KR101890192B1 - Method for preparing bone grafting substitutes comprising ceramic granules - Google Patents

Abstract

The present invention relates to a method for producing a bone graft material in which hyaluronic acid and gelatin are bonded to ceramic grains having a plurality of holes passing through the entire grains, and a bone graft material produced thereby.
According to the present invention, a hyaluronic acid-gelatin solution is mixed with ceramic particles including through-holes and then crosslinked to obtain a bone graft material having improved strength and excellent biocompatibility.
Further, the shape of the mold can be modified as desired, so that the shape of the bone graft material can be manufactured to match the shape of the treatment site, thereby providing convenience of the procedure.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for preparing bone grafting substitutes comprising ceramic particles,

The present invention relates to a method for producing a bone graft material in which hyaluronic acid and gelatin are bonded to ceramic grains having a plurality of holes passing through the entire grains, and a bone graft material produced thereby.

Hard tissue transplantation from the patient (autologous) or the donor (allograft or xenotransplantation) is needed to support mechanical stress while also increasing natural bone regeneration and therapy.

Recently, bone graft materials have attracted medical attention as a substitute for injured bone or as a filler for hard tissue joints.

 The best method of bone grafting is autotransplantation, that is, transplantation from other parts of the body, but there is a limit to the condition of the recipient and possibility of harvesting. Therefore, bone grafts made from bone of animals or cadavers are being applied to clinical practice. For example, Korean Patent Laid-Open Publication No. 2010-0080028 discloses a method of manufacturing a sponge-like bone graft material by collecting the same kind or different kind of bone tissue and performing degreasing, demineralization, washing and sterilization in a short time. However, bone grafts using homologous or heterogeneous bone tissue may be exposed to potential infections such as mad cow disease or AIDS.

Calcium-phosphate-based ceramic bone graft materials such as HAp (hydroxyapatite), alpha / beta-TCP (Tricalcium phosphate) and BCP (biphasic calcium phosphate) similar to those of natural bone have been developed to improve these problems. Ceramic is used in dentistry and orthopedics because of its balance of mechanical strength and biodegradability.

On the other hand, hard tissue replacement materials have different requirements depending on the clinical application.

Block type bone graft materials are required for large defect sites, but they require a long time for manufacturing. Therefore, there is a limit in manufacturing bone graft materials most suitable for the defect sites. For example, Korean Patent Publication No. 2010-0061649 discloses a process for producing a porous block-type synthetic ceramic support, which comprises the steps of preparing an aqueous solution of a multi-branched PEG-thiol and a multi-branched PEG-acrylate in which the total sum of branches is greater than or equal to 5 And mixing the porous block-type synthetic ceramic support with the mixture to thereby immerse the porous block-type synthetic ceramic support. However, these block-type bone graft materials have a disadvantage of slow biodegradation because they have a thick skeleton structure.

Injection - type bone graft materials can compensate for the shortcomings of the block - like bone graft materials. However, they can not control porosity, which is an important factor affecting retention and bone formation, and have a disadvantage of low strength.

The granular bone graft material has the potential to overcome the above drawbacks and has excellent strength and easy applicability, and thus can be used as an excellent bone graft material. However, since these granular bone graft materials are not separated from each other and are separated from each other, in order to fill the defects at the time of surgery, they are transported in units of grains or mixed with a viscous material in a certain space, There was an inconvenience that it was necessary to use the above.

Patent Document 1. Korean Patent Publication No. 2010-0080028 (public date July 8, 2010) Patent Document 2. Korean Patent Publication No. 2010-0061649 (Published on June 8, 2010)

It is an object of the present invention to provide a method of manufacturing a bone graft material which can be easily manufactured into a desired shape using a mold.

It is also an object of the present invention to provide a bone graft material having excellent strength and easy cell adhesion and improved bone formation.

