KR101776675B1 - Multi-syringe for producing collagen hydrogel - Google Patents

Abstract

Embodiments of the present invention relate to a method of manufacturing a collagen hydrogel and a multi-syringe capable of injecting a collagen hydrogel into a living body or an implant. The first syringe containing the collagen preparation and the second syringe containing the sodium hydrogen carbonate preparation And a multi-syringe.

Description

[0001] MULTI-SYRINGE FOR PRODUCING COLLAGEN HYDROGEL [0002]

The present invention relates to a method for producing a collagen hydrogel and a multi-syringe capable of injecting a collagen hydrogel into a living body or an implant.

Biomolecular engineering is a field of research that focuses on the repair of damaged tissues and organs in the event of injury to living tissues or organs due to disease or accident. In this regard, a method using a gel that can be injected as a supporter for regenerating a living tissue has been extensively studied. The method using the injectable gel can be easily injected into a necessary site without surgical procedures, , Cost, and so on. As the injectable gel, gels using natural polymers such as collagen gel, chitosan gel and alginic acid gel and gels prepared using synthetic polymers such as PGA, PLGA and polycaprolactam have been used.

However, conventional gels are produced by polymerizing synthetic polymers or natural polymers with thermosensitive polymers or pH-sensitive polymers, and there is a problem that can induce inflammation in the body. In order to solve this problem, there has been a problem in that direct introduction of natural polymers has been studied, but it has been difficult to inject into the body due to an initial high viscosity and cell growth factors and physiologically active substances are not uniformly mixed. Further, when the viscosity is lowered, there is a problem that the physical properties are lowered, and there is a problem that the efficiency of the gel is reduced.

Accordingly, the inventors of the present invention have conducted studies to develop a hydrogel preparation and injection method which is less likely to be implanted in the body and has better tissue affinity and physical properties than the biopsy tissue. As a result, the collagen preparation, A method of injecting a sodium bicarbonate preparation containing substances into each syringe and simultaneously injecting it into a body or an implant was devised. When this method is used, since the viscosity is not high at the time of injecting both preparations prior to gel formation, it is easy to inject into the body, and the sodium hydrogen carbonate preparation having a low viscosity can be freely used including growth factors and physiologically active substances . Also, since the viscosity of both preparations is low, both preparations can be uniformly mixed in the multi-syringe at the time of injection. Formation of the gel is carried out after injection of the positive agent and after treating it at 35 to 40 ° C for 5 to 30 minutes, and this process is naturally accompanied when the positive agent is injected into the body. The gel thus formed is excellent in physical properties and does not use a cross-linking agent and a synthetic polymer, so that it does not substantially cause an immune response after the transplantation.

The present invention seeks to provide a hydrogel having excellent physical properties and substantially not causing post-transplantation immune response.

In order to solve the above-described problems,

A first syringe containing a collagen preparation and a second syringe containing a sodium bicarbonate formulation.

Embodiments of the present invention also provide a method for preparing a hydrogel comprising injecting a first syringe containing a collagen preparation and a second syringe containing a sodium bicarbonate preparation simultaneously into an animal body or an implant.

The hydrogel according to the embodiments of the present invention is composed of only natural polymers and does not substantially cause post-transplantation immune reaction. Thus, it has excellent biocompatibility and can mix the physiologically active substance and the growth factor uniformly in the high-viscosity hydrogel, Or can be injected effectively into the implant.

1 is a schematic diagram of a multi-syringe.
FIG. 2 is a photograph of the result of gel formation test on the pH of various collagen. FIG.
FIG. 3 is a photograph of gel formation test results using a pH 3.0, pH 4.0 collagen preparation. FIG.
Figure 4 is a photograph of a biopsy result according to various sodium bicarbonate formulation.
5 is a photograph of MT staining for each experimental group of Example 2. Fig.
Fig. 6 shows the results of compressive strength measurement for each experimental group of Example 3. Fig.
7 is a photograph of the biopsy result of each experimental group of Example 4. Fig.
8 is a MT staining photograph for each experimental group of Example 4. Fig.
9 is a photograph of von-kossa staining for each experimental group of Example 4. Fig.
Fig. 10 is a photograph of gel-forming experiment results of Example 5. Fig.

Best Mode for Carrying Out the Invention

Embodiments of the present invention provide a multi-syringe comprising a first syringe containing a collagen formulation and a second syringe containing a sodium bicarbonate formulation.

