KR20200054578A - Preparation of cartilage acellular matrix hydrogel with controllable biodegradation and properties - Google Patents

Abstract

The present invention relates to hydrogel manufactured by crosslinking animal cartilage-derived tissue and a method for manufacturing the same. The biocompatible animal cartilage-derived tissue hydrogel according to the present invention can delay a biodegradation period by biochemical crosslinking according to a content of a crosslinking agent, etc., can increase mechanical properties, and can adjust the biodegradation period and the mechanical properties according to formation of a cross linking degree. In addition, the animal cartilage-derived tissue hydrogel according to the present invention is used as an injectable preparation, thereby being used for tissue repair, drug delivery, cell delivery, etc.

Description

Preparation of cartilage acellular matrix hydrogel with controllable biodegradation and properties}

The present invention relates to a hydrogel prepared by crosslinking tissues derived from animal cartilage and a method for manufacturing the same.

Cartilage, like other connective tissue, is composed of connective tissue cells and acellular matrix, but unlike the native connective tissue, it is a special connective tissue that contains a rigid yet somewhat flexible substrate. The connective tissue cells of cartilage are mostly composed of one chondrocyte, and these chondrocytes are located in the cartilage cavity in the cartilage matrix.

Cell-free substrates are divided into fibers and amorphous substances. Most fiber components are composed of type II collagen (collagen), but some cartilage is rich in elastic fibers. The intangible substance is composed mainly of protein sugar composed mainly of sulfated glycosaminoglycans. In the cartilage matrix, a large number of protein sugar molecules are linked by a linking protein by hyaluronic acid, one of glycosaminoglycans, to form a macromolecule. These macromolecules are also combined with glue fibers. The protein-sugar molecule is attached to the glue fiber by a non-morphic glycoprotein, chondronetin.

As other substrates, non-collagen proteins and glycoproteins account for 10-15% of the dry weight of cartilage, and these mainly stabilize the structure of the substrate macromolecule and help to form organic tissue.

The natural material derived from such animal cartilage tissue is composed of an extracellular matrix (ECM). The extracellular matrix provides an environment in which cells can stay while supplying the chemical factors necessary for cells to grow and differentiate, and has high biocompatibility and almost no immune response. However, the biodegradation period is short and the physical properties are low, which limits its practical application. Therefore, in order to solve these limitations, it is necessary to improve physical properties of cartilage-derived tissue through physical or chemical treatment.

Recently, injection-type hydrogels have received a lot of attention in the medical field, and are expected to be widely used, such as the release system of bioactive substances from medical fillers, and organ or tissue regeneration using a three-dimensional structure. The injection-type hydrogel has an advantage that it can be injected into a living body simply by using a syringe or the like without a surgical procedure. In general, injection-type hydrogels have fluid-like properties outside the body and can be implanted using a syringe, and gelation occurs after injection into the body. Therefore, after transplantation, it can serve as a drug delivery system for sustained release of drugs or bioactive substances or as a support for maintaining cell growth. In addition, by introducing various crosslinking methods, it is possible to control the physicochemical properties such as gelation time, degree of swelling, decomposition, and mechanical properties of the hydrogel, so it is a great advantage to manufacture the hydrogel according to the desired purpose and use it in the drug delivery system or tissue engineering. It becomes.

Accordingly, the present inventors tried to provide a hydrogel and a method for preparing the hydrogel that can control the physical properties, as a result of crosslinking the tissue derived from animal cartilage, which is a natural material, to produce a hydrogel. Since the physical properties can be controlled, the present invention was completed by confirming that it could be used as an injectable preparation for tissue repair, drug delivery, or cell delivery with biocompatibility.

In the case of natural materials, biocompatibility is excellent, but biodegradation rate and mechanical properties are difficult to control, so there are limitations in the application as natural materials, and it is necessary to increase physical properties and improve physical properties by chemical treatment.

Thus, the present inventors are biocompatible cartilage-derived tissues capable of forming a gel that is not easily degraded through a chemical reaction with a cross-linking agent using tissues derived from animal cartilage, which is a natural material, and can control biodegradation period and mechanical properties according to the content of the cross-linking agent. It is intended to provide a method of manufacturing a hydrogel.

The present invention comprises the steps of: (a) preparing a cartilage-derived tissue powder by pulverizing animal cartilage-derived tissue; (b) preparing a cross-linked cartilage-derived tissue by mixing and crosslinking the aqueous solution containing cartilage-derived tissue powder prepared in step (a) with a solution containing a cross-linking agent; And (c) drying the cross-linked cartilage-derived tissue prepared in step (b) to powder again, and then dispersing it in a buffer solution to prepare a hydrogel.

