WO2019124653A1 - Production method of oxygen-releasing hydrogel for bioinjection and biomedical use thereof - Google Patents

Abstract

The present invention relates to production of an oxygen-releasing hydrogel for bioinjection. A new type of oxygen-releasing in situ cross-linked hydrogel thus produced is formed as a hydrogel by inducing, by means of calcium ions released by decomposition of calcium peroxide, ion coupling with a negatively charged polymer, and generates and releases highly concentrated oxygen inside the hydrogel. Additionally, the present invention is novel in originating from the first of the studies into formation of a hydrogel using existing alginates to produce a hydrogel using calcium peroxide, is able to improve the limitations of existing oxygen-delivery systems, allows the production of oxygen-releasing hydrogel for bioinjection by a simple and rapid method without chemical synthesis, and has the further advantage of allowing the physical, chemical and biochemical characteristics of the hydrogel to be easily controlled in accordance with production conditions.

Description

METHOD FOR PREPARING BIO-INHIBITED OXYGEN-EMITTING HYDROGEL AND BIO-MEDICAL USE

The present invention relates to a method for producing a bioinjected oxygen-releasing hydrogel using calcium peroxide and a biomedical use thereof, and more particularly to a method for producing a bioinjected oxygen-releasing hydrogel using calcium peroxide. More particularly, the in situ bridged hydrogel is used in a previously reported method for preparing alginate- The present invention relates to a new type of injection-type polymer hydrogel which can be produced through ionic crosslinking of divalent cations using calcium peroxide and capable of releasing high-concentration oxygen in a formed hydrogel matrix.

In addition, the present invention can be manufactured by a simple method using a natural polymer, alginate, without chemical modification of a polymer, compared with a conventional method of manufacturing a bioinjectable hydrogel, and the physical / chemical / There is an advantage that the characteristic can be easily controlled.

Polymer hydrogels composed of a three-dimensional network of hydrophilic polymers are used in a variety of biomedical applications due to their biocompatibility, high water content, good permeability of nutrients and metabolites, structural similarity with natural tissues and multi-tunable properties Has been widely used.

In particular, in situ bridged hydrogels have been extensively studied as drug / cell carriers, tissue fillers, or tissue engineering supports based on minimally invasive techniques. These in situ bridged hydrogels can be prepared using natural and synthetic polymers, and hydrogels can be formed through various chemical and physical crosslinks.

Alginate is an anionic polysaccharide derived from brown algae, and is excellent in biocompatibility and biodegradability and has been applied in various fields in biomedical applications. Ionotropic crosslinking occurs in the presence of divalent cations such as calcium (Ca ²⁺ ), magnesium (Mg ²⁺ ), or zinc (Zn ²⁺ ) ions in the alginate polymer main chain to form a three-dimensional hydrogel network In recent years, calcium carbonate (CaCO ₃ ), calcium chloride (CaCl ₂ ), zinc carbonate (ZnCO ₃ ) and the like have been used as new crosslinking agents. It is known that calcium peroxide is decomposed into calcium hydroxide in the presence of water and generates hydrogen peroxide or oxygen, and is capable of releasing calcium ions. Taking advantage of these characteristics, calcium peroxide is widely used in various biomedical applications, but no alginate-based bioinjected oxygen-releasing hydrogel has been reported using calcium peroxide.

Oxygen acts as a metabolic substrate and a signal molecule, which plays an important role in homeostasis maintenance and wound healing. In particular, hyperbaric oxygen promotes cell proliferation by temporarily increasing oxygen partial pressure in the cell, increasing reactive oxygen species, and promoting collagen synthesis and processing by oxygen-based lysyl oxidase Promotes angiogenesis. It also promotes angiogenesis and wound healing by causing secretion of growth factors that promote angiogenesis or by allowing stem cells to migrate from the bone marrow. In this context, several technologies are recently being developed to transport oxygen.

This includes, for example, the use of hyperbaric oxygen therapy (HBOT) or an oxygen carrier such as hemoglobin-based oxygen carrier, perfluorocarbon (PFC) technology. High-dose oxygen therapy is technically simple and oxygen can be provided continuously during treatment, and the oxygen carrier has the advantage that it can release oxygen according to the oxygen partial pressure surrounding it.