The inventors of the present invention have studied to develop a hydrogel preparation and infusion method which is less susceptible to transplantation and is more similar to a biotissue and has better tissue affinity and physical properties. As a result, it has been found that collagen preparation, Injected into a syringe to form the multi-syringe, and then injecting the preparation into the body or implant at the same time using the multi-syringe, the injection of the hydrogel is easy, and each component Were mixed homogeneously and did not use a cross-linking agent and a synthetic polymer, so that the immune reaction did not substantially occur after the transplantation, thereby completing the present invention.

The fact that the immune response does not substantially occur means that no inflammatory reaction or exudate is found after the administration of the antibiotic during the normal period after the injection, and the engraftment in the living body is well performed.

Also, the multi-syringe means an apparatus in which a plurality of syringe internal solutions can be mixed and flowed out with one outlet. In one embodiment of the present invention, the number of syringes constituting the multi-syringe is 2 to 3 .

In another embodiment of the present invention, the multi-syringe may be a structure as shown in Fig.

1, a first syringe 10 including a collagen preparation and a second syringe 20 including a sodium bicarbonate preparation are connected to each other through a connection portion 30. In the connection portion 30, The inner solution and the solution in the second syringe are mixed and flowed out into one outlet

The outflow should be performed while maintaining the same proportion of the positive agent. An auxiliary device (40) for fixing the pistons of the both syringe may be used to equalize the injection rate.

The first syringe and the second syringe may be selected with appropriate dosages for control of the injection rate.

In one embodiment of the invention, the pH of the collagen preparation within the first syringe may be 2.5 to 4.5, 3 to 4.3 or 3.5 to 4.2.

In another embodiment of the present invention, the amount of collagen contained in the collagen preparation is 10 to 100 mg / ml, 10 to 70 mg / ml, 10 to 50 mg / ml, 10 to 30 mg / ml or 15 to 25 mg / ml. < / RTI >

The collagen promotes cell attachment, migration and proliferation. It is possible to use both insoluble collagen and soluble collagen. Specifically, it may be derived from a mammal such as cow, or derived from marine life such as skin of tibia fish.

Specific examples of the marine life include, but are not limited to, fishes having a non-pigmented skin, more specifically, sea urchins such as Seodaemun, Turmeric, Turbot, Brass and the like.

In still another embodiment of the present invention, in addition to the collagen, at least one selected from the group consisting of methylated collagen and succinylated collagen may be further used.

Since the methylated collagen has a positive polarity and the succinylated collagen has a negative polarity, the dissolution rate can be easily controlled according to the polarity of the mixed growth factor and the physiologically active substance.

When methylated collagen is used, it may be used in an amount of 5 to 40 parts by weight or 5 to 35 parts by weight based on 100 parts by weight of total collagen, and when succinylated collagen is used, 5 to 60 parts by weight, 5 to 55 parts by weight or 10 to 40 parts by weight may be used.

The collagen preparation is prepared by dissolving at least one selected from the group consisting of collagen, methylated collagen and succinylated collagen in an acidic solution. Acetic acid or hydrochloric acid aqueous solution may be used as the acidic solution. But is not limited to.

In one embodiment of the invention, the amount of sodium bicarbonate contained in the sodium bicarbonate formulation within the second syringe may be from 0.07 to 0.1 g / ml or from 0.08 to 0.09 g / ml. The sodium hydrogencarbonate preparation can be used by purchasing commercially available sodium hydrogencarbonate buffer solution for in vivo injection.

In another embodiment of the present invention, the sodium hydrogencarbonate preparation may further comprise, in addition to sodium hydrogencarbonate, a mixture of at least one selected from the group consisting of hydroxyapatite, osteogenic protein, hyaluronic acid and glycosaminoglycan .

Hydroxyapatite (HAp) is basic calcium phosphate, and its chemical formula is Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ . It is very similar to inorganic components of human bones and teeth. It is not bio-toxic and promotes bone induction at the interface. Therefore, it is a representative biomaterial widely used as a coating material for artificial implants.

In yet another embodiment of the present invention, the hydroxyapatite particles may have a diameter of 1 to 500 nm and may be used at a concentration of 0.0001 to 1 g / ml, 0.001 to 0.5 g / ml, or 0.005 to 0.2 g / ml.

