KR102100062B1 - Hyrogel for 3d printing and method thereof - Google Patents

Abstract

The present invention relates to a production method of a hydrogel for 3D printing, which comprises the step of: oxidizing carboxymethyl cellulose (CMC); and forming an imine bond by Schiff′s base reaction between the oxidized carboxymethyl cellulose and glycol chitosan (GC), wherein the step of forming the imine bond comprises a step of reacting and bonding the carboxymethyl cellulose and the glycol chitosan at the ratio of 2 : 1 to 2 : 3. The production method of a hydrogel for 3D printing can improve mechanical properties.

Description

Hydrogel for 3D printing and its manufacturing method {Hyrogel for 3d printing and method thereof}

In the present specification, a method for manufacturing a hydrogel for 3D printing that forms an imine bond between oxidized carboxymethyl cellulose (CMC) and glycol chitosan (GC), a hydrogel for 3D printing, and the same Disclosed is a bio ink for 3D printing.

Conventional hydrogels were manufactured using a method of synthesizing chemically by polymer bonding with a crosslinking agent. In this case, it is common to synthesize a hydrogel by crosslinking by chemically bonding a crosslinking agent to a polymer in advance to synthesize it using a reaction between modified polymers, or by combining a crosslinking agent with a polymer solution. Alternatively, a specific ion or organic compound may be added to induce the synthesis of a hydrogel by ion bonding, or a hydrophobic compound may be combined to induce hydrogel synthesis by hydrophobic bonding. In addition, a method for preparing a polymer gel using an enzyme and a catalyst for a functional group of a modified polymer is also generally used.

Hydrogels produced by ion bonding have the advantages of high reaction speed between reactants during manufacture and easy handling, while having low cell adhesion and weak viscoelasticity and mechanical properties. There is a method of using a high molecular weight alginic acid to overcome this problem, but in this case, the viscosity of the polymer increases as the molecular weight increases, and there is a disadvantage that a uniform structure is not formed through the manufacturing process.

In the case of synthesis of a polymer gel modified with a crosslinking agent, there is a disadvantage in that a problem due to toxicity of the crosslinking agent has to be solved, and when using an enzyme or catalyst, there is a problem of demonstrating human harmlessness of the enzyme and catalyst have. Recently, the synthesis of gels using poly (ethylene glycol diglycidyl ether), fumaric acid or citric acid using caffeine as a catalyst, and poly (propylene oxide), citric acid, mercapto receiving acid and poly (propylene dimethacrylate) Synthesis of used gels has been reported (polymeric materials for bio-applications, PCT / US2015 / 028311). Recently, many studies have been conducted to develop a self-bonding hydrogel through a Schiff's base reaction.

Commonly used biocompatible polymer materials for hydrogel production include natural polymers such as cellulose, chitosan, hyaluronic acid, and collagen. Of these, cellulose is environmentally friendly and is most commonly used in hydrogel production due to its high biodegradability and low toxicity. In addition, the hydrogel made of cellulose is suitable for use as a drug delivery system because it can absorb and deliver drugs in a short time due to its unique porous structure.

Cellulose is the most abundant biological resource on the earth, and has been provided as a functional and low-cost recyclable material to humans for a long time as it is an infinite resource that repeats the cycle of generation, decomposition, use, and generation by laws of nature. The cellulose is a resource for energy, food, etc., and the development of technology to utilize the cellulosic component of plants has been actively conducted. Recently, as the need for environmentally friendly polymer materials increases, cellulose is used in various functional polymer materials and industrial fields. Research is being actively conducted to replace the polymer resources.