However, the former requires specialized facilities for treatment, oxygen can only be provided by respiration, and the latter has the limitation of rapid release of oxygen.

To solve this problem, research has been conducted on biomaterials that produce oxygen in situ for local and sustained-release oxygen delivery. Hydrogen peroxide, sodium percarbonate, and calcium peroxide are representative examples of such oxygen-generating substances.

However, these materials have limited early oxygen release behavior and limited long-term oxygen release.

Accordingly, there is a great need to develop an in situ formed hydrogel having excellent biostability while releasing a high concentration of oxygen in a sustained release form.

The present invention meets the above-mentioned conventional demands and reports new researches that have not been reported so far. The new type of bioinjection-type alginate-based hydrogel that releases oxygen at a high concentration, the physical / chemical / The present invention provides a method for producing a sustained release oxygen-containing injection-type hydrogel which can easily control biological characteristics, and its various biomedical uses.

In order to achieve the above technical object, the present invention relates to a method for producing an ionic cross-linking agent, wherein the natural alginate polymer composed of mannuronic acid and glutaric acid, which is not chemically modified, Based cross-linked polymer hydrogel that produces a crosslinked alginate-based hydrogel and at the same time releases a high concentration of oxygen by decomposition of calcium peroxide in an aqueous solution.

In the present invention, the bioinjection type oxygen release which can easily control the physical / chemical / biological properties of hydrogels such as hydrogel mechanical strength, hydrogen peroxide release, oxygen release behavior and the like by controlling composition and concentration of alginate, calcium peroxide and catalase A method for producing a hydrogel is provided.

The present invention also provides an implant material for tissue regeneration and filling comprising an alginate-based in situ crosslinked oxygen releasing hydrogel.

The present invention also provides a material for tissue adhesion and hemostasis comprising an alginate-based in situ crosslinked oxygen releasing hydrogel.

The present invention also provides a carrier for a physiologically active substance or drug carrier, which comprises an alginate-based in situ crosslinked oxygen releasing hydrogel.

The new type of in situ crosslinked hydrogel according to the present invention is characterized in that hydrogels formed by calcium ions generated by the decomposition of calcium peroxide and the release behavior of high concentration oxygen generated in the hydrogel can be controlled.

In other words, the present invention has the novelty of inventing a new alginate hydrogel using calcium peroxide, which has not been reported in studies on the preparation of hydrogels using divalent cations, and can overcome the limitations of oxygen generating hydrogels Based on excellent biocompatibility, various biomedical applications (eg tissue regeneration, artificial tissue preparation, wound healing material, tissue adhesive material, drug delivery system, etc.) are possible.

FIG. 1 is a schematic view showing the formation of a hydrogel for generating oxygen based on alginate.

FIG. 2 is an image of decomposition of calcium peroxide and formation of a hydrogel according to a buffer solution.

FIG. 3 is a graph showing the hydrogel formation using calcium peroxide and the mechanical strength varying with the concentration of alginate and calcium peroxide (A: alginate, C: calcium peroxide).

4 is a graph showing hydrogen peroxide decomposition behavior using catalase (A: alginate, C: calcium peroxide).

5 is a graph showing the oxygen release behavior with addition of calcium oxide concentration and catalase (A: alginate, C: calcium peroxide).

Fig. 6 is a diagram showing cell fitness of a hydrogel (A: alginate, C: calcium peroxide).

Hereinafter, the present invention will be described in more detail.

As a result of intensive studies, the present inventors have found that a new type of in situ formed hydrogel using calcium peroxide is produced, and a high concentration of oxygen (more than 80%) is generated in such a hydrogel, 25% or more) continued for 48 hours. In addition, it has been found that a polymer hydrogel material capable of releasing oxygen at a high concentration in a simple manner without chemical modification of the polymer can be manufactured, and physical / chemical / biological properties can be easily controlled.

The present invention employs calcium peroxide for natural / synthetic polymers with anions to induce ionic interactions and thereby produce oxygen-releasing in situ hydrogels.

Referring to FIG. 1, alginate having a gluconic acid monomer unit having an anion is a natural polymer without chemical modification.

