KR102004861B1 - Preparation method of calcium peroxide-mediated in situ crosslinkable hydrogel as a sustained oxygen-generating matrix, and biomedical use thereof - Google Patents

Abstract

The present invention relates to a process for preparing a sustained-release oxygen-in-situ crosslinked hydrogel using calcium peroxide, and a novel type of oxygen-releasing in situ crosslinked hydrogel thus produced is characterized in that oxygen generated by the decomposition of calcium peroxide causes thiol A disulfide bond is induced in a polymer main chain having a reactor to form a hydrogel, and oxygen generated in the hydrogel is released in a sustained release form.
In addition, the present invention can improve the limitations of conventional oxygen delivery systems, and can produce a sustained release oxygen-in-situ crosslinked hydrogel in a simple and rapid manner. Further, according to the manufacturing conditions, It has the advantage of easy control of chemical / biological properties.

Description

TECHNICAL FIELD [0001] The present invention relates to a process for producing a sustained-release oxygen-containing in situ crosslinked hydrogel using calcium peroxide, and a biomedical use thereof, and a biomedical application thereof. BACKGROUND ART < RTI ID = 0.0 &

The present invention relates to a process for producing a sustained-release oxygen-containing in situ crosslinked hydrogel using calcium peroxide and a biomedical use thereof, and more particularly, to a new hydrogel crosslinking process using calcium peroxide in situ crosslinked hydrogel The present invention relates to a new type of injection-type polymer hydrogel capable of releasing high-concentration oxygen in a sustained-release form in a formed hydrogel matrix.

In addition, the present invention is advantageous in that the physical / chemical / biological properties of the in situ crosslinked hydrogel can be easily controlled according to the manufacturing conditions as well as the convenient manufacturing method as compared with the existing injection type hydrogel manufacturing method .

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.

Oxygen acts as a metabolic substrate and a signal molecule, which plays an important role in homeostasis maintenance and wound healing. In particular, high-density oxygen promotes new angiogenesis and wound healing in wounds by increasing cellular oxygen tension and increasing reactive oxygen species, thereby causing the cells to secrete growth factors that promote angiogenesis or to allow 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 to receive treatment, oxygen can only be provided by respiration, and the latter has the limitation that early release of oxygen occurs rapidly.

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.

Korean Patent No. 10-1573571

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned conventional problems, and it is an object of the present invention to provide a new type of in situ crosslinked polymer hydrogel for releasing a high concentration of oxygen in a sustained release form and a method for easily controlling the physical / chemical / A method for producing a sustained release oxygen-containing injection-type hydrogel, and a variety of biomedical uses thereof.

In order to accomplish the above object, the present invention provides a method for producing a natural / synthetic polymer having a thiol (-SH) reactor by causing disulfide bonds (SS-) formation by decomposition of calcium peroxide in an aqueous solution, situ crosslinked polymer hydrogel and simultaneously releases a high concentration of oxygen in a sustained release form by decomposition of calcium peroxide in an aqueous solution.

In the present invention, the natural / synthetic polymer containing a thiol group is prepared using Traut's reagent (TR), and the thiol reactant introduced into the polymer main chain can be controlled according to the amount of the initial TR used in the synthesis.

In addition, the present invention can easily control the physical / chemical / biological properties of in situ crosslinked hydrogels such as hydrogel formation time, mechanical strength, biodegradability, and oxygen release behavior by controlling the polymer, calcium peroxide and TR substitution degree Based oxygen-releasing in situ crosslinked hydrogel.

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

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

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

The new type of in situ crosslinked hydrogel according to the present invention is characterized in that hydrogel formation by oxygen generated by decomposition of calcium peroxide and controlled release behavior of oxygen generated in the hydrogel are controlled.

In other words, the present invention overcomes the limitations of existing oxygen generating hydrogels and can be applied to various biomedical applications based on superior biocompatibility (eg, tissue regeneration, artificial tissue preparation, wound healing material, tissue adhesive material, drug Carrier, etc.).

