Hydrogel Composition For Tissue Regeneration, And Support Prepared Using Same

Abstract

The present invention relates to a hydrogel composition for tissue regeneration, and a support prepared using same. More specifically, the present invention comprises anionic polysaccharides, aminated hyaluronic acid, and collagen. The hydrogel composition comprising the ingredients, and a support prepared using same can quickly form a three-dimensional structure through spontaneous cross-linking without the addition of a common cross-linking agent in which an epoxide group or an amine group is at a single end or both ends thereof, thereby providing a structure capable of soft tissue transplants.

Claims

1. A hydrogel composition for tissue regeneration comprising 1 to 10 wt % of an anionic polysaccharide, 0.1 to 2 wt % of aminated hyaluronic acid, and 0.1 to 3 wt % of collagen, and further comprising 150 to 400 parts by weight of a divalent cation and 0.1 to 2 parts by weight of methanol based on 100 parts by weight of the anionic polysaccharide.

2. The hydrogel composition according to claim 1, wherein the anionic polysaccharide has a weight average molecular weight of 100 to 500 kDa and comprises 50 to 70 wt % of (β-D-mannuronic acid and 30 to 50 wt % of α-L-guluronic acid.

3. The hydrogel composition according to claim 1, wherein the aminated hyaluronic acid has a weight average molecular weight of 100 to 500 kDa.

4. The hydrogel composition according to claim 1, wherein the collagen has a weight average molecular weight of 100 to 500 kDa and comprises atelocollagen.

5. The hydrogel composition according to claim 1, wherein the divalent cation has a mass concentration of 0.5 to 2% and comprises an alkaline earth metal or a compound thereof.

6. The hydrogel composition according to claim 1, wherein the methanol has a mass concentration of 40 to 60%.

7. The hydrogel composition according to claim 1, further comprising 0.1 to 1 parts by weight of an additive based on 100 parts by weight of the hydrogel composition, wherein the additive comprises at least one selected from the group consisting of a cell, cell growth factor, buffer, preservative, isotonicity regulator, salt, antioxidant, osmotic pressure regulator, emulsifier, wetting agent, sweetener, flavoring agent, and anesthetic agent.

8. The hydrogel composition according to claim 7, wherein the cell comprises at least one selected from the group consisting of a stem cell, sensory cell, brain cell, germ cell, epithelial cell, and immune cell.

9. A hydrogel support for tissue regeneration, prepared using the hydrogel composition according to claim 1.

Description

DESCRIPTION OF DRAWINGS

[0025] FIG. 1 is a photograph showing a hydrogel support prepared in Preparation Example 5 of the present invention;

[0026] FIG. 2 is a schematic view showing a process of preparing a hydrogel support in Preparation Example 5 of the present invention;

[0027] FIG. 3 is a graph showing the results of measurement of compressive strength depending on the concentration of a divalent cation;

[0028] FIG. 4 is a photograph showing a hydrogel composition prepared in Preparation Example 3 of the present invention;

[0029] FIG. 5 is an optical microscope image showing the hydrogel composition freeze-dried after preparation in Preparation Example 3; and

[0030] FIG. 6 is an optical microscope image showing the hydrogel support freeze-dried after preparation in Preparation Example 5 of the present invention.

MODE FOR INVENTION

[0031] Hereinafter, preferred embodiments of the present invention and the physical properties of individual components will be specifically described, which is intended to provide a sufficiently detailed explanation that those skilled in the art to which the present invention pertains could easily carry out the invention, and which is not to be construed as limiting the technical spirit and scope of the present invention.

[0032] According to the present invention, a hydrogel composition for tissue regeneration includes an anionic polysaccharide, aminated hyaluronic acid, and collagen, and preferably includes 1 to 10 wt % of an anionic polysaccharide, 0.1 to 2 wt % of aminated hyaluronic acid, and 0.1 to 3 wt % of collagen.