The polymer main chain may be selected from the group consisting of Arabic Gum, Tamarind Gum, Karaya Gum, Guar Gum, Locust Bean Gum, Quince Seed Gum, Xanthan Gum, Cyclodextrin, Casein, Tara Gum, Tragacanth Gum, Glucomannan, Glucosamine, Gum Ghatti, Furcelleran, Pullulan, Gellan Gum, Pectin, Agar, Alginate, Carrageenan, Dextrin, Poly Sodium alginate, sodium polyacrylate, propylene glycol alginate, methyl cellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethyl starch, ), Sodium alginate (Sodiu m Alginate, and sodium caseinate. However, the present invention is not limited thereto.

2, the oxygen-releasing hydrogel can be produced by in situ crosslinking of a polysaccharide main chain having an anionic glucuronic acid unit by inducing ionic interaction by release of calcium ions in a solution containing calcium peroxide have.

In the method of preparing the oxygen-releasing in situ fast-crosslinking hydrogel of the present invention, DPBS, which is a commonly used buffer solution, does not form a hydrogel because calcium ionization is limited, whereas in a Tris-HCl buffer solution , A hydrogel is formed by utilizing the fact that calcium ions are released. This was also applicable to calcium hydroxide with low solubility in water to form a hydrogel.

The present invention also provides a tissue regeneration, tissue engineering support and implant implant material comprising an oxygen-releasing in situ crosslinked hydrogel.

These materials include cartilage regeneration, bone regeneration, alveolar regeneration, skin regeneration, cardiac tissue regeneration, artificial intraocular lens, spinal nerve A regeneration and augmentation, a barrier for adhesion, a urinary incontinence treatment, a filler for wrinkle removal, a burn A wound dressing, a tissue augmentation, and an intervertebral disc treatment. However, the present invention is not limited thereto.

The present invention also provides a material for tissue adhesion and hemostasis comprising the oxygen-releasing in situ crosslinked hydrogel.

The material for hemostasis includes at least one of neurosurgical surgery including a vascular surgery area, orthopedic surgery including adhesion of bone, hemostasis of a laceration patient, suture of a femoral artery, cataract incision suture, cartilage healing, skin joining, Gastrointestinal tract differentiation, and tendon / ligament healing. However, the present invention is not limited thereto.

The present invention also provides a carrier for a physiologically active substance or a drug delivery carrier comprising the oxygen-releasing in situ crosslinked hydrogel.

The physiologically active substance or drug may be any one or more selected from the group consisting of a peptide or protein drug, an antibacterial agent, an anti-cancer agent, and an anti-inflammatory agent, but is not limited thereto.

The peptide or protein drug may be a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), a transforming growth factor (TGF), a bone morphogenetic factor protein, BMP), human growth hormone (hGH), porcine growth hormone (pGH), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO) , Macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), platelet-derived growth factor , Interferon-α, β, γ, interleukin-2 (IL-2), calcitonin, nerve growth factor (NGF), growth hormone Factor, angiotensin, luteinizing hormone releasing hormone (luteinizin (LHRH agonist), insulin, thyrotropin-releasing hormone (TRH), angiostatin, endostatin, somatostatin, glucagon, endorphin, But are not limited to, any one selected from the group consisting of neuron, mergein, cholestin, bacitracin, monoclonal antibody, vaccine, and mixture thereof.

Wherein said antimicrobial agent is selected from the group consisting of minocycline, tetracycline, oproxacin, phosphomycin, mergein, proproxacin, ampicillin, penicillin, doxycycline, thienamycin, cephalosporin, noradocin, gentamicin, neomycin, It may be advantageous to use a compound selected from the group consisting of paromomycin, micronomycin, amikacin, tobramycin, dibecasin, cytotoxic, sepracur, erythromycin, cyprofloxacin, levofloxacin, enoxasin, vancomycin, imipenem, foscidic acid and mixtures thereof , But the present invention is not limited thereto.

Wherein the anticancer agent is selected from the group consisting of paclitaxel, texothier, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cispletin, carboplatin, doxorubicin, daunorubicin, dirubicin, 5-fluorouracil, methotrexate, Actinomycin-D, and mixtures thereof, but is not limited thereto.