Brief Description of the Drawings Fig. 1 is a synthesis schematic diagram of GtnSH.
2 is a schematic diagram of the preparation of GtnSH hydrogel using calcium peroxide.
3 is a graph showing the content of thiol in the gelatin derivative according to the amount of TR introduced.
FIG. 4 is a graph showing the gelation time of the hydrogel according to the change of TR introduction amount, the concentration of calcium peroxide, and the concentration of polymer.
5 is a graph showing the oxygen release behavior of the hydrogel according to the concentration of calcium peroxide and the concentration of polymer.
6 is a graph showing the mechanical strength of the hydrogel.
FIG. 7 is a graph showing the degree of hydrolysis of the hydrogel. FIG.
Fig. 8 is a diagram showing cell fitness of the hydrogel. Fig.
9 is a graph showing the tissue adhesion of the hydrogel.

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 oxygen generated in such a hydrogel is released in a sustained form over a long period (14 days or more). In addition, it has been found that a polymer hydrogel material capable of releasing oxygen at a high concentration can be manufactured by a simple method, and physical / chemical / biological properties can be easily controlled.

The present invention applies calcium peroxide to natural / synthetic polymers having thiol reactors to induce disulfide bonds, thereby producing a sustained-release oxygen-containing in situ hydrogel.

Referring to FIG. 1, the polymer main chain having a thiol reactant is prepared using Traut's reagent (TR), and the thiol reactant introduced into the polymer main chain can be controlled according to the amount of the initial TR used in the synthesis.

The polymer main chain may be selected from the group consisting of gelatin, chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, , Fibrinogen, and Dodge-polymer, but is not limited thereto.

Here, the Dodge-polymer is a 3-arm-polyethylene glycol (3armPEG), a 4-arm-polyethylene glycol (4armPEG), a 6-arm polyethylene glycol (6armPEG) Polyethylene glycol (8-arPEG); at least one poly-ethylene glycol selected from 8-arm-polyethylene glycol (8arm PEG); And tetronic series (4arm-PPO-PEO); however, the present invention is not limited thereto.

Referring to FIG. 2, the sustained-release oxygen-releasing hydrogel may be prepared by in situ crosslinking of a polymer main chain having a thiol reactive group by inducing disulfide bond through an oxidation reaction in a solution containing calcium peroxide.

The method for preparing an oxygen-releasing in situ crosslinked hydrogel of the present invention is a method for preparing an oxygen-releasing in situ crosslinked hydrogel by adjusting the introduction amount of a thiol reactor, a concentration of a polymer, and a concentration of calcium peroxide to control physicochemical properties such as gelation time, mechanical strength, There are advantages.

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

Gelatin (type A from porcine skin,> 300 bloom), 2-iminothiolane hydrochloride (TR), calcium peroxide (CaO ₂ ), anhydrous Dimethyl sulfoxide (anhydrous DMSO ) And collagenase type II (collagenase type II) were purchased from Sigma Aldrich (St. Louis, Mo., USA).

Dulbecco's modified Eagle's Medium (DMEM), penicillin-streptomycin (P / S), trypsin / EDTA and DPBS (Dibbecco's phosphate buffered saline) Fetal Bovine Serum (FBS) was purchased from Research and Diagnostic Technology, and EGM-2 Single quots medium (EGM) was purchased from Lonza. 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 Synthesis and Structure Analysis of Gelatin Derivative Bonded with Thiol (FIG. 1)

100 mg of gelatin was dissolved in 10 ml of anhydrous DMSO, and this solution was mixed with a solution of TR (1-20 mg) dissolved in 1 ml of DMSO. At this time, the reaction temperature was 37 占 폚 and the reaction was carried out in a nitrogen atmosphere for 24 hours.

The reaction mixture was ultrafiltered from a dialysis tube with a molecular weight cut-off (MWCO) of 3.5 kD at 37 ° C and the medium was washed with 5 mM Hydrogen chloride (HCl) Solution and 1 mM HCl solution.

The thiol-bonded gelatin derivative (GtnSH) polymer was obtained by lyophilization.

Example 3 Hydrogel Formation (Figure 2)

A hydrogel was prepared by mixing GtnSH polymer dissolved in DPBS at 37 占 폚 with calcium peroxide (0-1 weight%).

EXPERIMENTAL EXAMPLE 1 Control of thiol content in gelatin derivatives according to the amount of 2-iminothiolane hydrochloride (TR) (FIG. 3)

The thiol content in GtnSH was measured by Ellman's assay.

