KR20190037504A - Cell therapy for treatment of bone disease of equine and method of manufacturing the same using 3-dimensional system - Google Patents

Abstract

The present invention relates to: a cell therapy product for treating and preventing bone diseases of equine animals by using cartilage and bone differentiation-induced mesenchymal stem cells of equine animals; and a method for manufacturing the same using a three-dimensional differentiation method. Specifically, the present invention relates to manufacturing cells having cartilage and bone regeneration ability by inducing early differentiation of mesenchymal stem cells of equine animals into osteocytes. In particular, the present invention relates to manufacturing cells having the ability to rapidly regenerate cartilage and bone in large quantities by forming a three-dimensional cell culture and differentiation environment using a hydrogel.

Description

TECHNICAL FIELD The present invention relates to a cell therapy agent for treating and preventing bone diseases in horses and animals, and a method for producing the same using the three-dimensional environment.

The present invention relates to a cell therapy agent for treating and preventing bone diseases in horses and animals, and a method for producing the same using a three-dimensional differentiation method using cartilage and bone differentiation-induced mesenchymal stem cells of equine animals.

There is a growing interest and need for veterinary drugs worldwide, and the global market size is increasing every year. In particular, demand for companion animals and leisure sports animals has exploded in recent years. In particular, horse and equine animals are increasingly valued as representatives of leisure sports animals, and so much attention and investment is being made in the treatment of horses and animals.

In the case of Gyeongju Mana Roadster, which is used in leisure sports, musculoskeletal fracture and inflammation are frequently occurred due to its characteristics. Because of its high maintenance cost during periods when it is not easy to treat and is not utilized, Is required. In addition, there is an increasing demand for new veterinary medicines that can minimize treatment time and effort by rapidly treating aftertreatment after treatment. In particular, it is required to develop a next-generation cell therapy agent for the effective treatment of musculoskeletal system capable of rapidly treating a lesion site.

As a solution to this problem, a stem cell treatment agent has been suggested. As a therapeutic agent using stem cell self-replicating ability and differentiation ability, stem cell treatment agent has been attracting attention as a treatment method for intractable diseases as a major axis of regenerative medicine. Although stem cell therapy is currently being carried out in a variety of studies and clinical trials for humans, research and development of stem cell treatment agents for animals has not been active yet. However, as interest in animal welfare has increased, the demand for and demand for stem cell therapies has increased greatly.

In particular, horses are highly valued for leisure sports, and the demand for treatment of musculoskeletal diseases is high. Therefore, there is a high demand for therapeutic agents with high cost and high efficacy such as stem cell therapy.

Korean Patent Publication No. 10-2011-0022759

A first object of the present invention is to establish three-dimensional culture and differentiation conditions optimized for stem cells of equine animals.

According to the existing bone cell differentiation technique, since the differentiation time from mesenchymal stem cells to bone cells takes about 3 to 6 weeks, it is difficult to secure sufficient amount of bone morphogenetic induction cells in a short period of time. Accordingly, the present invention provides a technique for rapid culture, proliferation and differentiation of stem cells in order to obtain a large amount of cells required for clinical treatment.

The second object of the present invention is to provide a method for treating and preventing osteoporosis of horses and animals by producing mesenchymal stem cells into osteoblasts by inducing differentiation of osteoblast cells using osteoblast- And to provide a therapeutic agent for cell for cell.

Accordingly, it is an object of the present invention to provide a therapeutic agent for bone diseases and a therapeutic agent for horses and animals, which have excellent cartilage-aggregate bioactivity based on the allograft method.

The present inventors completed the present invention based on the excellent cartilage-bone regeneration effect induced by differentiation of mesenchymal stem cells derived from equine animals into short-term bone cells in a three-dimensional differentiation environment.

As one embodiment of the present invention, mesenchymal stem cells derived from horses and animals are induced to differentiate into short-term osteoblasts in a three-dimensional differentiation environment to treat and prevent osteopathy of horses and animals including cells having cartilage-bone regeneration ability Cell therapeutic agent.

It is preferable that the mesenchymal stem cell is a umbilical cord mesenchymal stem cell (UCMSC) derived from horse and animal. The mesenchymal stem cell includes CD31 (negative), CD44 (positive), CD90 (positive), CD105 .