In step (a), the animal may be one or more selected from the group consisting of humans, pigs, cows, sheep, horses, and cats.

In the step (b), the crosslinking agent may have one or more functional groups selected from the group consisting of aldehyde groups, epoxy groups, imide groups, amine groups and carboxyl groups.

With respect to the total content of the cartilage-derived tissue powder and the crosslinking agent in step (b), the content of the crosslinking agent may be 0.05% to 10% by weight.

The dispersion in the step (c) may be dispersed in a crosslinked cartilage derived tissue powder in a buffer solution of 5% to 30% by weight.

In one embodiment of the present invention, a hydrogel prepared by the above method is provided.

In another embodiment of the present invention, there is provided an injectable formulation comprising the hydrogel.

The injectable preparation may be for tissue repair, drug delivery, or cell delivery.

Biocompatible animal cartilage-derived tissue hydrogels according to the present invention can delay biodegradation time by biochemical crosslinking depending on the content of crosslinking agent, etc., can increase mechanical properties, and biodegradation period and It has the advantage of being able to control mechanical properties.

In addition, the tissue cartilage derived from animal cartilage according to the present invention is used as an injectable preparation, and has characteristics that can be used for tissue repair, drug delivery, or cell delivery.

Figures 1a and b shows a method for producing a tissue powder derived from pig cartilage biochemically crosslinked according to Example 1.
Figure 2a shows a method of manufacturing IR783-COOH according to Examples 4-6, Figure 2b is a fluorescent material is introduced according to Examples 4-6, biochemically cross-linked method for producing a tissue powder derived from pig cartilage It is shown.
3 is a photograph showing a tissue hydrogel derived from pig cartilage prepared in Example 7.
Figures 4a to c is a graph comparing the results of measuring the rheological properties (storage modulus, loss modulus and composite viscosity) of the tissue cartilage derived from pig cartilage prepared in Examples 7-9.
Figure 5a to d is to evaluate the compressive strength and injectability of pig cartilage-derived tissue hydrogel prepared in Example 7.
FIG. 6 is a photograph of observation and evaluation of biodegradation behavior as a result of performing an animal experiment on a hydrogel using a tissue powder derived from pig cartilage in which a fluorescent substance was introduced and biochemically crosslinked according to Example 4.
Figure 7a and b is a fluorescent material is introduced according to Example 4, as a result of performing an animal experiment on a hydrogel using a tissue powder derived from pig cartilage biochemically crosslinked, hematoxylin and eosin (H & E) staining of the extracted tissue And ED1 (mouse anti rat CD68: Serotec, UK) staining pictures.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of: (a) preparing a cartilage-derived tissue powder by pulverizing animal cartilage-derived tissue; (b) preparing a cross-linked cartilage-derived tissue by mixing and crosslinking the aqueous solution containing cartilage-derived tissue powder prepared in step (a) with a solution containing a cross-linking agent; And (c) drying the cross-linked cartilage-derived tissue prepared in step (b) to powder again, and then dispersing it in a buffer solution to prepare a hydrogel.

“Hydrogel” in the present specification refers to a gel that uses water as a dispersion medium, and hydrosol loses fluidity due to cooling, or a hydrophilic polymer having a three-dimensional network structure and microcrystalline structure contains water and expands. Speak.

The hydrogel has a porous structure, and when used as a drug carrier carrying the drug, it is possible to effectively deliver the drug to target cells while minimizing the dispersion of the drug. In addition, when used as a support, including stem cells, it can have the advantage of increasing the survival rate of cells by smoothly supplying sufficient oxygen and nutrients to the cells.

First, the method of manufacturing a hydrogel according to the present invention includes the step of preparing a cartilage-derived tissue powder by powdering the tissue derived from animal cartilage [step (a)].

“Cartilage-derived tissue” in the present specification refers to an animal-derived cartilage tissue, preferably an animal-derived cartilage acellular matrix (CAM), and includes a fiber component composed mostly of collagen. The "animal" from which the cartilage tissue is derived is preferably a mammal, and more preferably, any one selected from the group consisting of humans, pigs, cows, sheep, horses, and cats, and more specifically in humans or pigs. It is preferably derived, but is not limited thereto.

Powdering the tissues derived from animal cartilage comprises: a) lyophilizing and crushing the tissues derived from animal cartilage; b) decellularizing the cartilage derived tissue powder crushed in step a); And c) treating the decellularized cartilage-derived tissue powder in step b) with an acidic solution and pepsin, followed by neutralizing with a basic solution to obtain a cartilage-derived tissue powder aqueous solution.