In order to analyze the thiol content according to the amount of TR, the amount of TR introduced was changed into 1 mg, 5 mg, 10 mg and 20 mg under the constant composition of gelatin (100 mg) and anhydrous DMSO (10 ml).

100 μl of cysteine (L-cystein) and 1 mg / ml GtnSH solution used as a standard was mixed with 100 μl of Ellman's reagent solution, and the mixture was allowed to stand at room temperature for 20 minutes.

The mixture was measured for absorbance at 405 nm using an absorption detector, and a cysteine standard curve was created to calculate the thiol content in GtnSH.

As a result, the thiol content in the gelatin derivatives could be controlled to 5.224 ~ 25.145 μmol / g (polymer) by controlling the introduction amount of TR. The thiol content in GtnSH was increased as the amount of TR was increased.

When the amount of TR was increased from 10 mg to 20 mg, the content of thiol in the gelatin derivative was decreased because the amount of thiol that could be introduced into the gelatin was saturated.

Experimental Example 2 Analysis of gelation time according to the amount of thiol (FIG. 4)

The vial tilting method was used to evaluate the gelation time with the amount of thiol introduced.

The amount of TR introduced was changed to 1 mg, 5 mg, 10 mg and 20 mg under a constant composition of GtnSH (5 wt%), CaO ₂ (1 wt%) and DPBS (100 μl).

As a result, the gelation time of the hydrogel could be adjusted from 3 to 42 minutes by controlling the concentration of TR. Gel formation time was decreased with increasing amount of TR. It can be explained that the amount of thiol in GtnSH is increased due to the increase of the amount of TR introduced, and the number of reactors capable of forming crosslinks is increased, so that the formation time of hydrogel is shortened.

Experimental Example 3 Analysis of gelation time according to polymer concentration (FIG. 4)

The gelation time was analyzed by vial tilting method.

The concentration of GtnSH was changed to 3 wt%, 5 wt%, and 7 wt% under the constant composition of TR introduction (10 mg), CaO ₂ (1 wt%) and DPBS (100 μl). The preparation conditions of the hydrogel in more detail are shown in Table 1.

[Table 1]

Experimental results show that gelation time of hydrogel can be adjusted from 2 to 6 minutes by controlling the concentration of polymer, and gel formation time is decreased as the concentration of polymer increases. It can be explained that the hydrogel formation time is shortened because the reactor attached to the gelatin is also increased due to the increase of the polymer concentration.

Experimental Example 4 Analysis of Gelation Time with Calcium Peroxide Concentration (FIG. 4)

To evaluate the gelation time according to the calcium peroxide concentration, the vial tilting method was used.

The concentration of calcium peroxide was adjusted to 0% by weight, 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight and 1% by weight under a constant composition of TR introduction amount (10 mg), GtnSH (5% by weight) and DPBS Change. Table 2 shows the preparation conditions of the hydrogels in more detail.

[Table 2]

As a result, it was possible to control the gelation time of the hydrogel to 5 ~ 329 minutes by adjusting the concentration of calcium peroxide. In the solution having the concentration of calcium peroxide of less than 0.01% by weight, the gel was not formed but the concentration of calcium peroxide The gel formation time of the hydrogel was shortened. The reason for this is that as the concentration of calcium peroxide increases, the production of hydrogen peroxide is accelerated and the formation of a disulfide bond by the thiol group is accelerated, shortening the gel formation time.

EXPERIMENTAL EXAMPLE 5 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 3 days using an oxygen sensor.

The concentration of calcium peroxide was changed to 0.5% by weight, 0.75% by weight and 1% by weight under a constant composition of TR introduction amount (10 mg) and GtnSH (5% by weight).

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

As a result of the experiment, it was possible to control the maximum dissolved oxygen amount of the medium containing the hydrogel to 46.22% ~ 61.74% by controlling the concentration of calcium peroxide. As the concentration of calcium peroxide increased, the maximum dissolved oxygen amount of the medium containing the hydrogel became higher . The reason for this is that as the concentration of calcium peroxide increases, the production of oxygen is accelerated and the amount of oxygen released from the hydrogel to the medium becomes large.

