JP7629608B2 - X-ray opaque microgel fiber - Google Patents

Description

The present invention relates to a microfiber made of a hydrogel and containing an X-ray opaque material.

Hydrogels are formed from three-dimensional polymer networks that contain water, and are highly biocompatible and biodegradable. By micro-processing these, it is possible to create effective tools for delivering cells and drugs into the body, such as microgel fibers. Accurate delivery of drugs to the therapeutic target site is effective not only in increasing the therapeutic effect but also in reducing side effects, and in recent years, various DDS (Drug Delivery System) technologies have been developed. In addition, treatment methods that transplant healthy cells are being developed to treat diseases that develop when tissues necessary for maintaining normal biological functions become dysfunctional.

As such cell transplantation therapy, for example, a study on transplanting endothelial cells for liver cirrhosis (Non-Patent Document 1) and a study on transplanting bone marrow mononuclear cells for ischemic heart disease (Non-Patent Document 2) have been reported. In performing such cell therapy, in vitro tissue transplantation represented by cell sheets and cell suspension injection have been known as cell delivery systems that deliver cells to a desired location in the body. Among them, many studies have been conducted on cell suspension injection therapy, since it can be performed minimally invasively. In the conventional cell suspension injection method, there was a problem that the injected cells were dispersed and long-term engraftment was difficult. As a means to solve this problem, a core-shell type microhydrogel fiber that can be injected by a catheter (Non-Patent Document 3 and Patent Document 1) was developed.
This core-shell type microhydrogel fiber has excellent mechanical strength, and it is possible to construct three-dimensional structures such as a cylinder structure or a tube structure. Cell transplantation is possible by encapsulating cells to be transplanted in the core of the core-shell type microhydrogel fiber and delivering the cells to the transplant site using a catheter or the like. In addition, by encapsulating cells that secrete physiologically active substances in the core, it is possible to release cytokines and the like at the treatment site.

In this way, core-shell type microhydrogel fibers are effective in cell transplantation therapy. However, because hydrogel fibers have no fixed structure, it is necessary to confirm that they have been delivered to the desired site, but there is no way to visualize the fiber. Therefore, in order to ensure the safety of treatment, the development of a way to visualize the fiber is awaited.

WO2011/046105

Lee et al., Radiology 278:333-353 2016. Mitsutake et al., Int Heart J 58:435-440 2017. Onoe et al., Nat. Mater. 12:584-590 2013.

In view of the above circumstances, the present invention aims to provide a visualized microfiber.

In order to solve the above problems, the inventors created a core-shell microfiber with an X-ray-opaque substance encapsulated in the shell (Figure 1, left). When the microfiber was placed in an artery using a catheter, it was confirmed that the shape of the microfiber was clearly visualized (Figure 1, right).

That is, the present invention relates to the following (1) to (4).
(1) A microfiber made of a hydrogel and containing an X-ray opaque substance.
(2) The microfiber according to (1) above, characterized in that the microfiber has a shell portion and a core portion.
(3) The microfiber according to (2) above, characterized in that the X-ray opaque material is contained in the shell portion.
(4) The microfiber according to any one of (1) to (3) above, characterized in that the X-ray opaque substance is one or more selected from the group consisting of zirconia, tungsten, titanium, iodine, aluminum, copper, tin, gold, palladium and ruthenium.
In this specification, the symbol "to" indicates a numerical range including both the values on either side of it.

The present invention provides a visualized microfiber, the placement of which can be easily confirmed in vivo.