Chitin is a tasteless / odorless natural polymer polysaccharide that is harmless to the human body and is mainly extracted from red crab shells, so it is a high value-added material that has various characteristics without depletion, and a chemically modified substance is chitosan. Chitosan is a polymer produced by removing (deacetylation) acetyl groups from the chemical structure of chitin, which has insoluble properties that are insoluble in water or alkali. Depending on the degree of deacetylation, it can be converted from insoluble to water-soluble polymers. It has properties such as soluble in weak acids such as lactic acid, citric acid, and acetic acid, and is easily absorbed by decomposition by the action of enzymes. It is used as a functional polymer by controlling the degree of deacetylation, and is used more industrially than chitin. Chitosan exhibits excellent biocompatibility in tissues or bodies of humans, animals, plants, etc., and is easily decomposed to be environmentally friendly and has little toxicity, so it is safe as a material, and has excellent moldability such as powder, fiber, membrane, and sponge-like structures, It has functions such as viscous, moisturizing, metal adsorption, antibacterial and antifungal activity. When ingesting chitosan, about 40% is absorbed from the body, and the remaining 60% activates cells until excretion, suppresses aging and strengthens immunity, anti-cancer action, decholesterol action, liver function improvement, heavy metal and pollutant discharge in the body It plays an important role in the body's biological control functions.

In the case of deacetyl chitosan, there are limitations in preparing a water-soluble chitosan solution, and chitosan solution is mainly produced using acetone as a solvent, which has been reported as a problem in application as a medical polymer. In order to prepare a water-soluble chitosan solution, ethylene glycol is combined with deacetyl chitosan to increase water solubility, and glycol chitosan utilizing side chain length of ethyl alcohol has been recently used.

In addition, when forming a gel using the cellulose and chitosan, it is difficult to prepare a hydrogel that uniformly exhibits porosity, and in some cases, an injection-type gel is manufactured using a sieve base reaction, however, the shape stability of the gel is There were no reports.

Particularly, when it is intended to be applied as a 3D printing material having a plurality of stacked structures, there is a problem in that its shape is not maintained after printing, so efforts to solve it are necessary.

Korean Registered Patent No. 1437082, “Mixed non-woven fabric containing carboxymethyl cellulose fiber, manufacturing method and use thereof” (published: 2014.07.01) Korean Registered Patent No. 1667778, “Porous Cellulose Hydrogel and Method for Producing It” (Publication Date: 2016.05.11)

An object of the present invention, the step of oxidizing carboxymethyl cellulose (Carboxymethyl cellulose, CMC); And forming an imine bond by Schiff's base reaction between the oxidized carboxymethylcellulose and glycol chitosan (GC); Including, The step of forming the imine bond (imine bond) comprises the step of reacting and bonding the carboxymethyl cellulose and the glycol chitosan in a ratio of 2: 1 to 2: 3, 3D printing hydrogel production Is to provide a way.

Another object of the present invention includes a structure imine bond by a Schiff base reaction between Carboxymethyl cellulose (CMC) and Glycol chitosan (GC), the structure comprising: It is to provide a hydrogel for 3D printing, wherein the content ratio of the carboxymethyl cellulose and glycol chitosan is 2: 1 to 2: 3.

Another object of the present invention is to provide a bio ink for 3D printing containing the hydrogel of the present invention.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

According to an embodiment of the present invention, the step of oxidizing carboxymethyl cellulose (Carboxymethyl cellulose, CMC); And forming an imine bond by Schiff's base reaction between the oxidized carboxymethylcellulose and glycol chitosan (GC); Including, The step of forming the imine bond (imine bond) comprises the step of reacting and bonding the carboxymethyl cellulose and the glycol chitosan in a ratio of 2: 1 to 2: 3, 3D printing hydrogel production Methods are provided.

According to one side, the method of manufacturing the hydrogel for 3D printing may include forming an imine bond represented by Chemical Formula 1 in the step of forming the imine bond.

[Formula 1]

According to another embodiment of the present invention, the structure comprises imine bonds by a Schiff base reaction between Carboxymethyl cellulose (CMC) and Glycol chitosan (GC), The structure is provided with a hydrogel for 3D printing, wherein the content ratio of the carboxymethylcellulose and glycol chitosan is 2: 1 to 2: 3.