The mesenchymal stem cells can be initially transformed into bone-differentiated cells in a three-dimensional environment, thereby producing cells having cartilage-bone regeneration ability at the same time. That is, stem cells induced by cartilage-bone morphogenesis to have aggregate bioactivity have some characteristics of cartilage cells as early differentiation-induced cells as bone cells. The cells can express collagen type I, positive expression of alkaline phosphatase, and positive expression of collagen type II and SOX9, which are characteristic of cartilage differentiated cells, by the characteristics of bone differentiated cells.

The mesenchymal stem cells are preferably cultured and differentiated in a three-dimensional environment. The induction of early differentiation into bone cells is a process of differentiation into osteocytes in the differentiation medium in a short period of time, and short-term differentiation of about 24 hours is preferable.

The conditions for culturing and differentiation in the three-dimensional environment are such that when the hydrogel is formed on the culture container with a proper thickness by coating the cell culture container with the hydrogel, if the mesenchymal stem cells are inoculated into the hydrogel, The air is passed through to create a three-dimensional environment.

The hydrogel forming the three-dimensional environment preferably has a stiffness of about 2 kPa or less.

In another aspect of the present invention, there is provided a method for preparing a cell therapy agent for treatment and prevention of osteopathy and equine and animal bone diseases using a three-dimensional differentiation environment, comprising the steps of:

(a) synthesizing a hydrogel and coating the cell culture container to an appropriate thickness;

(b) inoculating mesenchymal stem cells derived from horse and animal into the coated hydrogel;

(c) culturing the inoculated cells in a three-dimensional environment for 24 to 48 hours to stabilize the cells; And

(d) inducing early differentiation of the cultured cells in a three-dimensional environment for 24 to 48 hours.

The method of the present invention is characterized in that stem cells are initially differentiated into osteoblasts in a three-dimensional environment, thereby producing cells having cartilage-aggregate ability.

The stem cells are preferably umbilical cord mesenchymal stem cells (UCMSC) from horses and animals.

In the step (a), the hydrogel is made of a water-soluble polymer having high cell affinity and may be one or more of hyaluronic acid, poloxamer, alginate, gelatin, aggregate, fibrin gel, Can be selected and synthesized.

In the step (b), the coating of the hydrogel is preferably performed at 36 ° C for at least 30 minutes and at most 24 hours, preferably at least 2 hours. The hydrogel thus hardened has a hardness of about 2 kPa or less.

The three-dimensional culture and differentiation method according to the present invention can obtain cells three times or more the same area as the conventional two-dimensional method, and the cell differentiation period can be drastically shortened. Therefore, it can be a very effective core technology when a cell is extracted from a tissue, cultured in a large amount, and then cell differentiation is induced to develop a cell therapy agent specific to the application site.

Accordingly, the present invention can provide an effect of maintaining excellent horseshoe value and prolonging the life span of a horse race by providing a cell therapy agent effective for treating and preventing bone diseases in horses and animals.

1 is a schematic diagram of a three-dimensional cell culture and differentiation method according to the present invention.
FIG. 2 is a view showing a three-dimensional cell culture and differentiation method according to the present invention. FIG. 2 (a) is a view showing a three-dimensional cell culture and differentiation method according to the present invention in which a medium in which both a culture medium and air are permeated is coated with a hydrogel, (B) is a method in which a culture container formed of a membrane permeable only to air is coated with a hydrogel, and cells are inoculated and cultured. At this time, the water-soluble hydrogel can be prepared and coated by using the medium so that the cells three-dimensionally present in the hydrogel can be supplied with nutrients even if the medium is not supplied.
FIG. 3 is a graph showing the degree of expression of CD31 (negative), CD44 (positive), CD90 (positive) and CD105 (positive) by flow cytometry in order to confirm that it is a umbilical cord mesenchymal stem cell.
FIG. 4 shows an image according to the stiffens of the hydrogel used for three-dimensional differentiation (A). (B) A graph showing the degree of bone differentiation by selecting a soft gel having a hardness of 0.5 kPa or less and a heavy gel having a viscosity of 1-2 kPa in order to select a hydrogel to be applied to the three-dimensional differentiation. Wherein the hydrogel A is a soft gel and the hydrogel B is a heavy gel.
FIG. 5 is a graph showing APL activity for confirming the degree of bone differentiation.
FIG. 6 is an image observed after staining with Alizarin Red, which is a bone differentiation label staining method, in order to confirm the degree of bone differentiation induced by 24 hour-differentiated cells. The figure below shows the degree of calcium deposition according to differentiation by measuring the absorbance after dissolving in Alizar Red dye.
FIG. 7 shows the results of confirming the expression of Sox9, COL1A1 and COL2A1 in order to identify cartilage and bone differentiation markers expressed in cells induced to differentiate for 24 hours.
FIG. 8 is an image of the result of the confirmation test of the aggregate bioassay using the mouse skull defect model.
FIG. 9 is an image showing the results of confirmation of the aggregate bioavailability using a mouse radial defect model.