According to a preferred embodiment of the present invention, a method for producing a bone graft material comprises the steps of: preparing a hyaluronic acid-gelatin solution by mixing a hyaluronic acid solution and a gelatin solution; A method of manufacturing a ceramic particle, comprising: preparing ceramic particles having a plurality of holes penetrating the entire particles; Immersing the ceramic particles in the hyaluronic acid-gelatin solution to obtain ceramic particles having the hyaluronic acid-gelatin solution immersed therein; A step of injecting the ceramic particles into a mold, followed by freezing and lyophilization to obtain a bone graft material; Treating the bone graft material with a cross-linking solution to progress a cross-linking reaction; And freezing and lyophilizing the bone graft material having undergone the crosslinking reaction.

Also, the ceramic particles have a size of 0.1 to 10 mm and include 2 to 20 holes.

Further, the holes of the ceramic particles have a size of 100 to 1,000 mu m.

The ceramic particles of the present invention may be obtained by first extruding a composite comprising a carbon filament core and a ceramic shell to obtain a plurality of first filaments; Placing the plurality of composite filaments inside, placing the ceramic shell on the outside, and secondly extruding the filaments to obtain a second filament; And a step of burning out and sintering the secondary filaments to obtain ceramic particles.

Further, the ceramic particles are immersed in the hyaluronic acid-gelatin solution for 1 to 12 hours.

In addition, after the ceramic particles are injected into a mold, bubbles are removed under reduced pressure, and freeze-drying and freeze-drying are performed.

According to another preferred embodiment of the present invention, the bone graft material comprises: ceramic particles having a plurality of holes penetrating the whole of the grains; And crosslinked hyaluronic acid and gelatin.

Also, it is characterized in that the crosslinked hyaluronic acid and gelatin are contained in an amount of 1 to 10 parts by weight based on 100 parts by weight of the ceramic particles.

According to the present invention, a hyaluronic acid-gelatin solution is mixed with ceramic particles including through-holes and then crosslinked to obtain a bone graft material having improved strength and excellent biocompatibility.

Further, the shape of the mold can be modified as desired, so that the shape of the bone graft material can be manufactured to match the shape of the treatment site, thereby providing convenience of the procedure.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a sectional view of individual ceramic particles in a bone graft material produced by the present invention, which schematically illustrates a state in which a polymer is filled in (a) or partially adhered or coated (b) in a hole of a ceramic particle.
FIG. 2 is a schematic view of a method for producing a first filament and a second filament through two extrusion processes in the process of producing the ceramic particle of the present invention. ((A) First filament, (b) Section of second filament )
FIG. 3 is a photograph of ceramic particles immersed in a hyaluronic acid-gelatin solution in a wedge-shaped mold.
4 is a photograph showing the bone graft material produced by the present invention.
5 is a SEM photograph of the surface shape of the bone support produced by Example 1 (a) and Comparative Example 1 (b).

Hereinafter, the present invention will be described in detail. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately It should be interpreted in accordance with the meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined. Therefore, the constitutions described in the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the technical ideas of the present invention. Therefore, various equivalents which can be substituted at the time of application It should be understood that variations can be made.

In the present invention, the term "bone graft material" is also referred to as "bone filler", "bone substitute", "osteoporosis", and is defined as implantation when a part of bone tissue in a living body is deficient or needs reinforcement.

A method for manufacturing a bone graft material according to the present invention comprises:

Mixing a hyaluronic acid solution and a gelatin solution to prepare a hyaluronic acid-gelatin solution; A method of manufacturing a ceramic particle, comprising: preparing ceramic particles having a plurality of holes penetrating the entire particles; Immersing the ceramic particles in the hyaluronic acid-gelatin solution to obtain ceramic particles having the hyaluronic acid-gelatin solution immersed therein; A step of injecting the ceramic particles into a mold, followed by freezing and lyophilization to obtain a bone graft material; Treating the bone graft material with a cross-linking solution to progress a cross-linking reaction; And freezing and lyophilizing the bone graft material having undergone the crosslinking reaction.

Hereinafter, the method for producing the bone graft material of the present invention will be described in more detail.

First, a hyaluronic acid solution and a gelatin solution are mixed to prepare a hyaluronic acid-gelatin solution.

Herein, the gelatin solution may be prepared by dissolving gelatin having a strength of 100 to 500 bloom in distilled water (DI water), preferably 1 to 30 wt%, more preferably 5 to 20 wt% It is good to use.