A conceptual diagram of a core-shell type microfiber containing a radiopaque material is shown. The microfiber exemplified here has a shell made of alginate hydrogel containing zirconia particles as a radiopaque material, and a core made of collagen gel containing cells and extracellular matrix (ECM). An example of a microfluidic device and a method for fabricating a fiber are shown. The results of observation of microfibers containing mouse embryonic fibroblast cells (NIH/3T3) in the core are shown. "Day 0" is a photograph of the microfiber immediately after preparation, and "Day 5" is a photograph of the microfiber cultured for 5 days after preparation. The diameter of the core and the outer diameter of the shell are shown under each photograph. A phase contrast micrograph (left) and an X-ray contrast image (right) of core-shell microfibers containing zirconia particles in the shell are shown. Microfibers containing 20% (w/w), 30% (w/w), 40% (w/w), or 50% (w/w) zirconia were prepared. The graph shows the results of evaluating the visibility (X-ray transmittance) of a zirconia-containing microfiber by an aluminum step. The right graph shows an image of the zirconia-containing microfiber and the aluminum step (an index of X-ray transmittance) taken by an X-ray imaging device, and the left graph shows the relationship between the zirconia concentration and the aluminum equivalent. The results of X-ray imaging after delivering zirconia-containing microfibers to a rat renal artery using a catheter are shown in the figure on the right, which shows a schematic diagram of the experiment, and the figure on the left, which shows an X-ray image. The X-ray contrast photograph (right) shows a core-shell type microfiber containing tungsten particles in the shell. Microfibers containing 60% (w/w) tungsten were produced. A phase contrast microscope photograph (top) and an X-ray contrast photograph (bottom) of a core-shell type microfiber containing titanium powder in the shell are shown. Microfibers containing 38% (w/w) titanium were produced. The phase contrast microscope photographs show core-shell microfibers that contain mesenchymal stem cells (MSCs) in the core and zirconia particles in the shell. "Day 1" is a photograph of a microfiber that had been cultured for one day after production, and "Day 3" is a photograph of a microfiber that had been cultured for three days after production. The results of measuring the concentration of Vascular Endothelial Growth Factor (VEGF) released from MSCs into the culture medium are shown. The prepared MSCs and zirconia-containing fibers were cultured, and the culture medium was collected every day and replaced with fresh medium. "Day 1" is the VEGF concentration in the culture medium collected on the first day of culture, and "Day 3" is the VEGF concentration in the culture medium collected on the third day of culture. Confirmation of the effect of the X-ray opaque microfiber as an alternative to contrast media. The microfiber according to the present invention was inserted into a Y-connector with a hemostasis valve (A), and photographed by moving the microfiber back and forth (B). The photographed images were integrated to confirm the branching positions of blood vessels (C). A renal failure model (1) created by using a radiopaque microfiber as an embolic material. X-ray angiograms of the kidneys of the model rat are shown. Pre, Post, and Completion respectively show abdominal aortic angiograms before placement of the radiopaque microfiber, plain X-ray photography after placement of the radiopaque microfiber, and abdominal aortic angiograms after placement of the radiopaque microfiber. Renal failure model created using radiopaque microfibers as an embolic material (2). The number of radiopaque microfibers used was adjusted to achieve an embolic area of 70% or more or less than 70%. Blood urea nitrogen (BUN) and creatinine (Cr) values were measured over time in the model rats. A cerebral infarction model (1) created using a radiopaque microfiber as an embolic material. The head X-ray of the model rat is shown. The arrow indicates the inserted radiopaque microfiber. A cerebral infarction model created using radiopaque microfibers as an embolic material (2). This is an MRI (T2 weighted) image of a model rat 24 hours after middle cerebral artery embolization.

Hereinafter, an embodiment of the present invention will be described.
An embodiment of the present invention is a microfiber made of a hydrogel, the microfiber containing a radiopaque material.
The microfiber according to the embodiment of the present invention is characterized in that it contains a radiopaque material and can be visualized by X-ray imaging. Whether or not a material is radiopaque in the present embodiment can be evaluated by its mass absorption coefficient. The larger the mass absorption coefficient, the higher the radiopaqueness. For example, suitable radiopaque materials include zirconia (zirconia dioxide, ZrO ₂ ) containing zirconium, tungsten (W), titanium (Ti), iodine (I), aluminum (Al), copper (Cu), tin (Sn), gold (Au), palladium (Pd), and ruthenium (Ru). Here, the mass absorption coefficients of zirconia, tungsten, and titanium when irradiated with an electron beam of 1 KeV are 4210 cm ² /g, 3683 cm ² /g, and 5869 cm ² /g, respectively. Although not particularly limited, the mass absorption coefficient of the X-ray opaque material in this embodiment is, for example, 1000 ^cm2 /g or more, preferably 2000 ^cm2 /g or more, and more preferably 3000 ^cm2 /g or more when irradiated with an electron beam of 1 KeV. For details of the X-ray mass absorption coefficient, see, for example, Masato Kazuo, Journal of the Japan Institute of Metals, Vol. 38 (1974) No. 2, etc.