Hereinafter, the present invention will be described in detail.

The term " equine animal " as used herein includes an overhead belonging to the antelope, horse, donkey, zebra, mule and the like. All existing horses and animals belong to Equus.

As used herein, "Mesenchymal Stem Cells (MSC)" refers to mesenchymal stem cells (MSCs) derived from mesodermal cells and having cells such as mesenchymal tissue (eg, bones derived from osteoblasts), dental tissues, cartilage, tendons, Quot; means a proliferative progenitor cell or a partially differentiated cell. Mesenchymal stem cells can be obtained from various tissues such as bone marrow, periosteum, cavernous bone, fat, synovium, smooth muscle, lung, kidney, umbilical cord, umbilical cord and umbilical cord.

As used herein, the term "differentiation" refers to a phenomenon in which the structure or function of a cell is specialized and differentiated while it is growing and proliferating, that is, a cell or tissue of a living organism has a form or function It means changing. For example, there may be qualitative or quantitative differences between parts of a biological system that were initially homogeneous, such as head or trunk distinction between eggs that were initially homogeneous in origin, or distinctions of cells such as muscle cells or neurons The state in which there is a difference or, as a result, is divided into partial systems that can be distinguished qualitatively is called eruption. In particular, the differentiation of stem cells refers to a phenomenon in which stem cells develop directionally into cells having specific functions.

As used herein, the term " a cell that is initially induced to differentiate into bone cells " means a cell differentiated to the stage of differentiation of immature osteoblasts or pre-osteoblasts in a bone development step, Osteoblast precursor cells (pre-osteoblast) or mature osteoblasts. As bone morphogenesis progresses in stem cells, it begins to express characteristic factors of osteogenic cells. For example, the expression level of alkaline phosphatase, which is an early marker of osteoblast differentiation expressed by mesenchymal stem cells, is increased.

The term " bone regeneration ability " as used herein means regeneration or healing of damaged, missing or deficient bones. As used herein, the term " cartilage regeneration ability " refers to regeneration or healing of cartilage damaged, missing or deficient.

Hereinafter, mesenchymal stem cells having an ability to produce aggregates of horses, animals and animals according to the present invention and a method for producing the mesenchymal stem cells will be described in detail with reference to examples and drawings. However, the present invention is not limited to these embodiments and drawings.

One aspect of the present invention is to provide a therapeutic agent for treating and preventing bone diseases in horses and animals comprising cells having cartilage-bone regeneration ability by inducing early differentiation of mesenchymal stem cells into bone cells in a three-dimensional environment .

The cells having cartilage-bone regeneration ability constituting the cell therapy agent according to the present invention are cultured in a three-dimensional environment for about 24 hours to 48 hours and then subjected to differentiation for about 24 hours to 48 hours, To induce early differentiation into bone cells.

The mesenchymal stem cells according to the present invention may be derived from various tissues such as fat, bone marrow, size, umbilical cord, and umbilical cord, and are preferably derived from umbilical cord.

In the case of umbilical cord-derived stem cells, the umbilical cord tissue that is discarded after delivery is used, which facilitates harvesting and allows a large amount of stem cells to be readily obtained. Although stem cells derived from fat or bone marrow are limited in their proliferation and differentiation potential due to the influence of the donor and the health condition of the donor to be extracted and extracted, the umbilical cord stem cells can be obtained in the earliest stage of adult stem cells As a stem cell, it is hardly influenced by stem cell function depending on a donor's age and other variables, and has excellent proliferation and differentiation ability. In addition, umbilical cord stem cells can be used for various diseases such as neurological diseases, liver diseases, musculoskeletal diseases and the like.