[0033] The hydrogel composed of anionic polysaccharide, aminated hyaluronic acid, and collagen as described above is able to exhibit mechanical properties of rapidly realizing cross-linking and maintaining the printed structure, and also to be injected into the skin soft tissue, thereby overcoming conventional limitations on biomaterials.

[0034] Moreover, the hydrogel composition composed of the above components may be added with a tissue-derived extracellular matrix component or with a specific cell differentiation regulator to induce differentiation into specific tissues.

[0035] As used herein, the term “hydrogel” refers to a water-swellable polymer that is usable as a synthetic material for biotissue as well as drug delivery. Such a water-swellable polymer is a polymer that absorbs water but does not dissolve in water, namely a hydrophilic polymer having sufficient expansion in water, and may be blended with cells and growth factors to provide a more biocompatible support.

[0036] The anionic polysaccharide is contained in an amount of 1 to 10 wt %, has a weight average molecular weight of 100 to 500 kDa, and is composed of 50 to 70 wt % of β-D-mannuronic acid and 30 to 50 wt % of α-L-guluronic acid. The amount of the anionic polysaccharide accounts for 1 to 10 wt %, preferably 4 to 10 wt %, of the total amount of the hydrogel composition for tissue regeneration according to the present invention.

[0037] If the amount of the anionic polysaccharide is less than 1 wt % or exceeds 10 wt %, an egg box cannot be formed when a divalent cation is added, or a water-insoluble hydrogel cannot be formed even when an egg box is formed locally.

[0038] The aminated hyaluronic acid is contained in an amount of 0.1 to 2 wt % and has a weight average molecular weight of 100 to 500 kDa. It is preferable to use aminated hyaluronic acid in which at least a portion of the hydroxyl group hydrogen atoms of hyaluronic acid is substituted with a group having a quaternary ammonium cation group.

[0039] As described above, the hyaluronic acid having a quaternary ammonium cation group is contained in an amount of 0.1 to 2 wt %, preferably 0.5 to 1 wt %, in the first hydrogel. If the amount of aminated hyaluronic acid exceeds 2 wt %, a polyion complex may be formed by electrostatic interaction between the anionic polysaccharide of the hydrogel composition and the aminated hyaluronic acid due to coexistence, so a locally non-uniform water-insoluble gel may precipitate. On the other hand, if the amount of aminated hyaluronic acid is less than 0.1 wt %, a 13 sheet with collagen may not be properly formed in the solvent.

[0040] The collagen is contained in an amount of 0.1 to 3 wt %, has a weight average molecular weight of 100 to 500 kDa, and is composed of atelocollagen. Here, it may include any one or more selected from among type-1 atelocollagen, type-2 atelocollagen, and type-3 atelocollagen, and it is preferable to use collagen in which the end of the functional group thereof is substituted with succinic acid or a sulfide bond.

[0041] The collagen is contained in an amount of 0.1 to 3 wt %, preferably 0.5 to 1.5 wt % in the hydrogel composition for tissue regeneration. If the amount of collagen exceeds wt %, conversion into the 13 sheet may occur drastically due to the solvent and partial non-uniformity may result, making it impossible to prepare a uniform hydrogel composition and support. On the other hand, if the amount of collagen is less than 0.1 wt %, a uniform shape may be obtained in a hydrogel composition state, but non-uniform fibrosis may occur during preparation of the support.

[0042] Also, the hydrogel composition for tissue regeneration according to the present invention may further include 150 to 400 parts by weight of a divalent cation and 0.1 to 2 parts by weight of methanol based on 100 parts by weight of the anionic polysaccharide contained in the hydrogel composition for tissue regeneration. When the cation is further contained in this way, the hydrogel composition for tissue regeneration according to the present invention is insolubilized in water.

[0043] Here, the divalent cation has a mass concentration of 0.5 to 2%, and preferably includes an alkaline earth metal or a compound thereof. More preferably, the alkaline earth metal includes beryllium, magnesium, calcium, strontium, barium, and radium.