According to one side, the hydrogel may have shape stability in a plurality of stacked structures.

According to another embodiment of the present invention, a bio ink for 3D printing including any one of the hydrogels is provided.

According to another embodiment of the present invention, a bio ink for 3D printing including any one of the hydrogels is provided.

Using the manufacturing method of the present invention, it is possible to manufacture an injection-type hydrogel and a hydrogel for porous 3D printing of uniform shape. In addition, it is also possible to print a 3D printing structure having excellent mechanical properties by using the 3D printing bio ink containing the 3D printing hydrogel of the present invention.

In addition, by using glycol chitosan, which has improved water solubility by bonding glycol functional groups to the chitosan produced by controlling the degree of deacetylation, it is possible to improve the mechanical properties of the hydrogel by improving hydrogen bonding with carboxymethylcellulose.

In addition, before manufacturing the hydrogel of the present invention, a hydrogel containing a bioactive substance can be formed by mixing a carboxymethylcellulose solution containing a bioactive substance such as cells or growth factors or a glycol chitosan solution to form a hydrogel. It can be used as a tissue engineering support and bioactive carrier that can manufacture and induce their release to regenerate damaged tissue or treat diseases.

In particular, the hydrogel for 3D printing of the present invention, which is manufactured at an optimum content ratio between reactants, has a feature that is excellent in shape stability in which a printed gel maintains a printing shape even when printing in a multi-layered structure. Using these features, the hydrogel for 3D printing of the present invention can be used for tissue engineering purposes, drug carriers, and the like, and can be used to create various 3D printing structures that require biocompatibility.

However, it should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a reaction structure showing the mechanism of gel formation through a Schiff's base reaction between the aldehyde group of CMC-A and the amine group of GC.
Figure 2 shows the results of FTIR analysis using the hydrogel of the present invention.
3 shows an SEM image of each hydrogel of the present invention.
4 is a graph showing the results of analysis by measuring the swelling rate and porosity of the hydrogel of the present invention.
Figure 5 shows the TGA results of the hydrogel of the present invention.
Figure 6 shows the results of performing the MTT analysis according to the C: G ratio of the hydrogel of the present invention.
7 shows a digital image of a 3D printing structure printed using the hydrogel of the present invention.
Figure 8 shows the SEM image before and after the stability test of the 3D printing structure laminated in two layers using the hydrogel of the present invention.
Figure 9 shows the results of the stability test of the printed 3D printing structure using the hydrogel of the present invention.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments, and the scope of the patent application right is not limited or limited by these embodiments. It should be understood that all modifications, equivalents, or substitutes for the embodiments are included in the scope of rights.

The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, the terms "include" or "have" are intended to indicate the presence of features, numbers, steps, actions, components, parts or combinations thereof described herein, one or more other features. It should be understood that the existence or addition possibilities of fields or numbers, steps, operations, components, parts or combinations thereof are not excluded in advance.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the embodiment belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that detailed descriptions of related known technologies may unnecessarily obscure the subject matter of the embodiments, detailed descriptions thereof will be omitted.

According to an embodiment of the present invention, the step of oxidizing carboxymethyl cellulose (Carboxymethyl cellulose, CMC); And forming an imine bond by Schiff's base reaction between the oxidized carboxymethylcellulose and glycol chitosan (GC); Including, The step of forming the imine bond (imine bond) comprises the step of reacting and bonding the carboxymethyl cellulose and the glycol chitosan in a ratio of 2: 1 to 2: 3, 3D printing hydrogel production Methods are provided.

According to another embodiment of the present invention, the structure comprises imine bonds by a Schiff base reaction between Carboxymethyl cellulose (CMC) and Glycol chitosan (GC), The structure is provided with a hydrogel for 3D printing, wherein the content ratio of the carboxymethylcellulose and glycol chitosan is 2: 1 to 2: 3.