The mesenchymal stem cells are characterized by having CD31 (negative), CD44 (positive), CD90 (positive) and CD105 (positive) as expression factors (see FIG. 3).

The cells having the cartilage-bone regenerating ability can positively express collagen type I and alkaline phosphatase (Alkaline Phosphatase), which are characteristics of bone regeneration ability, while positively expressing collagen type II and SOX9, which are characteristics of cartilage cells And Fig. 7).

Another aspect of the present invention is a method for producing a cell therapeutic agent for treating and preventing bone diseases in an equine animal, characterized by using a three-dimensional culture and a differentiation method. Two-dimensional differentiation, which is an existing differentiation method, takes a long time to differentiate stem cells and is greatly influenced by differences between donors. Therefore, in the case of bone marrow differentiation using mesenchymal stem cells of horses and animals, a three-dimensional differentiation system which is optimized for horse and animal cells was established by adopting a three-dimensional differentiation system rather than a two-dimensional differentiation system having a large experimental deviation.

Specifically, the present invention provides a method for preparing a cell therapy agent for treating and preventing bone diseases in an equine animal, comprising the steps of:

(a) synthesizing a hydrogel and coating the cell culture container to an appropriate thickness;

(b) inoculating mesenchymal stem cells derived from horse and animal into the coated hydrogel;

(c) culturing the inoculated cells in a three-dimensional environment for 24 to 48 hours to stabilize the cells; And

(d) inducing early differentiation of the cultured cells in a three-dimensional environment for 24 to 48 hours.

The method for producing a cell therapeutic agent according to the present invention is characterized in that the induction of chondro-osteogenesis proceeds in a three-dimensional manner for a short period of time (see FIG. 1). As shown in FIG. 1, the desired stem cells can be cultured in a three-dimensional environment for a certain time and then differentiated by coating the hydrogel in an appropriate thickness in a cell culture medium.

Particularly, it is preferable that the present invention induces differentiation and induction of cells for a short period of time. In other words, the stem cells are differentiated in a three-dimensional environment for 24 to 48 hours, and then differentiated into bone cells for 24 to 48 hours. Through these induction of early differentiation, it is confirmed that stem cells simultaneously express cartilage markers and bone markers, thereby having improved cartilage-aggregate bioactivity (see FIGS. 5 and 7).

The three-dimensional culture and differentiation method according to the present invention uses a synthetic hydrogel. The hydrogel may be synthesized by one or more selected from the group consisting of hyaluronic acid, poloxamer, alginate, gelatin, collagen, fibrin gel and the equivalent thereof.

The synthesized hydrogel is coated in a cell culture container to an appropriate thickness to form a three-dimensional environment in the cell culture container. The hydrogel may be coated to such a thickness that the cells can be sufficiently cultured, and the thickness thereof is preferably about 5 mm or less.

The cell culture container may be an air permeable membrane or a culture vessel having an air permeable and a medium permeable quality membrane as a culture surface. The culture vessel may be made of polystyrene (PS), polytetrafluoroethylene (PTEF) And may be a polymer film such as polyethylene terephthalate (PET) or polycarbonate (PCF).

The three-dimensional environment of the hydrogel according to the present invention is characterized in that it creates an environment in which air can be passed through the gel in all directions (see FIG. 2). 2 (a) and 2 (b) are examples of a three-dimensional culture method in which air can pass through in all directions. FIG. 2 (a) is a view in which the medium is further supplied in addition to the cell culture fluid contained in the hydrogel, and the polymer membrane is suitable for culturing cells consuming large amounts of medium in a form capable of permeating the medium as well as air permeation. b) is a culture form composed of a membrane that can only permeate air. Both show a culture system capable of adjusting the three-dimensional environment according to the present invention.

In the three-dimensional differentiation method according to the present invention, it is preferable that the hydrogel is coated on a container and then allowed to harden for about 30 minutes to 24 hours at about 36 ° C, and to harden for 2 hours or more.