[0044] In addition, the methanol serves to change the structure of the hydrogel composition in the process of insolubilizing the hydrogel composition for tissue regeneration in water as described above, and preferably has a mass concentration of 40 to 60%.

[0045] The process of insolubilizing the hydrogel composition in water by mixing the divalent cation and methanol is not particularly limited, but it is preferable to select and use a preparation method suitable for realizing high product uniformity and mass production. More preferably, it is performed in a manner in which the hydrogel composition is prepared, a divalent cation is sprayed thereto, methanol is added dropwise thereto, and then a negative pressure of −0.05 MPa is applied thereto at a temperature of 0 to 60° C. to remove the methanol.

[0046] Here, the conditions of temperature and negative pressure do not limit the scope of the present invention, and may be changed in various ways, and the methanol removal process is not limited to the temperature and negative pressure conditions, and a removal process using a dialysis membrane is also possible.

[0047] Moreover, the hydrogel composition for tissue regeneration according to the present invention may further include 0.1 to 1 parts by weight of an additive based on 100 parts by weight of the hydrogel composition for tissue regeneration, and the additive includes at least one selected from the group consisting of a cell, cell growth factor, buffer, preservative, isotonicity regulator, salt, antioxidant, osmotic pressure regulator, emulsifier, wetting agent, sweetener, flavoring agent, and anesthetic agent.

[0048] Here, the cell may include at least one selected from the group consisting of a stem cell, sensory cell, brain cell, germ cell, epithelial cell, and immune cell.

[0049] Also, the anesthetic agent may be, for example, a local anesthetic agent such as an aminoamide local anesthetic agent or an aminoester local anesthetic agent. Examples of the local anesthetic agent may include, but are not limited to, lidocaine, ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cyclomethycaine, dibucaine, dimethisoquin, dimethocaine, diperodon, dicyclomine, ecgonidine, ecgonine, ethyl chloride, etidocaine, β-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, combinations thereof, and salts thereof. Examples of the aminoester local anesthetic agent may include, but are not limited to, procaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine (larocaine), propoxycaine, procaine (Novocain), proparacaine, and tetracaine (amethocaine). Non-limiting examples of the aminoamide local anesthetic agent include articaine, bupivacaine, cinchocaine (dibucaine), etidocaine, levobupivacaine, lidocaine (lignocaine), mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, and combinations thereof.

[0050] When the additive described above is contained, the hydrogel composition according to the present invention may be used for improving the condition of the skin, filling wrinkles, or shaping the face or body, and is also useful as a dermal filler.

[0051] Hereinafter, a method of preparing a hydrogel composition for tissue regeneration according to the present invention, a method of preparing a support using a hydrogel composition prepared through the above method, and individual physical properties will be described with reference to the following examples.

<Preparation Example 1> Preparation of Hydrogel Composition for Injection

[0052] A hydrogel composition for injection was prepared in a manner in which 7 wt % of an anionic polysaccharide having a weight average molecular weight of 100 kDa to 500 kDa (obtained by mixing β-D-mannuronic acid and α-L-guluronic acid at a ratio of 50:50 at room temperature) was mixed with 0.7 wt % of aminated hyaluronic acid having a weight average molecular weight of 100 kDa to 500 kDa to afford a mixture, and the pH of the mixture was maintained at 6.0, followed by adding 1 wt % of type-1 atelocollagen having a weight average molecular weight of 100 kDa to 500 kDa thereto.

<Preparation Example 2> Preparation of Hydrogel Composition for Injection Containing Additive

[0053] A hydrogel composition for injection containing an additive was prepared by mixing 100 parts by weight of the hydrogel composition for injection prepared in Preparation Example 1 with 0.5 parts by weight of an additive (including a buffer, an osmotic pressure regulator, an emulsifier, and lidocaine, which are mixed together).