The shape of the X-ray opaque substance contained in the microfiber is not particularly limited as long as the microfiber can be imaged, and examples thereof include particulate, powder, plate, string, fiber, porous, and liquid. The size of the X-ray opaque substance depends on the outer diameter of the microfiber to be produced, and can be easily selected by those skilled in the art. In addition, the size of the X-ray opaque substance can be easily evaluated by those skilled in the art, and can be measured, for example, under a microscope.
The amount of the X-ray opaque substance contained in the microfiber may be an amount that does not impair the shape or function of the microfiber and is clearly contrasted to a degree that allows visualization, and can be determined by a person skilled in the art by carrying out a preliminary experiment or the like. The appropriate content of the X-ray opaque substance varies depending on the substance used, but for example, when zirconia particles are used, the content is about 20% (w/w) to 70% (w/w), preferably about 30% (w/w) to 65% (w/w), and more preferably about 50% (w/w) to 60% (w/w) with respect to the hydrogel that constitutes the microfiber. When the amount of zirconia particles is 20% (w/w) or more, excellent X-ray visibility can be achieved, and when the amount is 70% (w/w) or less, excellent strength can be imparted for delivering the microgel fiber into a blood vessel or the like.

The shape of the microfiber of this embodiment is not particularly limited and can be appropriately selected depending on the application. For example, the microfiber has a fibrous shape, and the cross-sectional shape may be any shape, such as a circle, an ellipse, or a polygon such as a square or a pentagon. The outer diameter may be about 10 μm to 1000 μm for delivery into a blood vessel. The length is also not particularly limited and may be 100 nm to 3000 μm.
In the "microfiber made of hydrogel" of this embodiment, the type of hydrogel used as the substrate is not particularly limited, and may be, for example, a hydrogel formed by irradiating a water-soluble polymer such as collagen gel, chitosan gel, gelatin, peptide gel, fibrin gel, alginic acid gel, agarose gel, polyvinyl alcohol, polyethylene oxide, or polyvinylpyrrolidone with ultraviolet light, or a supramolecular hydrogel (see, for example, Dojin News, 118, pp.1-17 2006). The hydrogel of this embodiment may contain an aqueous organic solvent such as ethanol, acetone, ethylene glycol, propylene glycol, glycerin, dimethyl sulfoxide, or a substance that increases the strength of the hydrogel (for example, a crosslinking agent).
Furthermore, the microfiber of this embodiment may contain biological substances such as cells, proteins, lipids, sugars, and nucleic acids.

The microfiber made of the hydrogel according to this embodiment may have a multi-layer structure made of multiple types of hydrogels. Furthermore, the microfiber according to this embodiment may have a structure with a shell part (outer hydrogel) and a core part (inner hydrogel) by providing a layer made of another hydrogel on the outside (hereinafter also described as "core-shell type microfiber of this embodiment"). For example, in the left diagram of FIG. 1, the part "barium alginate gel + zirconia particles" corresponds to the shell part, and the part "cells + collagen" corresponds to the core part. The hydrogels constituting the shell part and the core part may be made of different types of hydrogels, or, in the case of the same type of hydrogel, may be made of hydrogels with different concentrations or strengths. Here, "different strengths" means that the mechanical strengths of the hydrogel constituting the shell part and the hydrogel constituting the core part are different. Examples of the base material of the shell part of the hydrogel constituting the core-shell microfiber of this embodiment include, for example, when the core part is collagen gel, chitosan gel, gelatin gel, polyvinyl alcohol hydrogel, etc., a gel of a different type and strength from these hydrogels, such as alginate gel, agarose gel, etc. Alternatively, when the shell part and the core part are the same type of hydrogel, such as alginate gel, the concentration of alginate may be different between the shell part and the core part.

When the microfiber of this embodiment is a core-shell type microfiber, the radiopaque substance may be contained in either the core portion or the shell portion depending on the purpose of use. When the main purpose is to confirm that the microfiber placed in the living body has been delivered to the desired site, the radiopaque substance may be contained only in the shell portion, and after the placement position is confirmed, the shell portion may be removed by the method described below. Alternatively, when cells or the like are contained in the core portion and placed in the desired position in the living body, and the dynamics of the cells are subsequently traced, the radiopaque substance may be contained in the core portion.