Wherein the anti-inflammatory agent is selected from the group consisting of acetaminophen, aspirin, ibuprofen, diclofenac, indomethacin, piroxycam, fenoprofen, plubiprofen, ketoprofen, naproxen, Camphor, tenoxicam, and mixtures thereof, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Example 1 Preparation of Material

Alginic acid sodium salt, calcium peroxide (CaO ₂ ), calcium hydroxide (Ca (OH) ₂ ) and catalase were purchased from Sigma Aldrich (St. Louis, Mo., USA) , UltraPure ™ 1M Tris-HCI (pH 8.0) buffer was purchased from Invitrogen (Carlsbad, CA, USA).

 (DMEM), penicillin-streptomycin (P / S), trypsin / EDTA and DPBS (Dibbecco's modified phosphate buffered saline (DPBS), newborn fetal serum Calf Serum (NBCS) solution was purchased from Gibco (Grand Island, NY, USA). Cell Proliferation reagent WST-1 was also purchased from Roche Diagnostics, and the Live / Dead Viability / Cytotoxicity Kit was purchased from Life science.

Other chemicals and solvents were used without further purification.

Example 2 Hydrogel Formation (Figure 1)

The hydrogel was prepared by mixing a solution of the alginate polymer dissolved in DPBS at 37 DEG C and calcium peroxide (0 to 0.75 wt%).

EXPERIMENTAL EXAMPLE 1 Formation of hydrogel according to buffer solution DPBS vs Tris-HCl (FIG. 2)

The reason why the use of existing calcium peroxide has not been reported is because the decomposition of calcium peroxide and the release of calcium ions are limited in commonly used aqueous solutions such as DPBS. To demonstrate that this problem can be solved by using Tris-HCl buffer, the vial tilting method was used to evaluate different hydrogels in the conventional hydrogel manufacturing process.

The type of solvent was changed to DPBS (250 ㎕) or Tris-HCl (250 ㎕) under a constant composition of Alginate (2 wt%) and CaO ₂ (0.75 wt%).

As a result of the experiment, it was confirmed that when using DPBS, the hydrogel was not formed and flowed down. On the other hand, when Tris-HCl was used, it was confirmed that the hydrogel was formed and maintained its shape. This is because calcium peroxide is not decomposed in DPBS but decomposes into calcium hydroxide in Tris-HCl, releasing calcium ions as bivalent ions and releasing the calcium ions to ionic interaction with carboxylic acids having anions of alginate, It can be explained that the gel is formed.

Experimental Example 2 Preparation of Hydrogel and Mechanical Strength (Fig. 3)

The mechanical strength of the alginate hydrogel was measured using a rheometer, and the change in mechanical strength was evaluated by adjusting the concentrations of alginate and calcium peroxide.

The concentration of alginate was varied to 0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, and 3.0 wt% under a constant composition of the concentration of calcium peroxide (0.3 wt%).

It was confirmed that the mechanical strength of the hydrogel can be controlled by controlling the concentration of the alginate, and the range was 4 to 1600 Pa.

The concentration of calcium peroxide was changed to 0 wt%, 0.15 wt%, 0.3 wt%, 0.45 wt%, 0.6 wt%, 0.75 wt%, and 0.9 wt% under a constant composition of alginate concentration (2.0 wt%). It was confirmed that the mechanical strength of the hydrogel was controllable by controlling the concentration of calcium peroxide, and the range was from 200 to 4000 Pa. On the other hand, when the concentration of calcium peroxide was set to 0 wt%, the hydrogel could not be formed. This is because there is no calcium peroxide and the formation of ionic bond between the calcium ion and the carboxylate functional group of glutaric acid is not achieved.

The concentration of calcium peroxide was changed to 0% by weight, 0.15% by weight, 0.45% by weight and 0.75% by weight under a constant composition of alginate (2.0% by weight) %, 0.6 wt%, 0.3 wt%, 0 wt%. It was confirmed that the mechanical strength of the hydrogel can be similarly controlled by adjusting the concentration of calcium peroxide and the amount of calcium ion released by the supplement of calcium hydroxide, and the range was from 1800 to 2100 Pa.

Experimental Example 3 Analysis of decomposition of hydrogen peroxide using catalase (Fig. 4)

The concentration of hydrogen peroxide generated in the hydrogel according to the concentration of calcium peroxide and the decomposition behavior of hydrogen peroxide using catalase were confirmed by Cu (II) -neocuproine assay.