<Preparation Example 3> Preparation of Water-Insoluble Hydrogel Composition for Injection

[0054] A water-insoluble hydrogel composition for injection was prepared in a manner in which the hydrogel composition prepared in Preparation Example 1 was mixed with calcium chloride having a mass concentration of 1% at room temperature, allowed to react with stirring at a rate of 100 rpm, added with methanol having a mass concentration of 60%, and then stirred at room temperature at a rate of 500 rpm, followed by removal of residual methanol using a dialysis membrane or a rotary vacuum evaporator.

<Preparation Example 4> Preparation of Water-Insoluble Hydrogel Composition for Injection Containing Additive

[0055] A water-insoluble hydrogel composition for injection was prepared in a manner in which the hydrogel composition prepared in Preparation Example 2 was mixed with calcium chloride having a mass concentration of 1% at room temperature, allowed to react with stirring at a rate of 100 rpm, added with methanol having a mass concentration of 60%, and then stirred at room temperature at a rate of 500 rpm, followed by removal of residual methanol using a dialysis membrane or a rotary vacuum evaporator.

<Preparation Example 5> Preparation of Hydrogel Support for 3D Printer

[0056] A hydrogel support for a 3D printer was prepared in a manner in which 7 wt % of a first solution (obtained by mixing β-D-mannuronic acid and α-L-guluronic acid having a weight average molecular weight of 100 kDa to 500 kDa at a rate of 50:50 at room temperature) was mixed with calcium chloride having a mass concentration of 1% at room temperature, allowed to react with stirring at a rate of 100 rpm, and dried to afford a support, and the support thus obtained was immersed in a second solution (obtained by mixing aminated hyaluronic acid having a weight average molecular weight of 100 kDa to 500 kDa, the pH of which was maintained at 6.0, with 1 wt % of type-1 atelocollagen having a weight average molecular weight of 100 kDa to 500 kDa) so that the second solution penetrated the support, after which the support having the second solution therein was immersed in methanol having a mass concentration of 60% and dried in a vacuum.

<Preparation Example 6> Preparation of Hydrogel Support for 3D Printer Containing Additive

[0057] A hydrogel support for a 3D printer containing an additive was prepared in the same manner as Preparation Example 5, with the exception that the first solution was prepared by adding a mixture, obtained by mixing β-D-mannuronic acid and α-L-guluronic acid having a weight average molecular weight of 100 kDa to 500 kDa at a ratio of 50:50 at room temperature, with 0.5 parts by weight of an additive (including a buffer, an osmotic pressure regulator, an emulsifier, and lidocaine, which are mixed together).

<Example 1> Preparation Depending on Concentration of Divalent Cation

[0058] Changes in a solid state depending on the concentration of the divalent cation were measured, and the results thereof are shown in Table 1 below.

TABLE-US-00001 TABLE 1 A Classification 0.0% 0.5% 1.0% 1.5% 2.0% B 4% 1 sec  5 secs 6% 1 sec  3 secs 5+ secs 8% 3 secs 5+ secs 10%  5 secs A: concentration of CaC1.sub.2 (w/v) B: concentration of mixture of β-D-mannuronic acid and α-L-guluronic acid (w/v) Table color: White is a liquid state, and as the color darkens, the liquid changes to a solid state. (sol −> sold)

[0059] Table 1 shows the state change depending on the concentration of the divalent cation and the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid. When the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid was 4 wt % in the first hydrogel and the mass concentration of the calcium ion was 1.0 to 1.5%, the state shown in FIG. 4 resulted.

[0060] In addition, when the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid was 6 wt % in the hydrogel composition and the mass concentration of the calcium ion was 0.5 to 1.0%, the state shown in FIG. 4 resulted. When the mass concentration of the calcium ion was 1.5%, a hard gel was formed, as shown in FIG. 1.

[0061] In addition, when the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid was 8 wt % in the hydrogel composition and the mass concentration of the calcium ion was 0.5%, the state shown in FIG. 4 resulted. When the concentration of the calcium ion was 1.0%, a hard gel was formed, as shown in FIG. 1.

[0062] In addition, when the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid was 10 wt % in the hydrogel composition and the mass concentration of the calcium ion was 0.5%, the state shown in FIG. 4 resulted.