The microfiber of this embodiment can be produced using a microfluidic device or the like. For example, in the production of a core-shell type microfiber, a double coaxial microfluidic device (see FIG. 2 or Non-Patent Document 3, etc.) can be used. For example, in the case of producing a microfiber in which the shell part is made of alginate gel and the core part is made of collagen gel containing cells, as shown in FIG. 2, an alginate containing an X-ray opaque substance (e.g., zirconia particles) is injected into 2 (circle 2) and a collagen solution is injected into 1 (circle 1), and then ejected so as to be coaxial as shown in the figure. When an aqueous solution containing a crosslinking agent (e.g., divalent metal ions such as calcium salts and barium salts) for gelling the alginate is injected into 3 (circle 3) and ejected, the alginate is introduced into the aqueous solution containing the crosslinking agent and gelled. The collagen in the core part can be gelled by heating to an appropriate temperature (e.g., about 37°C).

When producing the microfiber according to this embodiment, the speed at which the solution for preparing the hydrogel (e.g., an alginate solution, a collagen solution, etc.) is injected may be, for example, about 5 μL to 600 μL/min when the diameter of the microfluidic device is about 40 μm to 3 mm, in order to control the shape as desired. The speed at which the solution for gelation (e.g., an aqueous solution containing a divalent metal ion, etc.) is injected may be, for example, about 1 to 10 mL/min.

After the microfiber according to this embodiment is produced, the hydrogel in the shell or core portion can be removed as necessary, leaving only the shell or core portion. The hydrogel can be removed, for example, by contacting the hydrogel with a substance that destroys the gel structure. For example, in the case of a hydrogel made of alginate, the hydrogel can be removed by contacting the hydrogel with a chelating agent at an appropriate concentration to remove the divalent metal ions that serve as crosslinking agents for gelation.

The microfibers according to this embodiment, particularly the core-shell type microfibers, have excellent strength and can be used to construct three-dimensional structures such as twisted yarn structures, woven fabric structures, helical structures, and tube structures. For the preparation method of the three-dimensional structures, see Non-Patent Document 3, etc.

Application 1. Delivery system for cell therapy
The microgel fiber of this embodiment can be used, for example, as a delivery system for cell therapy. Specifically, cells or the like can be contained in the microfiber (particularly in the core portion) and delivered to a transplantation site by a catheter or the like to perform cell transplantation. In addition, cells that secrete physiologically active substances can be encapsulated in the microfiber (particularly in the core portion) to release cytokines or the like at the treatment site.

2. Use as a contrast agent A contrast agent is usually a liquid substance that contains an X-ray opaque material. By injecting a contrast agent into a blood vessel and taking a digital subtraction angiogram (DSA) image, an angiographic image can be obtained by accumulating the acquired images.
Here, the microfiber of this embodiment can function similarly to a contrast agent by using not only the above-mentioned X-ray imaging but also a digital subtraction angiography (DSA) function in combination. Specifically, the microfiber of this embodiment can be moved within a blood vessel by relying on the blood flow like a conventional contrast agent, or can be moved within a blood vessel intentionally. By performing DSA imaging in this state, a blood vessel branching image can be obtained like when a contrast agent is used.
In most endovascular treatment procedures, the branching of blood vessels is identified and the target blood vessel is cannulated, and contrast media is used to identify the branching position of the blood vessel. If the microfiber of this embodiment is used in this procedure, the microfiber is inserted into the blood vessel only during DSA imaging, and can be removed from the blood vessel to the outside of the body immediately after imaging is completed, reducing the amount of contrast media used and avoiding the physical burden of contrast media (risk of contrast media-related diseases such as contrast media nephropathy and contrast media allergy).

3. Use as an embolic material The microfiber of the present embodiment can also be used as an embolic material that blocks blood flow by being placed in a blood vessel. For example, it can be used to create the following disease models.
Renal failure model By applying an intravascular treatment technique by simply puncturing the tail artery of an experimental animal such as a rat and selectively cannulating the renal artery, the microfiber of this embodiment can be placed in the renal artery to create a renal failure model.
3-2. Cerebral infarction model As in the renal failure model in 3-1 above, a selective middle cerebral artery occlusion cerebral infarction model can be produced by puncturing the tail artery of an experimental animal such as a rat, guiding a catheter under X-ray fluoroscopy guidance, and placing the microfiber of this embodiment as an embolic substance in the blood vessel.
As described above in 3-1 and 3-2, any organ can be approached as long as the blood vessel can be cannulated by an intravascular treatment technique or can be directly reached. Therefore, the microfiber of this embodiment can also be used for embolization therapy for various cancers and inflammatory diseases.