Bone morphogenetic protein (BMP) is used to promote osseointegration. It may be used in an amount of 0.1 to 10 μg / ml, 0.1 to 5 μg / ml, or 0.1 to 2 μg / ml, 2 or BMP-12 may be used, but is not limited thereto.

Hyaluronic acid is a kind of acidic mucopolysaccharide in which n-acetylglucosamine and glucronic acid bind to each other and is widely distributed in the connective tissues of mammals together with chondroitin sulfate and the like. It is known to maintain moisture in the cell gap in the connective tissue, to form a gel-like matrix in the tissue to maintain the cells, to provide skin lubrication and flexibility, and to prevent external force and bacterial infection.

In another embodiment of the present invention, the hyaluronic acid may be used in an amount of 0.1 to 30 mg / ml, 1 to 20 mg / ml or 7 to 20 mg / ml.

Glycosaminoglycan has a cross-linking role between collagens. But are not limited to, chondroitin, chondroitin sulfate, heparan, heparan sulfate and dermatan sulfate, and the like.

In another embodiment of the present invention, the glycosaminoglycan may be used in an amount of 0.1 to 20 mg / ml, 1 to 8 mg / ml, or 3 to 7 mg / ml.

In an embodiment of the invention, the volume of the first syringe may be 2 to 15 times, 3 to 12 times, or 5 to 10 times the volume of the second syringe.

The embodiments of the present invention also include a method of injecting a collagen preparation contained in a first syringe and a sodium bicarbonate preparation contained in a second syringe into a body or an implant of an animal including a human, Gel. ≪ / RTI >

In one embodiment of the invention, simultaneous infusion means that infusions of the first syringe and the second syringe contents are injected at the same time in an arbitrary ratio, the ratio being determined by the ratio between the sodium bicarbonate preparation 1 part skin collagen preparation 2 To 15 parts skin, from 3 to 12 parts skin, or from 5 to 10 parts by volume.

The animal is preferably all vertebrate animals, including humans, and more preferably mammals.

Such injection may be made in the body of the animal, or may be internal to the implant.

And further treating the gelatinized material by treatment at 35 to 40 DEG C or 36 to 38 DEG C for 5 to 30 minutes or 10 to 20 minutes after the injection to induce gelation of the injected material.

In one embodiment of the invention, collagen preparations; Sodium hydrogencarbonate preparations; A first syringe; And a second syringe. The volume of the collagen preparation in the kit may be 2 to 15 times, 3 to 12 times, or 5 to 10 times the volume of the sodium hydrogencarbonate preparation. The kit may be assembled in the form of a multi-syringe immediately prior to use and may be used to inject both contents at the same rate.

DETAILED DESCRIPTION OF THE INVENTION

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Optimization of collagen preparation pH  Selection

A collagen preparation of pH 2.0, pH 3.0, pH 4.0, pH 5.0, pH 7.0 and pH 8.0 was prepared by dissolving 20 mg / ml of Atelocollagen (Bio Land, Korea) in an aqueous hydrochloric acid solution.

The 8.4% sodium hydrogencarbonate (NaHCO ₃ ) buffer solution, which is currently in clinical use, was used as the sodium hydrogencarbonate preparation.

The collagen of each pH was put into a syringe for 10 ml, and a sodium bicarbonate preparation was put in a syringe for 1 ml. Then, two syringes were attached to a glue adapter to form a multi-syringe.

The pistons of both syringes were pressed at the same time to mix the two preparations at the same volume ratio of 10: 1 and injected through the injection needle. The gelation was induced by treatment at 37 ° C for 15 minutes.

Referring to FIG. 2, gels were formed only in the experimental group using collagen of pH 3 and pH 4 in the collagen of various pH, and no gel was formed at the other pH.

FIG. 3 is a graph _{showing the results obtained} by adding 0.015 g / ml hydroxyapatite and 1 g / ml BMP-2 to 8.4% sodium hydrogencarbonate (NaHCO ₃ ) buffer under the same conditions as above, To form a gel. As a result, it can be confirmed that the gel is formed.