The hydrogel has a specific thickness and a specific stiffness property, and the stiffness of the hydrogel determines the mode of differentiation of the cells. In the present invention, a test was conducted to induce cartilage-bone differentiation using a soft gel (0.5 kPa or less), a heavy gel (1-2 kPa), a hard gel (5 kPa or more) and a soft gel (2 kPa or less) was preferred for cartilage-bone differentiation, and more preferably was a soft gel (see Fig. 4).

In the present invention, the expression of cartilage and bone markers in the cells induced to differentiate for 24 hours is checked, and both cartilage and bone markers are expressed. The markers are preferably collagen I and collagen II. In the present invention, the expression of SOX9 and collagen II, which are cartilage markers, is still expressed, but the expression of collagen I, a bone marker, is increasing. Thus, in the case of cartilage-bone differentiation, the expression level of the marker may vary but it is preferable that all of them express positive (see FIG. 7).

In the present invention, the degree of bone induction was determined by measuring the activity of alkaline phosphatase (ALP) to establish the differentiation conditions. In the two-dimensional bone differentiation method (general bone differentiation method), there was no change in ALP activity, but in the three-dimensional bone differentiation method, increase of ALP activity was confirmed from 24 hours (see FIGS. 5 and 6). The bone regeneration effectiveness of umbilical cord-derived mesenchymal stem cells according to the present invention was confirmed by a rat skull model and a mouse radial model (see FIGS. 8 and 9).

< Example  1>

Three-dimensional differentiation of umbilical cord stem cells

1-1. Acquisition (Establishment - Isolation) of umbilical cord stem cells

Obtained maternal wastes such as placenta released at the time of horse delivery, and umbilical cord was separated from this maternal waste. The arterial and venous blood vessels were first removed from the umbilical cord and the remaining tissue was minced and reacted with AdiCol ™ (CEFO Co.), a recombinant collagenase, at 37 ° C for at least 4 hours before the cells were extracted. The extracted cells were cultured in the medium of CEFOgro ™ eUCMSC (CEFO Co.) at 37 ° C and 5% CO2 to obtain mesenchymal stem cells.

1-2. The step of inducing the differentiation of umbilical cord stem cells

For induction of 2-D bone differentiation, 20,000 cells per cm 2 were inoculated into a culture dish and the medium was added with CEFOgro ™ eUCMSC to stabilize the cells for 24 hours. Then, the cells were exchanged with the differentiation medium CEFOgro (TM) DM Osteo (CEFO Co.) for induction of differentiation for 24 hours.

To induce three-dimensional differentiation, poloxamer gel (BASF, Germany) was completely dissolved in CEFOgro ™ eUCMSC medium to prepare bone differentiation induction. Hydrogel prepared in an air permeable culture container (Corning Co.) And coated with 55,000 cells per ㎠. The medium was supplemented with CEFOgro ™ eUCMSC and the cells were cultured for 24 hours to stabilize. Then, the cells were exchanged with the differentiation medium CEFOgro (TM) DM Osteo (CEFO Co.) for induction of differentiation for 24 hours.

< Example  2>

Identification of surface expression proteins of umbilical cord-derived mesenchymal stem cells

First, the cultured cells were collected, washed once with phosphate buffer saline (PBS), and further washed once with bovine serum albumin phosphate buffer saline (PBA) containing 2% bovine serum albumin protein The CD31, CD44, CD90, and CD105 antibodies were diluted 1: 100 in 2% PBA solution and reacted with the prepared cells for 1 hour. After the primary antibody reaction was completed, the sample was reacted with a secondary antibody attached to a fluorescent light for 1 hour, washed twice with PBS, and fixed with 2% formaldehyde to prepare a sample. The degree of surface expression of the protein was analyzed using BD Calibur (see FIG. 3).

< Example  3>

Identification of umbilical cord-derived mesenchymal stem cells into bone cells

3-1. Bone differentiation  Confirmation of ALP activity by induction time

Alkaline phosphatase (ALP) activity was measured for the cells induced to differentiate over a period of time according to the protocol provided by using a measurement kit, and the degree of induction of differentiation was determined by measuring alkaline phosphatase activity.

As a result, in the two - dimensional bone differentiation method (general bone differentiation method), it was judged that the ALP activity was almost not changed and the differentiation was not progressed. However, in the 3 - dimensional bone differentiation method, the ALP activity was increased from the 24th hour.