Experimental Example 6 Analysis of oxygen release behavior according to polymer concentration (FIG. 5)

The oxygen release behavior of the hydrogel according to the polymer concentration was confirmed for 3 days using an oxygen sensor.

The concentration of the polymer was changed to 0 wt%, 3 wt%, 5 wt%, and 7 wt% under a constant composition of TR introduction (10 mg) and calcium peroxide (1 wt%).

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

As a result of experiments, it was possible to control the maximum dissolved oxygen amount of the medium containing hydrogel to 51.49% ~ 86.12% by controlling the polymer concentration. As the polymer concentration increased, the maximum dissolved oxygen amount of the medium containing the hydrogel decreased, It has been maintained for a long time. As the concentration of the polymer increases, the permeability of the hydrogel is decreased due to the increase of the degree of crosslinking. Therefore, oxygen is not released rapidly but released into the sustained release.

EXPERIMENTAL EXAMPLE 7 Long term analysis of oxygen release behavior with respect to calcium peroxide concentration (FIG. 5)

The oxygen release behavior of the hydrogel was measured for 14 days using an oxygen sensor.

The concentration of calcium peroxide was changed to 0 wt%, 0.5 wt%, 0.75 wt%, and 1 wt% under a constant composition of TR introduction amount (10 mg) and GtnSH (5 wt%).

Hydrogel (300 μl) was prepared to measure the amount of dissolved oxygen in the media containing the hydrogel, and the amount of dissolved oxygen in the medium was measured at specific time intervals after adding the medium (600 μl) 10 minutes later.

As a result of the experiment, it was possible to control the maximum dissolved oxygen amount of the medium containing hydrogel to 23.68% ~ 51.62% by controlling the concentration of calcium peroxide. As the concentration of calcium peroxide increased, the maximum dissolved oxygen amount of the medium containing the hydrogel became higher . The reason for this is that as the concentration of calcium peroxide increases, the production of oxygen is promoted and the amount of oxygen released from the hydrogel to the medium is increased.

Also, it was confirmed that the oxygen generated in the hydrogel was released for 14 days.

<Experimental Example 8> Elastic modulus analysis (FIG. 6)

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

The concentration of calcium peroxide was changed to 0 wt%, 0.5 wt%, 0.75 wt%, and 1 wt% under a constant composition of TR introduction amount (10 mg) and GtnSH (5 wt%).

It was confirmed that the mechanical strength of the hydrogel can be controlled by adjusting the concentration of calcium peroxide, and the range was from 100 to 810 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 the absence of calcium peroxide did not promote disulfide bond formation between thiol groups.

Experimental Example 9 Evaluation of Biodegradability of Hydrogel (FIG. 7)

To evaluate the biodegradability of the hydrogel, a hydrogel was prepared in a microtube by the same production method as in the gelation time test.

After standing for 30 minutes to stabilize the hydrogel sample at room temperature, the weight (W _i ) of each hydrogel sample was recorded and then treated with DPBS or collagenase (0.01 mg / ml) enzyme.

After removing the supernatant completely at specific time intervals, the weight of the hydrogel was weighed, and then 200 μl fresh medium was added.

The degree of biodegradability was calculated using the following equation (1).

[Equation 1]

(Where W _d and W _i represent the weight of the deteriorated hydrogel and the original hydrogel, respectively).

As a result, the sample treated with DPBS did not decompose the hydrogel over time, whereas the hydrogel treated with the 0.01 mg / ml collagenase enzyme decomposed the sample over time. It is confirmed that even when a thiol group is introduced into gelatin, it can still be decomposed by collagenase.

<Experimental Example 10> Evaluation of cytotoxicity of hydrogel (FIG. 8)

To evaluate the cytotoxicity of GtnSH, GtnSH solution was added to a 96-well plate, and human dermal fibroblast (hDFBs) was cultured to perform cytotoxicity evaluation.

Cell viability was measured using the WST-1 assay after culturing for 24 hours at 37 ° C and 5% CO ₂ atmosphere at a cell concentration of 2.0 × 10 ³ cells / wells.

To evaluate the cytotoxicity of the hydrogel, a hydrogel carrying human umbilical vein endothelial cells (HUVEC) was formed on a 96-well plate three-dimensionally and then cultured to perform cytotoxicity evaluation.