The hyaluronic acid-gelatin solution may be prepared by slowly adding a hyaluronic acid solution to the gelatin solution. However, the gelatin solution and the hyaluronic acid solution may be separately prepared and mixed.

At this time, the hyaluronic acid solution preferably shows a concentration of 0.1 to 10% by weight, more preferably 0.5 to 5% by weight.

The gelatin solution and the hyaluronic acid solution may be mixed in a volume ratio of 70:30 to 95: 5, more preferably 80:20 to 90:10, , The mechanical strength, biocompatibility and shape of the extracellular matrix show excellent results.

Next, ceramic particles having a plurality of holes penetrating the entire particles are produced.

Here, the ceramic particles may preferably be formed to have a size of 0.1 to 10 mm, preferably 0.2 to 5 mm, and most preferably 0.5 to 3 mm. Here, if the size of the ceramic particles is too small, it is difficult to form holes penetrating the particles. If the size of the ceramic particles is too large, space between the particles becomes large and the strength may be deteriorated.

Further, the ceramic particles are characterized by having a plurality of holes passing through the whole of the particles. It is preferable to use two to twenty holes, more preferably three to ten holes .

In addition, the hole of the ceramic particles preferably has a diameter of 100 to 1,000 mu m, more preferably 200 to 700 mu m, and most preferably 300 to 500 mu m. When the holes of the ceramic particles are formed below the diameters described above, hyaluronic acid and gelatin adhering to the inside of the holes are hardly filled, so that the proliferation rate of the osteocytes is lowered. When the holes are formed in excess of the diameters described above, The lowering of the bending strength may be a problem.

The ceramic particles may have a cylindrical shape, for example. The plurality of holes may be vertically formed in the form of a channel so as to penetrate the upper surface and the lower surface of the column, but a plurality of holes may be formed The shape of the ceramic particles is not limited thereto.

The ceramic particles may be obtained by first extruding a composite material composed of a carbon filament core and a ceramic shell to obtain a plurality of first filaments; Placing the plurality of composite filaments inside, placing the ceramic shell on the outside, and secondly extruding the filaments to obtain a second filament; And burning-out and sintering the secondary filaments to obtain ceramic particles.

Hereinafter, the method of producing the ceramic particles of the present invention will be described in more detail.

First, a plurality of primary filaments are obtained.

Here, a ceramic shell, which is a biomaterial, is placed around the carbon filament core for forming a hole, and primary extruded to form a first filament having one carbon core. The ceramic shell may be composed of a mixture of a ceramic powder and a binder, and the mixture is preferably mixed in a shear mixer.

The type of the ceramic powder as the biomaterial is not particularly limited, but it is preferable to use hydroxyapatite (HAp), tricalcium phosphate (TCP), or a mixture of biphasic calcium phosphate (biphasic calciumphosphate, BCP) can be used.

The type of the binder is not particularly limited, but preferably ethylene vinyl acetate (EVA), polyethylene, wax, polypropylene, and praffin can be used. Most preferably, EVA can be used.

Also, the mixture may be selected from the group consisting of stearic acid (CH ₃ (CH ₂ ) ₁₆ COOH), polyethylene waxes, oxidized polyethylene waxes, esters, amides such as oleamide, erucamide, , Fatty acids (e.g., lauric acid, myristic acid, palmitic acid).

On the other hand, the carbon filament may be prepared by mixing the carbon powder with the binder or the lubricant and then extruding the carbon filament.

The binder or lubricant is used to maintain the viscosity of the carbon filament and the ceramic shell and can contribute to making the microstructure during extrusion.

Next, the plurality of primary filaments prepared above are extruded to obtain a secondary filament.

Here, it is preferable that the plurality of first filaments are placed in the interior, the ceramic shell is positioned outside, and then the second filaments are obtained by secondary extrusion. At this time, the number of holes of the ceramic particles can be adjusted by changing the number of the first filaments.

Next, the above-prepared secondary filament is burned out and sintered to obtain ceramic particles.

At this time, the secondary filaments can be cut to a desired size and then burned out and sintered, but it is also possible to cut after burning out and sintering in some cases.