Carboxymethylcellulose replaces the hydroxy group of glucose constituting an anionic polysaccharide, cellulose, as a carboxymethyl group, and is also commonly used as an abbreviation for CMC.

In one case, the step of oxidizing carboxymethylcellulose may be by sodium periodate (periodic acid). Specifically, sodium periodate may include a step of generating two aldehyde functional groups by oxidizing a diol group of carboxymethylcellulose.

According to one side, the oxidized carboxymethylcellulose is composed of the following structural formula 1, and is described herein by mixing CMC-A with a term.

[Structural Formula 1]

At this time, the two glycol functional groups of the carboxymethylcellulose repeat structure can be converted to all aldehyde functional groups by an oxidation reaction.

Polysaccharides used in hydrogels include alginic acid, gellan gum, k-carrageenan, xanthan gum, chitosan, dextran, hyaluronic acid, etc., in addition to the aforementioned cellulose. Heavy chitosan is a cationic polysaccharide with excellent non-toxicity, biocompatibility and biodegradability.

The glycol chitosan of the present invention is one of the water-soluble chitosan derivatives of chitosan that exhibits water solubility at neutral pH by introduction of a hydrophilic ethylene glycol functional group, and an amine group present along the backbone of glycol chitosan can be modified according to the application. It plays a role and also has the characteristic of forming hydrogen bonds with the carboxy group. In particular, conventional glycol chitosan can introduce various types of functional groups to improve its properties, and it is also possible to control the hydrophobic properties of chitosan by leaving or removing 100% of acetyl groups in the process of deacetylating chitin.

In one case, the hydrogel production method of the present invention may include a step of deacetylating chitin. The degree of deacetylation of the chitosan may be 40% to 100%, specifically 50% to 70%, but most preferably 60%.

The hydrogel of the present invention may use glycol chitosan which improves water solubility of chitosan by binding a glycol functional group to chitosan obtained by varying the degree of deacetylation from chitin. By using the glycol chitosan, hydrogen bonds with carboxymethylcellulose can be improved, and the activity of bioactive substances such as cells and growth factors can be maintained.

According to one side, the method of manufacturing the hydrogel for 3D printing may include forming an imine bond represented by Chemical Formula 1 in the step of forming the imine bond.

[Formula 1]

In the hydrogel for 3D printing of the present invention, a chemical bond is formed by an imine bond between an aldehyde group of carboxymethyl cellulose and an amine group of glycol chitosan, which are oxidized using a sieve base reaction. do.

The Schiff's base reaction means a reaction in which an imine bond (C = N) is formed between an aldehyde group of an anionic polymer and an amine group of a cationic polymer. As an aldehyde and a primary amine functional group is used as a generic term for the azomethine compound produced while undergoing a dehydration condensation reaction. At this time, imine bond (imine bond) means a functional group or compound containing a double bond (C = N) between carbon-nitrogen.

Figure 1 shows the mechanism of gel formation through a Schiff's base reaction between the aldehyde group of CMC-A and the amine group of GC, as shown in Figure 1, as shown in Figure 1, carboxymethylcellulose (CMC ) Is oxidized by sodium periodate (NaIO ₄ ) to add glycidyl acid (GC) to carboxymethylcellulose-aldehyde (CMC-A) where aldehyde groups are formed, and reacts to form imine bonds (C = N Bond). Intermediate CMC-x-GC is produced. At this time, the added glycol chitosan (GC) was 60% deacetylated. If the reaction is continued through this process, hydrogels having different number of crosslinks may be formed depending on the ratio and amount of functional groups of oxidized carboxymethylcellulose and glycol chitosan. Will increase.

In one case, when the intermediate is formed, the formation of the imine bond and the ion interaction (hydrogen bond) of the polymer act as a double to form a double cross-link.

This double cross-linking has different degradability depending on the pH, reduces the effect of hydrolysis, can improve the stability of the hydrogel in water or cell culture medium, and can maintain the same stability during or after 3D printing. have.