< Example  2> Evaluation of animal efficacy according to composition and mixing ratio of sodium hydrogencarbonate preparation

2% (w / v) Hyaluronic Acid (pH 3.0, Bio Land, Korea), 8.4% sodium hydrogencarbonate buffer solution, 2% (w / v) Atelocollagen (pH 4.0, (Hartman injection) were prepared. The Hartmann's solution containing sodium bicarbonate was prepared by mixing 10% (v / v) 8.4% sodium bicarbonate buffer solution in Hartmann's solution, and 2% hyaluronic acid and 8.4% sodium hydrogencarbonate buffer in sodium bicarbonate containing hyaluronic acid Sodium hydrogencarbonate and hyaluronic acid-containing water were prepared by mixing 10% (v / v) 8.4% sodium hydrogencarbonate buffer solution with Hartmann's solution and then adding 2% hyaluronic acid to 50% (v / v) % (V / v).

A: 2% collagen: 8.4% sodium hydrogencarbonate buffer solution = 10 ml: 1 ml injection

B: 2% collagen: 8.4% sodium hydrogencarbonate buffer solution = 1 ml: injected at a ratio of 1 ml

C: 2% collagen: sodium hydrogencarbonate Hartmann injection = 10 ml: 1 ml injection

D: 2% collagen: sodium hydrogencarbonate Hartmann injection = 1 ml: 1 ml injection

E: 2% Collagen: Hyaluronic acid-containing sodium hydrogencarbonate = 10 ml: 1 ml injection

F: 2% Collagen: Hyaluronic acid-containing sodium hydrogencarbonate = 1 ml: 1 ml injection

G: 2% collagen: injected with a ratio of sodium hydrogencarbonate and hyaluronic acid: Hartman injection water = 10 ml: 1 ml

H: 2% collagen: injected with a ratio of sodium hydrogencarbonate and hyaluronic acid = 1 ml: 1 ml

Fig. 4 shows the result of biopsy after subcutaneous injection for evaluation of inflammation and degradation according to various sodium hydrogencarbonate preparations. As a result, the composition (left) mixed with 10% of buffer solution (left) Of the total amount of water.

FIG. 5 shows the results of MT staining 1 week after the injection of the sample. As a result, the buffer solution was mixed with 10% (v / v) in 50% (v / v) mixed group (B, D, (V / v) mixed group (A, C, E, G) and 50% (v / v) , C, E, and G).

< Example 3 > Measurement of compressive strength according to sodium bicarbonate preparation and mixing ratio

The injected collagen hydrogel was extracted after 2 weeks according to the composition and mixing ratio of the various buffer solutions prepared in Example 2, cut into a size of 2 × 2 mm, and then measured with a universal testing machine (HOUSFIELD, UK) The compression test was performed by applying pressure at a rate of mm / min.

As shown in FIG. 6, it was confirmed that the mechanical compression strength was the most improved in the experimental group (E) in which 10% (v / v) of sodium hydrogencarbonate containing hyaluronic acid was injected together.

<Room Sime  4> Evaluation of animal efficacy according to composition and mixing ratio of sodium hydrogencarbonate preparation

To evaluate the efficacy of collagen hydrogel prepared by adding various mixtures to a sodium bicarbonate preparation, a bone morphogenetic effect was evaluated using a mouse as a model.

After injecting a sodium bicarbonate formulation consisting of the following combination of A to F into a 1 ml syringe, 2% (w / v) Atelocollagen (pH 4.0, Bio Land, Korea) was injected into a 5 ml syringe, To prepare a multi-syringe and injected subcutaneously in the mouse while maintaining the ratio of both compositions constant. Biopsies were performed approximately two weeks after injection.

A: 8.4% sodium hydrogencarbonate

B: 8.4% sodium hydrogencarbonate + hydroxyapatite 0.015 g / ml

C: 8.4% sodium hydrogencarbonate + hydroxy apatite 0.015 g / ml + BMP-2 0.1 g / ml

D: 8.4% sodium hydrogencarbonate + 20% (v / v) hyaluronic acid

E: 8.4% Sodium bicarbonate + 20% (v / v) of hyaluronic acid + 0.015 g / ml of hydroxyapatite

F: 8.4% sodium hydrogencarbonate + 20% (v / v) hyaluronic acid + hydroxyapatite 0.015 g / ml + BMP-2 0.1 g / ml

As a result of observing the hydrogel prepared according to the above combination, as shown in FIG. 7, the group in which sodium hydrogen carbonate (A) and sodium hydrogencarbonate (D) containing 10% of hyaluronic acid were injected with the buffer solution, (B, E) and / or BMP-2 (C, F) in the buffer solution injection group showed strong physical properties.