The burning-out process may be performed in two steps to remove the binder and carbon, respectively. First, in order to remove the binder, the primary burn-out is preferably performed by slowly raising the temperature to 650 to 900 ° C. for 100 to 120 hours in an inert atmosphere of N ₂ atmosphere to degrease the binder. The secondary burn-out can also be performed by slowly raising the temperature to 1000 ° C in the atmosphere to remove carbon.

By maintaining the time required to reach the above-mentioned temperature range and temperature range, it is possible to remove the binder and the carbon component while preventing the deformation of the particles. Through the removal of the binder, fine pores in the particles can be formed, and holes through the particles can be formed through removal of the carbon component.

Next, the sintering process is performed at 1200 to 1400 ° C to obtain ceramic particles containing a plurality of holes while maintaining a constant strength.

Next, the ceramic particles are immersed in the hyaluronic acid-gelatin solution prepared above to obtain the ceramic particles in which the hyaluronic acid-gelatin solution is immersed.

Here, the ceramic particles are preferably immersed at about 30 to 40 DEG C for 1 to 12 hours. When the immersion time of the ceramic particles is less than 1 hour, the degree of adhesion of the hyaluronic acid-gelatin solution in the holes of the ceramic particles is lowered, and when it is more than 12 hours, the gelatin may be hardened or denatured.

The method of immersing the hyaluronic acid-gelatin solution in the ceramic particles is not particularly limited, and as an exemplary embodiment, a hyaluronic acid-gelatin solution may be added to the cylinder, followed by the addition of ceramic particles. In addition, the ceramic particles can be placed on the bottom of the vacuum container and the hyaluronic acid-gelatin solution mounted on the syringe can be slowly injected onto the ceramic particles through the filter.

The ceramic particles are preferably immersed in 1 to 2 g of 1 mL of the hyaluronic acid-gelatin solution, but the present invention is not limited thereto.

On the other hand, after the ceramic particles are injected into the mold, the bubbles are formed to lower the strength of the bone graft material. The bubbles may be removed. Preferably, the bubbles are removed under reduced pressure .

Next, the ceramic particles immersed in the hyaluronic acid-gelatin solution are injected into the mold, and freeze-dried and freeze-dried are carried out to obtain the bone graft material.

The freezing process may be performed at a temperature of -50 to -100 ° C for 12 to 36 hours, and the freeze-drying process may be performed at a temperature of -50 to -100 ° C for 24 to 60 hours to remove moisture.

Next, the bone graft material is treated with a crosslinking solution to proceed the crosslinking reaction. Since hyaluronic acid-gelatin is easily dissolved in water itself, a crosslinking reaction is required to maintain the shape of the support.

The crosslinking solution may be 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) solution, but is not limited thereto as long as it can crosslink the hyaluronic acid-gelatin. The treatment of the crosslinking solution is preferably performed at a temperature of 1 to 10 DEG C for 12 to 36 hours.

Since the cross-linking solution such as EDC is a harmful substance to the human body, it is preferable to wash it with distilled water or the like and ultrasonically treat it in order to remove it.

Next, the bone graft material treated with the crosslinking solution is preferably frozen and lyophilized, and the freezing process may be performed at a temperature of -50 to -100 ° C. for 1 to 10 hours, At a temperature of -50 to -100 < 0 > C for 12 to 36 hours.

Meanwhile, the bone graft material according to the present invention is characterized in that it comprises ceramic particles having a plurality of holes penetrating the entire grains, and crosslinked hyaluronic acid and gelatin.

The bone graft material can be manufactured by the above-described manufacturing method, and the constitution of the ceramic particles, the size of the holes, and the number of the bone grains are the same as those described above.

The bone graft material preferably comprises 1 to 10 parts by weight of the cross-linked hyaluronic acid and gelatin with respect to 100 parts by weight of the ceramic grains. The moisture content is removed through drying in a hyaluronic acid-gelatin solution, it means.