Using the above-described preparation method, a stable gel can be formed by the cationic and anionic polymers participating in the reaction. In addition, when cells are seeded on a hydrogel or printed with a hydrogel, the cells tend to release wastes that can change the acidic pH typically seen in cell culture media in vitro. The hydrogel forms a polymer electrolyte complex to suppress hydrolysis of the hydrogel and to maintain the network inside the gel for a long time, thereby causing an effect of stabilizing the structure of the gel.

At this time, mechanical properties can be controlled by adjusting the functional group ratio and the content ratio of the Schiff reaction of carboxymethyl cellulose and glycol chitosan.

In order to prepare the hydrogel for 3D printing of the present invention, the ratio of carboxymethylcellulose: glycol chitosan (hereinafter, C: G) is preferably 2: 1 to 2: 3, and most preferably 1: 1. In one case, when biocompatibility is required for the gel of the present invention, as the content of carboxymethylcellulose decreases and the content of glycol chitosan increases, the cytotoxicity is low and the viability of cells appears high, thereby increasing the content of glycol chitosan. It can improve biocompatibility.

In addition, the higher the density of the network of gels produced by the sieve base reaction, the more the structural stability of the gel can be secured, so it is preferable to induce the reaction to have uniform and small-sized pores. The induction is possible by controlling the ratio, molecular weight, content, etc. of the sip reaction functional groups of carboxymethyl cellulose and glycol chitosan participating in the reaction, and considering both biocompatibility and structural stability, the C: G is 2: 1 to 2 It is preferable to be: 3, and in particular, when C: G is 1: 1, a hydrogel that is optimal for 3D printing in terms of biocompatibility, swelling rate, and form stability can be prepared.

The hydrogel of the present invention prepared by the above method has an ionic bond by a glycol and alcohol group, and has structural stability by forming a hydrogen bond by introducing a glycol functional group. This structural stability contributes to the shape stability of the structure when forming a 3D printing structure.

According to one side, the hydrogel may have shape stability in a plurality of stacked structures.

If, if the hydrogel does not have a ratio of C: G in the above range, the form stability is low, it is difficult to form a laminated structure, and even if laminated, the form may not be maintained for a long time. That is, even if the hydrogel of the present invention is stacked in a plurality of layers, the shape stability is remarkably improved. Thus, a 3D printed multi-layer stacked structure can be formed using the hydrogel. The structure, the content ratio of the carboxymethyl cellulose and glycol chitosan may be 2: 1 to 2: 3.

As described above, in the hydrogel for 3D printing of the present invention, when carboxymethylcellulose is oxidized by sodium periodate and has an aldehyde group, when glycol chitosan having an amine group is added, there is a shift between the aldehyde group and the amine group. It may have a stable intermediate (unit) in which imine bonds are formed by a base reaction.

According to one side, the hydrogel of the present invention may have form stability in a plurality of stacked structures.

The hydrogel prepared by the above-described method may be included in bio-inks used for injection and 3D printing, and in this case, it may also be used as a support for tissue engineering and a drug carrier. At this time, the hydrogel may be prepared by using a carboxymethylcellulose solution or glycol chitosan solution containing cells, bioactive molecules, etc., depending on the application.

A more specific description will be described later through the following examples.

Example 1. Oxidation of carboxymethylcellulose

Prior to the synthesis of the hydrogel of the present invention, carboxymethylcellulose was oxidized to facilitate reaction.

First, 1 g of carboxymethylcellulose sodium salt (medium viscosity, 400-800 cps, Sigma Aldrich) was dissolved in 100 ml distilled water double-distilled at room temperature for 12 hours. Thereafter, 0.5 g of sodium periodate (Wako Pure Chemical Industries Ltd, Korea) dissolved in 5 ml of water was added dropwise to the dissolved solution, and the mixture was stirred at 400 rpm at room temperature for 12 hours at room temperature. To remove unreacted sodium periodate, 1 ml of ethylene glycol (Sigma Aldrich) was added to the stirred solution and further stirred for 2 hours to quench the reaction.