[0063] As is apparent from Table 1, application of the hydrogel composition to a support for injection or a support for a 3D printer is determined depending on the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid and the concentration of the divalent cation. Here, the calcium ion was added at a ratio of 3:7 relative to the mixture of β-D-mannuronic acid and α-L-guluronic acid.

<Example 2> Change in Morphology Depending on Concentration of Divalent Cation

[0064] When the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid was each of 4, 6, 8, and 10 wt % in the hydrogel composition in Example 1, changes in morphology depending on the concentration of the divalent cation were measured.

TABLE-US-00002 TABLE 2 B 50 100 150 200 250 300 A mM mM mM mM mM mM 4% 5 kPa  5 kPa  6 kPa 21 kPa 21 kPa 26 kPa 6% 5 kPa 20 kPa 21 kPa 21 kPa 25 kPa 29 kPa 8% 28 kPa 34 kPa — — — 10%  — 35 kPa — — — — A: concentration of mixture of β-D-mannuronic acid and α-L-guluronic acid B: concentration mM of calcium ion —: Impossible to measure due to non-uniform gel state

[0065] As is apparent from Table 2, the tensile strength of the gel increased in most cases depending on the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid and the concentration of the divalent cation. However, when both the concentration of the mixture and the concentration of the divalent cation were increased, non-uniform gelation progressed due to drastic egg box formation between the internal carboxyl groups of β-D-mannuronic acid and α-L-guluronic acid and the divalent cation.

<Example 3> Change in Morphology of Mixture of β-D-Mannuronic Acid, α-L-Guluronic Acid, Aminated Hyaluronic Acid, and Collagen

[0066]

TABLE-US-00003 TABLE 3 B 50 100 150 200 250 300 A mM mM mM mM mM mM 4% 6 kPa  6 kPa  7 kPa 22 kPa 23 kPa 26 kPa (+0.5% + 1.0%) 6% 6 kPa 20 kPa 21 kPa 22 kPa 25 kPa 30 kPa (+0.5% + 1.0%) 8% 27 kPa 35 kPa — — — (+0.5% + 1.0%) 10% — 34 kPa — — — — (+0.5% + 1.0%) A: concentration of mixture of β-D-mannuronic acid and α-L-guluronic acid uniformly added with 0.5% aminated hyaluronic acid and 1.0% collagen B: concentration mM of calcium ion —: Impossible to measure due to non-uniform gel state

[0067] As is apparent from Table 3, the tensile strength of the gel increased in most cases depending on the concentration of the mixture of β-D-mannuronic acid and α-L-guluronic acid and the concentration of the divalent cation, like Table 2. However, when both the concentration of the mixture and the concentration of the divalent cation were increased, non-uniform gelation progressed due to drastic egg box formation between the internal carboxyl groups of β-D-mannuronic acid and α-L-guluronic acid and the divalent cation, indicating that egg box formation is not affected by the divalent cation concentration.

<Test Example 1> Cytotoxicity Test of Hydrogel of Preparation Example 3

[0068] The hydrogel composition prepared in Preparation Example 3 was dissolved in an amount of 10 wt % in PBS, and this mixture was added with 1×10.sup.6/100 μl of fibroblasts. The solution containing cells was cultured for 3 days in a DEME medium (10% FBS and 1% antibiotic), after which a cytotoxicity test was performed thereon using a Live and Dead kit.

[0069] Consequently, the average cell viability after 3 days was observed to be 96% or more.

<Test Example 2> Cytotoxicity Test of Hydrogel Support of Preparation Example 5

[0070] The hydrogel support prepared in Preparation Example 5 was dissolved in an amount of 10 wt % in PBS, and this mixture was added with 1×10.sup.6/100 μl of fibroblasts. The solution containing cells was cultured for 3 days in a DEME medium (10% FBS and 1% antibiotic), after which a cytotoxicity test was performed thereon using a Live and Dead kit.

[0071] Consequently, the average cell viability after 3 days was observed to be 98% or more.