Particularly, the crosslinked hyaluronic acid and gelatin can be partially adhered or coated inside the holes of the ceramic particles, and in some cases, they may be filled in the holes. Cross-linked hyaluronic acid and gelatin formed inside the holes of the ceramic particles improve cell adhesion and make the bonding between the ceramic particles more robust (see FIG. 1).

The foregoing description is merely illustrative of the technical idea of the present invention, and various changes and modifications may be made by those skilled in the art without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the scope of the present invention but to limit the scope of the technical idea of the present invention. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas within the scope of equivalents should be construed as falling within the scope of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Manufacturing example  1. Preparation of ceramic particles

HAp and TCP were prepared as initial powders and carbon powder (15 ㎛, Aldrich, USA) was prepared for carbon filament production.

In addition, EVA (ethylene vinyl acetate) (ELVAX 210 and 250, Dupont, USA) and stearic acid (Daejung Chemicals & Metals Co. Korea) were used as binders and lubricants.

(HAv 24 vol% / TCP 24 vol% / EVA 42 vol% / stearic acid 10 vol%) and carbon filament core (carbon 48 vol% / EVA) using a shear mixer process (Shina Platec, Korea) 42 vol% / stearic acid 10 vol%).

Next, the ceramic shell was placed in the form of wrapping the carbon filament core, and extrusion was performed on the extrusion die to extrude the first filament.

The next seven primary filaments were wrapped with two ceramic shells and then subjected to secondary extrusion to obtain a secondary filament (see Fig. 2). (See Fig. 2)

The secondary filament was cut to a length of 0.7 mm.

Subsequently, the binder was removed through a first heat treatment process. For this purpose, the temperature was slowly raised to 800 ° C in an N ₂ atmosphere, followed by a primary heat treatment for 5 days to degrease.

The carbon component forming the hole was completely removed through the second heat treatment process. For this purpose, heat treatment was performed at 1000 ° C in an air atmosphere. In this process, through holes were formed with the removal of carbon.

The strength and shape of HAp / TCP powders were grown by sintering of the third heat treatment (maintained at 1200 ~ 1400 ℃ for 1 ~ 4 hours).

Through this, it was possible to obtain 0.7 - mm - sized ceramic particles with seven through - holes with a diameter of about 300 ㎛.

Example  1. Ceramic particles Combined Bone graft  Produce

Gelatin (10% by weight, 300 bloom) is dissolved in distilled water (DI water) at 30 ° C to prepare a gelatin solution. To the prepared gelatin solution, hyaluronic acid solution (0.5% by weight) was mixed at 37 캜 for 1 hour. At this time, the mixing ratio of the gelatin solution to the hyaluronic acid solution was adjusted to 85: 15 by volume. Then, 2.5 mL of the resulting hyaluronic acid-gelatin solution was added to the cylinder.

Next, 3.8 g of the ceramic particles prepared in Preparation Example 1 was added to the cylinder and immersed at 37 캜 for 3 hours to prepare a bone graft material.

The prepared bone graft material was filled into a wedge-shaped mold (see Fig. 3)

Then frozen at a temperature of -80 DEG C for 2 hours, and lyophilized overnight at a temperature of -70 DEG C for removing water.

Then, 4.79 g of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) and 1.15 g of N-hydroxysulfosuccinimide (NHS) were mixed with 500 ml of 80 wt% ethanol to prepare a crosslinking solution. The lyophilized bone graft material was cross-linked with the cross-linking solution at a temperature of 4 ° C overnight, washed with distilled water to remove the remaining cross-linking solution, and ultrasonicated for 10 minutes.

And frozen at a temperature of -20 DEG C for 2 hours and lyophilized at a temperature of -70 DEG C to finally obtain a bone graft (see FIG. 4)

Comparative Example  One. Sponge type Bone graft  Produce

Polyurethane sponge (60ppi, HD sponge, Korea) was repeatedly added to a calcium phosphate (BCP) slurry (5% PVB, 10% BCP powder) and dried to form a coating layer on the sponge.

The thickness of the coating layer was arbitrarily selectable. The coated sponge was maintained in a sintering furnace at 1,300 캜 for 10 minutes to prepare a calcium phosphate scaffold (BCP scaffold).