Where this specification is translated into English and contains the singular words "a,""an," and "the," these are intended to include the plural as well as the singular, unless the context clearly indicates otherwise.
The following examples are given, but are not intended to limit the scope of the present invention.

1. Method 1-1. Device fabrication The microfluidic device used to fabricate the microfiber is shown in Figure 2. A glass tube with a square cross section and a glass tube with a circular cross section and an inner diameter of about 300 μm at the tip were combined using a connector fabricated with a 3D printer and fixed onto a glass slide with adhesive.

1-2. Preparation of NIH/3T3 cell-containing fiber A 2.5% (w/w) sodium alginate solution dissolved in pure water was prepared as a reagent for the shell of the microfiber, and a 100 mM barium chloride solution was prepared as a crosslinking agent for gelling sodium alginate. At that time, sucrose was added to the barium chloride solution to make it 3% (w/w) of the total in order to adjust the osmotic pressure. For the core, a solution in which NIH/3T3 cells were suspended in a 4 mg/mL reconstituted collagen solution to a cell concentration of 1 x 10 ⁸ cells/mL was used. Each of the prepared solutions was filled into a syringe and attached to a syringe pump, and the prepared device and each syringe were connected with a Teflon (registered trademark) tube. Each solution was injected into a 145 mM sodium chloride solution to prepare a cell fiber. The flow rates of the core, shell, and cross-linking agent were set to Qcore, Qshell, and Qsheath, respectively, at 30 μL/min, 170 μL/min, and 2000 μL/min. The fabricated fibers were cultured in an incubator at 37°C and 5 vol% _CO2 , and the medium was replaced once every three days.

1-3. Preparation of zirconia particle-containing microfiber and tungsten particle-containing microfiber As a reagent for the shell of the microfiber, 2.5% (w/w) sodium alginate solution was added with φ30μm zirconia particles at concentrations of 20% (w/w), 30% (w/w), 40% (w/w), 50% (w/w) and 60% (w/w) or φ1μm tungsten particles at a concentration of 60% (w/w) and stirred using a vortex mixer, 1.5% (w/w) sodium alginate solution was prepared as a reagent for the core, and 100 mM barium chloride solution was prepared as a reagent for the crosslinking agent. Each solution was filled into a syringe and then placed on a syringe pump, and the prepared microfluidic device and each syringe were connected with a tube. In order to prevent the precipitation of the zirconia particles, the zirconia particles contained in the 2.5% sodium alginate solution were stirred with a homogenizer immediately before. The flow rate of the core part was Qcore, the flow rate of the shell part was Qshell, and the flow rate of the crosslinker was Qsheath. The flow rate of the Qcore was fixed at 30 μL/min, the Qshell at 170 μL/min, and the Qsheath at 2000 μL/min to prepare the microfiber. The prepared zirconia-containing microfiber was immersed in phosphate buffered saline, then sucked into a syringe and ejected through a catheter to check whether it could withstand shear force.

1-4. Preparation of titanium powder-containing microfibers As a reagent for the shell part of the microfiber, 325 mesh titanium powder was added to 2.5% (w/w) sodium alginate solution at a concentration of 38% (w/w) and stirred using a vortex mixer. As a reagent for the core part, 1.5% (w/w) sodium alginate solution was prepared, and as a reagent for the crosslinking agent, 100 mM barium chloride solution was prepared. Each solution was filled into a syringe and then placed on a syringe pump, and the prepared microfluidic device and each syringe were connected with a tube. In order to prevent the precipitation of titanium powder, the titanium powder contained in the 2.5% (w/w) sodium alginate solution was stirred with a homogenizer immediately before. The flow rate of the core part was Qcore, the flow rate of the shell part was Qshell, and the flow rate of the crosslinking agent was Qsheath. The microfibers were prepared by fixing Qcore at 15 μL/min, Qshell at 85 μL/min, and Qsheath at 2000 μL/min.

1-5. X-ray photography of microfiber
The prepared microfibers with zirconia contents of 20% (w/w), 30% (w/w), 40% (w/w), and 50% (w/w), tungsten content of 60% (w/w), and titanium content of 38% (w/w) were transferred to centrifuge tubes or dishes, respectively, and it was confirmed whether they could be imaged by an X-ray imaging device (Artis zee, Siemens Healthineers). Furthermore, the prepared microfiber with zirconia content of 60% (w/w) was delivered to the renal artery of a rat using a catheter, and it was confirmed whether X-ray imaging was possible. The imaging was performed by digital subtraction angiography (DSA). The imaging conditions were tube voltage of 60-70 kV, tube current of 100-200 mA, X-ray exposure time of 2-30 s, and number of frames of imaging of 15-225.