The solution after the reaction was dialyzed for 7 days by exchanging every 24 hours using distilled distilled water. The dialysis solution was freeze-dried for 7 days in a freeze dryer to obtain oxidized carboxymethylcellulose (CMC-A), and the yield for the entire reaction was confirmed to be in the range of 50 to 55%.

Example 2. Synthesis of oxidized carboxymethylcellulose (CMC-A) and glycol chitosan (GC)

Samples of CMC-A obtained in Example 1 were added to PBS buffer (pH 7.4) at various concentrations and stirred at room temperature for 4 hours. In the same way, different concentrations of glycol chitosan (Sigma Aldrich) were added to PBS buffer (pH 7.4), respectively, and each of them was individually vortexed at 1000 rpm for 4 hours at room temperature.

After the CMC-A solution and the GC solution were gently mixed using vortex, a uniform gel was formed, and the time required for this was measured. Whether the final gel formation was confirmed by inverting the tube containing the mixed solution.

Measurement records for gel formation and time are shown in Table 1 below.

No CMC-A GC Sample code
(Sample composition) Gel formation
Time (sec) Sample 1 50 mg / ml 50 mg / ml C50: G50 30-40 s Sample 2 30 mg / ml 70 mg / ml C30: G70 30-40 s Sample 3 70 mg / ml 30 mg / ml C70: G30 30-40 s Sample 4 10 mg / ml 90 mg / ml C10: G90 30-40 s Sample 5 90 mg / ml 10 mg / ml C90: G10 30-40 s

As shown in Table 1, a total of 5 gel samples mixed were checked for stability in water in different pH environments, and the results showed that they were stable at all pH levels even after 50 days.

Example 3. Check the physical properties of the hydrogel

The gel samples of Example 2 were performed using an ATR-FTIR spectrometer (Travel IR, Smiths Detection, USA).

Experimental Example 1: FTIR analysis

The FTIR spectrum was obtained by scanning 32 times for each sample from 500 to 4000 cm ^-1 . The hydrogel sample was frozen at -80 ° C for 24 hours, dried in a freeze dryer for 3 days, and coated with gold using SEM (TESCAN VEGA3, South Korea), followed by observing the cross-sectional shape of the hydrogel sample. Using the SEM image, the morphological appearance of the sample was observed using ImageJ software, such as pore diameter, pore wall thickness, and pore size of the gel sample. On the other hand, 700 MHz NMR (Agilent, USA) was used. The ¹ H NMR spectrum was recorded to analyze the chemical structure.

Figure 2 shows the results of FTIR analysis using the hydrogel of the present invention, each component such as CMC, GC, CMC-A and CMC-GC hydrogel was confirmed through FTIR spectrum. As a result of the confirmation, it was confirmed that a characteristic aldehyde peak was found around 1735 cm ^-1 , and that there was an oxidation reaction generated by the CMC-A reaction in the CMC. By observing the peak around 1640 cm ^-1 corresponding to C = N binding vibration, the formation of the sieve base in CMC-GC was confirmed. . In addition, another broad peak around 3400 cm-1 was found to correspond to NH and OH bonds shifted to higher frequencies. Through this FTIR analysis, binding between the -CHO group of CMC-A and the -NH ₂ group of glycol chitosan was confirmed.

Experimental Example 2: Porosity Verification

The porosity of the hydrogel sample was quantified using a previously reported method (Ming et al., 2016). In detail, the following Equation 1 was used.

[Equation 1]

Porosity (%) = (V ₁ -V ₃ ) / (V ₂ -V ₃ ) * 100 (%)

After the hydrogel sample was lyophilized, the dry weight of the sample was measured. The volume of the solvent to be immersed in the dried sample was first measured, and the total volume was measured after immersing the sample for about 1 hour. Then, the volume of the solution after removing the sample was measured to quantify porosity by using Equation 1.

