish (Asterina pectinifera), sun starfish (Solarstar borealis), rod starfish (Solarstar paxillatus), A method of at least one starfish selected from the group consisting of Astropecten polyacantbus and Marthasterias glaciallis. The method according to claim 1, wherein the acidic aqueous solution is hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, Which is an aqueous solution of maleic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or p-toluenesulfonic acid. The method according to claim 1, wherein step (b) is to react for 10 to 50 hours by adding an acidic aqueous solution to the separated bone fragments. The method according to claim 1, wherein step (b) is to react by adding an acidic aqueous solution to the separated bone fragments and then treating them with ultrasonic waves. The method according to claim 5, wherein the ultrasonic treatment is to process ultrasonic waves of 10 to 70 kHz, the method. The method according to claim 1, wherein step (c) comprises performing a hydrothermal reaction by treating the bone fragment obtained in step (b) with microwaves. The method according to claim 1, wherein the hydrothermalization reaction of step (c) is performed at 30 to 600 ° C. for 2 to 40 hours. The method according to claim 1, wherein the sintering process of step (d) comprises: raising the temperature to 400 to 800 °C at a rate of 1 to 20 °C/min and maintaining the temperature for 1 to 8 hours; and
The method comprising raising the temperature to 600 to 1500 °C at a rate of 1 to 20 °C/min and maintaining the temperature for 1 to 8 hours. The method according to claim 1, wherein the starfish-derived bone graft material has a porosity of 50 to 80%. The method according to claim 1, wherein the starfish-derived bone graft material has a pore diameter of 15 to 50 um. The method according to claim 1, wherein the starfish-derived bone graft material exhibits an effect of promoting stem cell proliferation, an effect of promoting the differentiation of stem cells into osteoblasts, or an effect of promoting bone regeneration or formation. A bone graft material derived from starfish having porosity manufactured by the method of any one of claims 1 to 12. A bone graft material comprising a porous bone fragment derived from a starfish. The bone graft material according to claim 14, wherein the bone graft material has a porosity of 50 to 80%. The bone graft material according to claim 14, wherein a pore diameter of the bone graft material is 15 to 50 um. A pharmaceutical composition for preventing or treating bone disease comprising the bone graft material of any one of claims 14 to 16 as an active ingredient.

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