3-2. Bone differentiation  Determination of Calcium Deposition of Induction Cells

 In order to confirm the degree of calcium deposition in the oocyte - derived cells, it was confirmed that the osteogenesis was well induced by confirming the degree of calcium deposition by alizarin red staining induced by differentiation for 24 hours.

< Example  4>

Cartilage and Bone differentiation  Identify markers

24 hour-differentiated cells and undifferentiated cells were collected, treated with Trizol ™ (Invitrogen Co.) and chloroform (Sigma Co.), separated by centrifugation, and only mRNA was obtained. Transcriptor Universal cDNA Master Kit (Roche Co.) was used to synthesize mRNA obtained from cDNA. DNA was amplified by real-time polymerase chain reaction (RT-PCR) on SOX9, COL1A1 and COL2A1. The polymerase chain reaction conditions were 35 cycles of 95 ° C for 10 seconds, 54 ° C for 10 seconds, and 72 ° C for 30 seconds.

< Example  5>

Using the rat skull defect model Bone regeneration  Ability confirmation test

A 4 mm diameter bone defect model was made in the rat's calvaria. Three experimental groups were constructed to treat the cells and bone regeneration effect of each experimental group was confirmed for 8 weeks. The cells were obtained from adipose-derived mesenchymal stem cells (ADMSC) and umbilical cord mesenchymal stem cells (UCMSC). Three experimental groups were undifferentiated, osteogenic differentiated, undifferentiated and osteogenic differentiated group. The degree of bone regeneration was confirmed by μCT images.

< Example  6>

rat radius (radius) defect model Aggregate bio-function  Confirmation test

A 2 mm diameter bone defect model was made in the radius of the rat. Three experimental groups were constructed and treated with 1X106 cells per mouse to confirm the bone regeneration effect of each experimental group for 12 weeks. The cells were obtained from umbilical cord mesenchymal stem cells (UCMSC). The three experimental groups were control group treated with only scaffold, undifferentiated group, and bone differentiation induction group. The degree of bone regeneration was confirmed by μCT images and the amount of regenerated new bone was quantitated.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (11)

An agent for treating and preventing osteoporosis of horses and animals, comprising cells having cartilage-bone regeneration ability by inducing early differentiation of mesenchymal stem cells derived from an equine animal into osteoblasts in a three-dimensional environment. The method according to claim 1,
Wherein said mesenchymal stem cells are umbilical cord mesenchymal stem cells (UCMSC). The method according to claim 1,
Wherein said mesenchymal stem cells have CD31 (negative), CD44 (positive), CD90 (positive) and CD105 (positive) as expression factors. The method according to claim 1,
The cells having the cartilage-bone regenerating ability are characterized by positive expression of collagen type I and alkaline phosphatase, which are characteristic of bone regeneration ability, and positive expression of collagen type II and SOX9, which are characteristics of cartilage cells. Cell therapy. The method according to claim 1,
Wherein the three-dimensional environment is constituted by a hydrogel having a hardness of 2 kPa or less, and air flows in all directions. (a) synthesizing a hydrogel and coating the cell culture container to an appropriate thickness;
(b) inoculating mesenchymal stem cells derived from horse and animal into the coated hydrogel;
(c) culturing the inoculated cells in a three-dimensional environment for 24 to 48 hours to stabilize the cells; And
(d) inducing early differentiation of the cultured cells in a three-dimensional environment for 24 hours to 48 hours, thereby producing a cell therapeutic agent for treating and preventing bone diseases in equine animals. The method according to claim 6,
Wherein the hydrogel is selected from one or more selected from the group consisting of hyaluronic acid, poloxamer, alginate, gelatin, collagen, fibrin gel and its equivalent. The method according to claim 6,
Wherein the step (a) comprises coating the synthesized hydrogel on the cell culture container, and then solidifying the mixture at 36 DEG C for 2 hours or more. The method according to claim 6,
Wherein the hydrogel has a hardness of 2 kPa or less. The method according to claim 6,
Wherein said initial differentiation-induced cells have cartilage-bone regeneration ability. The method according to claim 6,
Wherein the three-dimensional environment is omnidirectional air is passed into the hydrogel.

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