The prepared BCP support was placed on the bottom of the vacuum container and the hyaluronic acid-gelatin solution mounted on the syringe was slowly injected through the filter onto the BCP support. When the sponge BCP support was completely filled with hyaluronic acid-gelatin solution, it was frozen at -20 ° C for 4 hours. Thereafter, freeze-drying was carried out at -80 캜 for 48 hours to remove moisture.

Next, the crosslinking solution in Example 1 was crosslinked overnight at a temperature of 4 ° C., washed with distilled water to remove the remaining crosslinking solution, and ultrasonicated for 10 minutes to remove hyaluronic acid-gelatin-loaded Sponge-type bone graft materials were prepared.

Experimental Example  1. Measurement of surface shape of bone support

The surface morphology of bone graft materials prepared by Example 1 and Comparative Example 1 was measured by SEM and is shown in Fig.

Here, the surface shape of the bone graft material prepared in Example 1 is (a), and the surface shape of the bone graft material prepared in Comparative Example 1 is (b).

 5, the bone graft material prepared in Example 1 showed uniform distribution of hyaluronic acid-gelatin crosslinked in the through-hole and the surface, but the bone graft material prepared in Comparative Example 1 contained hyaluronic acid- It was found that the degree of adhesion of the gelatin inside the pores was small.

Experimental Example  2. Measurement of bone strength

The compressive strengths of the bone graft materials prepared in Example 1 and Comparative Example 1 were measured.

As a result, the compressive strength of the bone graft material prepared in Example 1 was 4.24 MPa, and the compressive strength of the bone graft material prepared in Comparative Example 1 was 3.6 MPa.

Therefore, it was confirmed that the strength of the bone graft material using the ceramic particles of the present invention is superior to that of the known sponge bone graft material.

100: Ceramic particles
200: Crosslinked hyaluronic acid-gelatin

Claims (9)

Mixing a hyaluronic acid solution and a gelatin solution to prepare a hyaluronic acid-gelatin solution;
A method of manufacturing a ceramic particle, comprising: preparing ceramic particles having a plurality of holes penetrating the entire particles;
Immersing the ceramic particles in the hyaluronic acid-gelatin solution to obtain ceramic particles having the hyaluronic acid-gelatin solution immersed therein;
A step of injecting the ceramic particles into a mold, followed by freezing and lyophilization to obtain a bone graft material;
Treating the bone graft material with a cross-linking solution to progress the cross-linking reaction of the hyaluronic acid-gelatin; And
Freezing and lyophilizing the bone graft material subjected to the cross-linking reaction
≪ / RTI >
The method according to claim 1,
Wherein the ceramic particles have a size of 0.1 to 10 mm.
The method according to claim 1,
Wherein the ceramic particles comprise 2 to 20 holes. ≪ RTI ID = 0.0 > 15. < / RTI >
The method according to claim 1,
Wherein the holes of the ceramic particles have a size of 100 to 1,000 mu m.
The method according to claim 1,
The ceramic particles
A step of first extruding a composite composed of a carbon filament core and a ceramic shell to obtain a plurality of first filaments;
Placing the plurality of composite filaments inside, placing the ceramic shell on the outside, and secondly extruding the filaments to obtain a second filament; And
And then obtaining the ceramic particles by burning out and sintering the secondary filaments.
The method according to claim 1,
Wherein the ceramic particles are immersed in the hyaluronic acid-gelatin solution for 1 to 12 hours.
The method according to claim 1,
Wherein the ceramic particles are injected into a mold, and the bubbles are removed under a reduced pressure condition, followed by freezing and freeze-drying.
And 1 to 10 parts by weight of crosslinked hyaluronic acid and gelatin, based on 100 parts by weight of the ceramic particles having a plurality of holes penetrating the whole of the particles,
Wherein the crosslinked hyaluronic acid and gelatin are adhered or coated inside the hole of the ceramic particle.
9. The method of claim 8,
The crosslinked hyaluronic acid and gelatin are characterized in that 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) crosslinked solution is treated with hyaluronic acid and gelatin at 1 to 10 ° C for 12 to 36 hours Bone graft material.

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