1-6. Evaluation of X-ray opacity due to aluminum step The fabricated fiber was transferred to a 100 mm diameter dish together with 25 mL of ultrapure water, and an aluminum step (A1050) with a thickness varying from 2 mm to 16 mm in 2 mm increments was placed next to the dish, and an X-ray fluoroscopic image was obtained. Using image analysis software, the brightness of the acquired image was measured to determine how many mm of aluminum the fiber was equivalent to, and the X-ray opacity was evaluated.

1-7. Measurement of Vascular Endothelial Growth Factor (VEGF) Released from Mesenchymal Stem Cells (MSCs) in Zirconia-Containing Fibers 1-7-1. Preparation of MSCs and Zirconia-Containing Fibers As a reagent for the shell of the microfiber, 30% (w/w) zirconia particles of φ30 μm were added to a 2.5% (w/w) sodium alginate solution dissolved in 145 mM sodium chloride solution, and the solution was stirred with a vortex mixer to prepare a 100 mM barium chloride solution as a crosslinking agent to gel the sodium alginate. At that time, sucrose was added to the barium chloride solution to adjust the osmotic pressure to 3% (w/w) of the total. For the core, a solution of human-derived mesenchymal stem cells (MSCs) suspended in 4 mg/mL reconstituted collagen solution to a cell concentration of 1 × 10 ⁸ cells/mL was used. Each prepared solution was filled into a syringe, attached to a syringe pump, and a device made using a glass tube with a circular cross section and an inner diameter of about 200 μm at the tip was connected to each syringe with a Teflon (registered trademark) tube. Each solution was injected into a 145 mM sodium chloride solution to prepare a zirconia-containing cell fiber. At this time, the flow rate of the core part was Qcore, the flow rate of the shell part was Qshell, and the flow rate of the crosslinker was Qsheath, with Qcore being 15 μL/min, Qshell being 85 μL/min, and Qsheath being 2000 μL/min. The prepared fiber was immersed in 10 mL of medium in an incubator at 37 °C and CO ₂ 5 vol % and cultured, and 1.75 mL of medium was collected every day and 1.75 mL of new medium was added to replace the medium. The collected medium was stored at -30 °C until use.

Measurement of VEGF concentration released from MSCs into the medium The VEGF concentration in the collected medium was measured by ELISA using the Human VEGF Quantikine ELISA Kit (R&D Systems). According to the instructions attached to the kit, a microplate containing the measurement sample was prepared, and measurements were performed using a plate reader (Thermo Scientific Multiskan FC, Thermo Fisher Scientific).
First, the plate reader was set up as follows: absorbance was measured at two wavelengths, 450 nm (main wavelength) and 570 nm (secondary wavelength), and the absorbance was calculated by subtracting the measurement value at the secondary wavelength from the measurement value at the main wavelength. To calculate the concentration using a calibration curve with Multiscan FC, the concentration was set in the calibrator wells in the plate layout settings, and the calculation method for creating the calibration curve was set to four-parameter logistic.
The microplate was loaded into the prepared plate reader and measurements were performed using the above settings.

2. Results 2-1. Cultivation of fibrous cells using barium alginate Figure 3 shows the observation results of the produced cell fiber on days 0 and 5. It can be seen that cells were encapsulated in the core on day 0, and that a fibrous tissue had been constructed in the center of the fiber on day 5. These results confirmed that it is possible to cultivate NIH/3T3 cells inside barium alginate gel.