FIG. 3 shows SEM images of each of the hydrogels of the present invention, and uses samples 1 to 3 of Example 2. 3, the pore sizes of Samples 1 to 3 were measured to be 100.86 ± 37.38 μm and 197.35 ± 84.07 μm and 150.25 ± 48.39 μm, respectively. It was confirmed that the pore size of the hydrogel varied as the concentration changed. Particularly, it was found that Sample 1 exhibited a small pore size due to a high covalent bond and high crosslinking density due to the similar number of aldehyde functional groups and imine functional groups forming imine bonds compared to the other two concentrations.

Experimental Example 3: Analysis of swelling ratio

The swelling rate analysis of the hydrogel sample was measured by the same method as previously known (Das et al., 2018; Huang et al., 2016). In detail, the following Equation 2 was used.

[Equation 2]

Swelling ratio (%) = [(W ₄₈ -W- ₀ ) / W ₀ ] x 100%

The lyophilized hydrogel was weighed (W ₀ ) and the hydrogel was immersed in 10 ml PBS solution (ph 7.4) in a 60 ml glass bottle. The soaked sample was incubated at room temperature for 48 hours, and after removing only water present on the surface, the weight of the hydrated sample was weighed (W48).

Figure 4 shows a graph of the results of measuring and analyzing the swelling rate and porosity of the hydrogel of the present invention, using samples 1 to 3. Referring to Figure 4 (a), the sample 2 of the hydrogel showed a relatively high swelling rate compared to other samples. In the case of Sample 1, it was confirmed that the swelling rate is not too high, so that the shape of the gel in environmental changes can be stabilized. FIG. 4 (b) is a graph showing the porosity percentage value of a hydrogel sample measured using a liquid displacement method. As a result of the measurement, more than 75% porosity was observed in all samples, and in particular, the porosity of Sample 2 was measured to be the highest, but the difference was not large with other samples.

These analysis results show the same results as those analyzed in the SEM image confirmed in FIG. 3.

Experimental Example 4: Thermogravimetric Analysis (TGA)

The cross-linked hydrogel sample was dried in a lyophilizer, and the sample was subjected to nitrogen treatment for thermal weight analysis. Thermogravimetric analysis was performed using a TGA analyzer (DTG-60, Shimadzu, Japan) at a scan rate of 5 ° C / min. TGA was performed to confirm and compare the stability of the hydrogel produced by crosslinking the two functional groups of the aldehyde group and the amine group.

5 shows the TGA result of the hydrogel of the present invention, and the graph of FIG. 5 analyzed the stability of the sample using the TGA result measured from room temperature to 700 ° C. Looking at the graph shown in Figure 5, it can be seen that the samples 1 to 3 and the TGA values of CMC and GC showed similar weight loss patterns during the analysis. Zones in the range of 175 to 250 ° C and 250 to 550 ° C to 650 ° C are weight loss due to decomposition of the main chain of the polymer and are mainly due to evaporation of carbon dioxide. Particularly, Sample 1 had a weight reduction specific gravity of 77%, and showed less weight loss due to decomposition compared to Sample 2 (90%) and Sample 3 (84%). This is because in the case of the hydrogel of Sample 1, there were more crosslinked sites, so the network was stably formed.

Experimental Example 5: Cytotoxicity evaluation

Cytotoxicity studies on hydrogel samples with different proportions of participation in the reactions prepared in Example 2 were performed using known methods (Das et al., 2018). Specifically, 300 μl hydrogel / 1 ml medium was placed in a 96 well plate containing 10,000 MC3T3 cells / well. MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) solution was added to the plate for 1, 3 and 5 days, and the optical density of the sample was at 570 nm. Cytotoxicity was assessed by recording through a microplate reader (Tecan GENios FL., GMI, USA).