0.75 wt.%, 0.3 wt.%, 0 wt.% Of calcium hydroxide, and 0.75 wt.% Of calcium hydroxide were added to adjust the concentration of calcium peroxide to 0 wt%, 0.15 wt%, 0.45 wt% and 0.75 wt% %.

The concentration of catalase was changed to 0 U / ml, 50 U / ml, and 100 U / ml to prepare a polymer solution for the hydrogel preparation.

To measure the concentration of hydrogen peroxide in the medium containing the hydrogel, hydrogel (100 μl) was prepared using a uniform mold, and 10 minutes later, it was placed in a 1.5 ml EP tube together with the medium (600 μl) The amount of hydrogen peroxide after 24 hours was measured.

As a result, the amount of hydrogen peroxide in the medium containing hydrogel could be controlled to 10 ~ 520μM by controlling the concentration of calcium peroxide. As the concentration of calcium peroxide increased, the concentration of hydrogen peroxide in the medium containing hydrogel increased, because the concentration of calcium peroxide, which is the cause of hydrogen peroxide, increased, and more hydrogen peroxide was generated inside the hydrogel.

The amount of hydrogen peroxide in the hydrogel containing 50 U / ml and 100 U / ml catalase could be adjusted to 9 ~ 16 μM, and no significant difference was observed between the groups regardless of the increase in the concentration of calcium peroxide Respectively. This is because hydrogen peroxide produced by calcium peroxide is decomposed into water and oxygen by catalysis.

EXPERIMENTAL EXAMPLE 4 Analysis of oxygen release behavior according to calcium peroxide concentration (FIG. 5)

The oxygen release behavior of the hydrogel according to the concentration of calcium peroxide was confirmed for 2 days using an oxygen sensor. In addition, the effect of decomposition of hydrogen peroxide generated by the decomposition of calcium peroxide on the oxygen release behavior was analyzed by using catalysis.

0.75 wt.%, 0.3 wt.%, 0 wt.% Of calcium hydroxide, and 0.75 wt.% Of calcium hydroxide were added to adjust the concentration of calcium peroxide to 0 wt%, 0.15 wt%, 0.45 wt% and 0.75 wt% %.

The concentration of catalase was changed to 0 U / ml and 50 U / ml to prepare a polymer solution for the hydrogel preparation.

Hydrogel (300 μl) was prepared to measure the amount of dissolved oxygen in the media containing the hydrogel, and the medium (600 μl) was added 10 minutes later, and the amount of dissolved oxygen in the medium was measured.

As a result of experiment, it was possible to control the maximum dissolved oxygen amount of the medium containing hydrogel to 22.8 ~ 42.27% by controlling the concentration of calcium peroxide without catalase. As the concentration of calcium peroxide increased, the maximum dissolved oxygen level of the medium containing hydrogel was increased, and the high oxygen environment was maintained for 9 to 24 hours. This is because the concentration of calcium peroxide, which is the cause of oxygen generation, is increased and more oxygen is generated in the hydrogel and then released.

The maximum dissolved oxygen level of the medium containing hydrogel prepared with 50 U / ml of catalase could be adjusted to 22.83 ~ 81.03%. As the concentration of calcium peroxide increased, the maximum dissolved oxygen amount of the medium containing hydrogel increased, The oxygen environment was maintained for 24 hours to 48 hours. This is because the concentration of calcium peroxide, which is the oxygen generating factor, increases, and the catalysis increases the oxygen concentration in the hydrogel while decomposing the hydrogen peroxide produced by the calcium peroxide into water and oxygen. As the oxygen inside the hydrogel increases, the oxygen release time is prolonged and the oxygen concentration is maintained for a long time.

Experimental Example 5 Evaluation of Cell Suitability of Hydrogel (FIG. 6)

To evaluate the cell fitness of the hydrogel, human dermal fibroblasts (hHDFs) were cultured in a two-dimensional manner on a 96-well plate, and a hydrogel and a medium were injected into another 96-well plate to form an eluate To perform cell fitness evaluation.

The concentration of the cells used in the experiment was 3.0 × 10 ³ cells / well. The cells were cultured for 24 hours at 37 ° C. in 5% CO ₂ atmosphere. After 24 hours of hydrogel-loaded eluate was diluted 10 times in the medium, After 24 hours, cell viability was measured using WST-1 assay and Live / Dead Viability / Cytotoxicity Kit.