2-2. X-ray transmission of zirconia-containing microfibers The left side of Figure 4 shows a micrograph of the prepared zirconia-containing microfiber. This result confirmed that increasing the concentration of zirconia particles improved the visibility of the microfiber. Next, the right side of Figure 4 shows the results of X-ray photography of the prepared microfibers with zirconia contents of 20% (w/w), 30% (w/w), 40% (w/w), and 50% (w/w) placed in centrifuge tubes. In all cases, the visibility was good in the overlapping parts of the fibers. On the other hand, when focusing on a single fiber that did not overlap, it was confirmed that the fiber was clearly visible in the transmission by adding zirconia particles at a rate of 50% (w/w) or more.
A microfiber with a zirconia content of 50% (w/w) was evaluated for visibility by X-ray fluoroscopy using an aluminum step. The results confirmed that it exhibited X-ray opacity equivalent to 3.2 mm aluminum (Figure 5). It was also found that zirconia particles with a particle concentration of approximately 30% (w/w) or more were visible through X-ray fluoroscopy.
Next, the results of X-ray imaging of the fiber delivered to the renal artery of a rat are shown in Figure 6. From this result, it was confirmed that the microfiber containing 60% (w/w) zirconia particles can be localized even in an in vivo environment.

2-3. X-ray fluoroscopy of tungsten-containing microfiber The X-ray contrast results of the prepared tungsten-containing microfiber are shown in Figure 7. It was confirmed that the microfiber with a tungsten content of 60% (w/w) was clearly visualized, even for individual fibers, as was the case with the microfiber containing zirconia particles.

2-4. X-ray fluoroscopy of titanium-containing microfiber The X-ray contrast results of the prepared titanium-containing microfiber are shown in Figure 8. It was confirmed that the fiber portion of the microfiber with a titanium content of 38% (w/w) was clearly visible, similar to the zirconia-containing microfiber and tungsten-containing microfiber.

2-5. VEGF concentration released into the culture medium from MSC- and zirconia-containing fibers Figure 9 shows phase contrast microscope images of the fabricated MSC- and zirconia-containing fibers on Day 1 and Day 3. It was confirmed that a fiber was constructed with MSCs encapsulated within it. In addition, VEGF released from MSCs was detected in the culture medium in which the fiber was cultured, and it was confirmed that the concentration increased over the course of culture time (see Figure 10).

2-6. Confirmation of the effect of radiopaque microfiber as a substitute for contrast media A microfiber with a zirconia content of 60% (w/w) was inserted into a structure simulating a blood vessel (angioplasty accessory kit, manufactured by Abbott, certification number: 20600BZY01008000) (Fig. 11A), and images were taken under DSA by moving the microfiber back and forth in the same way as wire manipulation (Fig. 11B). The branching positions of blood vessels could be confirmed by integrating the images (Fig. 11C).

2-7. Use of radiopaque microfibers as an embolic material 2-7-1. Renal failure model A renal failure model was created by selectively cannulating the renal artery through the application of an intravascular treatment technique by simply puncturing the tail artery of a rat and placing a microfiber with a zirconia content of 60% (w/w) in the renal artery (Fig. 12). It was also confirmed that the embolization area could be adjusted arbitrarily by changing the number of microfibers used when inserting the microfibers under X-ray fluoroscopy. By this, a "severe model" could be created by embolizing the embolization area to 70% or more in both kidneys, and the values of BUN (urea nitrogen) and Cr (creatinine), which reflect renal function, increased significantly. On the other hand, when the embolization area was less than 70%, a "mild model" with mild renal dysfunction was created (Fig. 13).

2-7-2. Cerebral infarction model As in the renal failure model, a rat's tail artery was punctured, a catheter was introduced under X-ray fluoroscopy guidance, and a microfiber with a zirconia content of 60% (w/w) was placed in the blood vessel as an embolic material to create a rat model of selective middle cerebral artery occlusion cerebral infarction (Figure 14). Since the material can be placed under X-ray fluoroscopy, it can be placed in the optimal position, making it possible to create a model with a stable infarct area (Figure 15).

The microfiber of the present embodiment has excellent visibility, can be placed in a living body and its position can be identified, and allows for safe and secure treatment. Therefore, the present invention is expected to be used in the medical field.

Claims (4)

A microfiber made of alginate gel, containing an X-ray opaque substance in an amount that allows X-ray imaging, characterized in that the mass absorption coefficient of the X-ray opaque substance is 3000 ^cm2 /g or more when irradiated with an electron beam of 1 keV, the microfiber having a shell portion and a core portion, and the content of the X-ray opaque substance is 20% (w/w) or more and 70% (w/w) or less . The microfiber according to claim 1, characterized in that the X-ray opaque material is dispersed and contained in a three-dimensional manner. 2. The microfiber of claim 1 , wherein the radiopaque material is included in a shell portion. 4. The microfiber according to claim 1 , wherein the radiopaque material is one or more selected from the group consisting of zirconia, tungsten, and titanium .