The concentration of the cells used in the experiment is 2 ㅧ 10 ⁵ cells / wells, and, 37 ℃, 7 days after cultured in the 5% CO ₂ atmosphere to measure the cell viability by using the Live / Dead Viability / Cytotoxicity Kit.

Hydrogel was prepared by mixing catalase (0 ~ 50,000 U / mL) for hydrolysis of hydrogen peroxide generated 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, GtnSH showed more than 90% cell viability as compared with the control group, and 70 ~ 80% of living cells were observed in the hydrogel according to the concentration of calcium peroxide as a result of Live / Dead assay. That is, it was confirmed that the hydrogel had excellent cell suitability.

&Lt; Experimental Example 11 > Evaluation of tissue adhesiveness of hydrogel (Fig. 9)

Based on the evaluation method of ASTM F2255-03 ("Test Method for Strength Properties of Tissue Adhesives in Lip-Shear by Tension Loading"), the tissue of hydrogel in swine skin using a universal testing machine (UTM) The tissue adhesive strength was measured.

The porcine skin had a diameter of 2.5 cm and all parts of the pig skin were adhered onto a rectangular plastic substrate by an ethyl cyanoacrylate glue.

Prior to the experiment, the skin surface was cleaned for decellularization using 70% isopropanol in deionized water, and after 15 minutes after the washing procedure, 100 [mu] l of adhesive was applied to the area of the pig skin, Were wrapped together.

The bond was maintained at room temperature in a wet environment for 60 minutes by applying a force of 100 g.

The sample was then loaded with poor delivery at a crosshead speed of 10 mm / min.

The maximum strength for displacement was measured and shear stress was used at break (final bond strength) to characterize the adhesion of each sample.

At least five samples were used for the measurement.

As a result of evaluating the adhesion of tissue using pig skin, the adhesion strength was improved as the concentration of calcium peroxide was increased.

When the concentration of calcium peroxide was changed to 0.5% by weight, 0.75% by weight and 1% by weight under a constant composition of TR introduction amount (10 mg) and GtnSH (5% by weight), it was possible to control from 9.9 to 28.8 kPa, Showed the highest adhesive strength.

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 (15)

a) reacting gelatin and Traut's reagent (TR) in a DMSO solvent to synthesize a gelatin having a thiol group introduced into the main chain; And
b) dissolving the thiol-introduced gelatin in DPBS (Dulbecco's phosphate buffered saline; DPBS), and mixing and reacting calcium peroxide (CaO ₂ ) to form a hydrogel;
In step a), the mixture is mixed in a weight ratio of gelatin: Traut's reagent (TR) = 100: 1 to 10,
Wherein the gelatin to which the thiol group is introduced in step b) is 5 to 7% by weight in the reaction solution.
A method for producing a sustained release oxygenated in situ crosslinked hydrogel using calcium peroxide.
The method according to claim 1,
Characterized in that the oxygen generated by the decomposition of the calcium peroxide is released in a sustained manner from the hydrogel.
A method for producing a sustained release oxygenated in situ crosslinked hydrogel using calcium peroxide.
delete delete delete delete delete delete The method according to claim 1,
Wherein the calcium peroxide in the step b) is used in an amount of 0.01 to 1% by weight in the reaction solution.
A method for producing a sustained release oxygenated in situ crosslinked hydrogel using calcium peroxide.
A process for the preparation of a compound of formula (I) according to claim 1, 2 or 9,
Sustained oxygen release in situ crosslinked hydrogel.
11. The method of claim 10,
Characterized in that the hydrogel is an injection type hydrogel.
Sustained oxygen release in situ crosslinked hydrogel.
A sustained release oxygen-containing in situ crosslinked hydrogel according to claim 10,
Tissue regeneration, support for tissue engineering.
A sustained release oxygen-containing in situ crosslinked hydrogel according to claim 10,
Tissue adhesion, wound healing or hemostasis material.
A sustained release oxygen-containing in situ crosslinked hydrogel according to claim 10,
Carrier for a physiologically active substance or drug.
A sustained release oxygen-containing in situ crosslinked hydrogel according to claim 10,
Implant implant material.

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