Hydrogel disks were prepared by mixing catalase (0 ~ 100 U / ml) for the decomposition of hydrogen peroxide produced during hydrogel formation.

Live / Dead analysis is a method of assessing the cytotoxicity by showing the cells killed by cytotoxicity as red and living cells as green.

As a result, the cell viability was decreased as the concentration of calcium peroxide was increased in the hydrogel at 0 U / ml of catalase, and decreased to 22.29% as compared with the control. These results were similar to those observed in Live / Dead assay results. This is because the amount of hydrogen peroxide generated by the increase of calcium peroxide has increased to toxicity to cells.

At 50 U / ml of catalase, the hydrogel showed similar cell densities regardless of calcium peroxide concentration, and showed cell viability above 105% of that of the control. These results show that the ratio of living cells is similar in Live / Dead assay results.

At 100 U / ml of catalase, the hydrogel showed similar cell densities regardless of calcium peroxide concentration, and showed cell viability of over 98% compared to the control. These results show that the ratio of living cells is similar in Live / Dead assay results.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be understood that various modifications and variations can be made within the scope of equivalents of the claims.

Claims (16)

1. Wherein the natural or synthetic polymer and calcium peroxide (CaO ₂ ) are mixed in a Tris-HCl buffer.
2. The method according to claim 1,

   Wherein the polymer mixture solution further comprises a catalase.
3. The method according to claim 1,

   The calcium oxide (CaO ₂ ) is decomposed into calcium hydroxide in a Tris-HCl buffer, and the calcium ion (Ca ²⁺ ) released from the calcium hydroxide forms an ionic interaction with the anion of the polymer to form a hydrogel (EN) METHOD FOR MANUFACTURING BIO - INHIBITED OXYGENED HYDROGEL.
4. The method according to claim 1,

   Wherein the natural or synthetic polymer is an anionic polymer that is not chemically modified. ≪ RTI ID = 0.0 > 21. < / RTI >
5. 5. The method of claim 4,

   The anionic polymer may be selected from the group consisting of Arabic Gum, Tamarind Gum, Karaya Gum, Guar Gum, Locust Bean Gum, Quince Sword, Seed Gum, Xanthan Gum, Cyclodextrin, Casein, Tara Gum, Tragacanth Gum, Glucomannan, Glucosamine, Gatineau, Such as Gum Ghatti, Furcelleran, Pullulan, Gellan Gum, Pectin, Agar, Alginate, Carrageenan, Dextrin, Sodium polyacrylate, propylene glycol alginate, methyl cellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, Starch), sodium alginate ( Sodium alginate, and sodium caseinate. The present invention also provides a method for producing a bio-injectable oxygen-releasing hydrogel.
6. The method according to claim 1,

   Wherein the mechanical strength of the hydrogel is adjustable by the concentration of the polymer or calcium peroxide.
7. The method according to claim 6,

   Wherein the mechanical strength is increased with increasing concentration of the polymer or calcium peroxide.
8. The method according to claim 1,

   Wherein the maximum amount of dissolved oxygen in the medium containing the hydrogel is adjustable by the concentration of the calcium peroxide.
9. 9. The method of claim 8,

   Wherein the maximum dissolved oxygen amount of the medium containing the hydrogel is increased as the concentration of the calcium peroxide is increased.
10. 10. The method of claim 9,

    Wherein the maximum dissolved oxygen amount is 22.83 to 81.03%.
11. The method according to claim 1,

    Wherein the hydrogel releases oxygen for at least 48 hours. ≪ RTI ID = 0.0 > 21. < / RTI >
12. 12. A bioinjected oxygen-releasing hydrogel prepared according to the method of any one of claims 1 to 11.
13. 13. The method of claim 12,

    Wherein the hydrogel is a bio-injectable hydrogel.
14. A tissue regeneration, tissue engineering support or filling implant material comprising the bio-injectable oxygen-releasing hydrogel according to claim 12.
15. A tissue adherence, wound healing or hemostasis material comprising a bio-injectable oxygen-releasing hydrogel according to claim 12.
16. 13. A carrier for a physiologically active substance or a drug delivery body comprising the bio-injectable oxygen-releasing hydrogel according to claim 12.

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