Figure 8 As a result of MT biopsy after 2 weeks of transplantation, some mononuclear cells were observed in nanohydroxyapatite (B, E) and BMP-2 added groups (C, F), but mild histologic results (A, D) mixed with only buffer solution showed almost no inflammatory reaction.

9, As a result of von-kossa staining after biopsy 2 weeks after transplantation, many calcium deposits were observed in nanohydroxyapatite added group (B, C, E, F).

< Example  5 &gt; methylated collagen ( 메틸 collagen ) And Succinylated  Collagen( succinylated collagen)  Mixed Hydrogel  Produce

To control the polarity of the surface of the collagen, methylated collagen and succinylated collagen were prepared and hydrogels containing these collagens were prepared. First, the collagen was titrated to pH 9.0 with NaOH to prepare a succinylated collagen with negative charge. Then, acetone and succinic anhydrous were added, and the mixture was titrated to pH 4.2 and then lyophilized. Positively charged methylated collagen was prepared by dissolving collagen in methanol, adding sodiumsulfate and lyophilization. Each of the prepared polar collagens was dissolved at a concentration of 10 mg / ml and then mixed with collagen at pH 4.0 in various ratios. Then, the collagen preparation and the sodium hydrogen carbonate preparation of the present invention were injected at a ratio of 7: 3.

As shown in FIG. 10, when the methylated collagen (A, B) was mixed with collagen at 30% or more, the gelation speed was slowed. However, even if the collagen was mixed with collagen up to 50% (AD), respectively. Hydrogel containing 30% of collagen and methylated collagen (E) or succinylated collagen (F), respectively, and hydrogel containing hyaluronic acid, nanohydroxyapatite and BMP-2 were also treated well at 37 ° C for 15 minutes. It can be observed that it is formed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

A first syringe containing a collagen preparation and a second syringe containing a sodium bicarbonate formulation,
Wherein the volume of the first syringe is 2 to 15 times the volume of the second syringe. The method according to claim 1,
Wherein the pH of said collagen preparation is 2.5 to 4.5. The method according to claim 1,
Wherein the amount of collagen contained in the collagen preparation is 10 to 100 mg / ml. The method according to claim 1,
Wherein the collagen of the collagen preparation further comprises at least one selected from the group consisting of methylated collagen and succinylated collagen. 5. The method of claim 4,
Wherein the amount of methylated collagen comprises 5 to 40 parts by weight based on 100 parts by weight of total collagen, and the amount of succinylated collagen is 5 to 60 parts by weight based on 100 parts by weight of total collagen. The method according to claim 1,
Wherein the amount of sodium hydrogencarbonate contained in the sodium hydrogen carbonate preparation is 0.07 to 0.1 g / ml. The method according to claim 1,
Wherein the sodium hydrogencarbonate preparation further comprises, in addition to sodium hydrogen carbonate, at least one selected from the group consisting of hydroxyapatite, osteogenic protein, hyaluronic acid, and glycosaminoglycan. delete The multi-syringe according to claim 1, wherein the multi-syringe comprises a structure in which a first syringe and a second syringe are connected through a connection portion, and a collagen preparation and a sodium bicarbonate preparation are mixed in a connection portion, . 10. The multi-syringe of claim 9, further comprising an auxiliary mechanism connecting the piston of the first syringe and the piston of the second syringe. Injecting the sodium hydrogen carbonate preparation contained in the first syringe and the collagen preparation and the second syringe contained in the first syringe while maintaining an arbitrary ratio in the body of an animal other than a human,
Wherein the volume of the first syringe is 2 to 15 times the volume of the second syringe and the volume of the collagen preparation is injected into 2 to 15 parts of the volume of the sodium bicarbonate formulation. Implanting the collagen preparation contained in the first syringe and the sodium bicarbonate formulation contained in the second syringe into the implant while maintaining an arbitrary ratio,
Wherein the volume of the first syringe is 2 to 15 times the volume of the second syringe and the volume of the collagen preparation is injected into 2 to 15 parts of the volume of the sodium bicarbonate formulation. 13. The method according to claim 11 or 12,
Treating the injected material by treating the mixture at 35 to 40 DEG C for 5 to 30 minutes after the injection, thereby inducing gelation of the injected material. delete delete

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