Figure 6 shows the results of performing the MTT analysis according to the C: G ratio of the hydrogel of the present invention, and the latex was used as a control to evaluate the cytotoxicity of samples 1 to 5 of Example 2. 7, it was confirmed that all of samples 1 to 5 exhibited cell viability of 75% or more. In particular, Sample 1 showed a cell viability of 90%. That is, it was confirmed that Sample 1 evaluated through the previous experimental example has stability in cell survival.

Experimental Example 6: 3D printing structure shape stability verification

To print the prepared hydrogel, a custom rotating 3D printer (SeoulTech) introduced in a known paper was used (Lee et al., 2017). Solidworks software (Dassault Systems SolidWorks Corp, USA) was used to design 3D structures using different templates and fillings.

When the hydrogel was used for 3D printing, the hydrogel was produced more stably and firmly than normal extrusion by sedimentation immediately after gelation. For injection needles used for printing, adjust the X and Y-axis stages using software, and after checking the parameters of optimal pressure, temperature, and printing speed for printing, the optimum pressure is 50 kPa and the optimum temperature is 35 °. C, the optimum printing speed was confirmed to be 180 mm / min.

Figure 7 shows a digital image of a 3D printing structure printed using the hydrogel of the present invention, a to e using the hydrogel of Sample 1 of Example 2 2, 4, 6, 8 These are structures made of layers and 16 layers. Although no separate crosslinking agent, polymer, or support was used during printing of the structure, it exhibited excellent mechanical properties supporting the layer itself, and as shown in FIG. 8, in the case of a to e, all were stable at regular intervals. It was confirmed that a structure was formed.

The structures having different numbers of layers were printed to confirm whether the hydrogel of the present invention is suitable for 3D printing, and in particular, whether the form stability is excellent when forming a plurality of layered structures. In order to test the stability of the printed structure, UV light exposure was performed for 1 hour in a PBS solution having a pH of 7.4, followed by steam sterilization at high pressure.

FIG. 8 shows SEM images before and after the stability test of the 3D printed structure stacked in two layers of the structure of FIG. 8, wherein a to c are different magnifications (a-2 mm; b-500 μm; c-50 μm) ) Shows the SEM image before the stability evaluation, and d to f represent the SEM image after the stability evaluation of each of the a to c.

As a result of testing the stability by the above-described method, SEM-2L, SEM-4L, and SEM-UV images were confirmed, as shown in FIG. 9, and it was confirmed that the structural spacing or shape of the printed structure was maintained.

As described above, although the embodiments have been described with limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order than the described method, and / or the described components are combined or combined in a different form from the described method, or replaced or replaced by another component or equivalent Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (5)

Oxidizing carboxymethyl cellulose (CMC); And
Forming an imine bond by Schiff's base reaction between the oxidized carboxymethylcellulose and glycol chitosan (GC);
Including,
The step of forming the imine bond includes the step of reacting and bonding the carboxymethyl cellulose and the glycol chitosan at a concentration ratio of 2: 1 to 2: 3,
The oxidizing step comprises the steps of oxidizing a diol group of carboxymethylcellulose with sodium periodate to generate two aldehyde functional groups, a method for producing a hydrogel for 3D printing. According to claim 1,
The step of forming the imine bond (imine bond), including the formation of an imine-bonded intermediate represented by the formula (1), 3D printing method for producing a hydrogel.
[Formula 1]

Contain imine bond structure by Schiff base reaction between Carboxymethyl cellulose (CMC) and Glycol chitosan (GC),
The structure, the concentration ratio of the carboxymethyl cellulose and glycol chitosan is 2: 1 to 2: 3,
The carboxymethylcellulose is a hydrogel for 3D printing in which two aldehyde functional groups are generated by oxidizing a diol group by sodium periodate. According to claim 3,
The hydrogel has a form stability in a plurality of stacked structures, 3D printing hydrogel. A bio ink for 3D printing, comprising the hydrogel of claim 3 or 4.

Images