Specifically, the powdering of the tissues derived from animal cartilage may mean freeze-drying and lysing the tissues derived from animal cartilage. The lyophilization and freeze-grinding may be performed under cryogenic conditions using pre-treated animal cartilage-derived tissue, wherein the cryogenic conditions refer to -100 to -50 ° C and 0.01 mTorr to 10 mTorr conditions. In addition, freeze crushing may be performed using a known grinder, and the prepared cartilage-derived tissue powder may have a size of 10 μm to 100 μm.

Thereafter, after dissolving the prepared cartilage-derived tissue powder in an aqueous solution containing a proteolytic enzyme, neutralizing, and then removing the proteolytic enzyme by a method such as dialysis, water-soluble cartilage-derived tissue powder can be prepared. Thereafter, lyophilization may be added at an extremely low temperature. At this time, as a proteolytic enzyme, an enzyme capable of degrading collagen constituting cartilage-derived tissue powder may be used, and pepsin is preferable, but is not limited thereto.

Next, the method of manufacturing a hydrogel according to the present invention comprises mixing and crosslinking the aqueous solution containing cartilage-derived tissue powder prepared in step (a) with a solution containing a crosslinking agent to prepare a cross-linked cartilage-derived tissue [(b) Step].

It is preferable to use a chemical crosslinking agent as the “crosslinking agent” in the present specification, but is not limited thereto. The chemical crosslinking agent inhibits decomposition by body fluids by forming crosslinks between polymer chains, thereby enabling control of a period during which the hydrogel according to the present invention decomposes in the human body. That is, the support using a natural material can improve mechanical properties through the treatment of a crosslinking agent.

The crosslinking agent preferably has at least one functional group selected from the group consisting of aldehyde groups, epoxy groups, imide groups, amine groups and carboxyl groups, but is not limited thereto.

Specifically, formaldehyde, glutaraldehyde or dextrin aldehyde may be used as a crosslinking agent having an aldehyde group as a functional group. In addition, as a crosslinking agent having an epoxy group as a functional group, butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE), ethylene glycol diglycidyl ether (EGDGE), hexanediol diglycidyl Ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether ( polytetramethylene glycol). Moreover, as a crosslinking agent which has an imide group, 1-ethyl-3- (3-dimethyl aminopropylcarboimide), 1-ethyl-3- (2-morpholinyl-4-ethyl) carboimide or dicyclohexylcarboimide Etc. can be used. In addition, as a crosslinking agent having an amine group or a carboxyl group as a functional group, a biocompatible polymer having an amine group or a carboxyl group at the side chain or terminal may be used. The biocompatible polymer may include polycaprolactone based on polyethylene glycol, and may be one or more selected from the group consisting of copolymers of monomers such as glycolide and lactide, but is not limited thereto. The aldehyde group, epoxy group or carboxyl group of the crosslinking agent can be chemically reacted with the amine group of collagen, and the imide group or amine group of the crosslinking agent can be crosslinked by chemical reaction with the carboxyl group of collagen.

More specifically, as the most preferred example of the crosslinking agent, an aqueous glutaraldehyde solution may be used, but is not limited thereto.

Further, with respect to the total content of the cartilage-derived tissue powder and the crosslinking agent, the content of the crosslinking agent is preferably 0.05% by weight to 10% by weight, more preferably 0.05% by weight to less than 1% by weight, but is not limited thereto. As the content of the crosslinking agent increases, the storage modulus and composite viscosity tend to increase. At this time, when the crosslinking agent is excessively mixed compared to the cartilage-derived tissue powder, there may be a limit in the degree of injectability when applying a hydrogel prepared by excessively forming cross-linking between the cartilage-derived tissue powder, and the internal space This decrease, and the rate at which it breaks down in the body may be delayed, so restrictions may apply when using it as an injectable formulation. Conversely, when the crosslinking agent is mixed with too little compared to the cartilage-derived tissue powder, since significant crosslinking is not formed, mechanical characteristics such as elastic modulus, compressive strength, and / or rheological properties may not be significant.

The mixing and crosslinking can be performed for 1 hour to 20 hours at a rate of 10 rpm to 500 rpm at 20 ° C to 50 ° C, and at 50 ° C to 200 rpm at 30 ° C to 40 ° C, for 5 hours to 20 hours. It is preferably performed for a period of time, but is not limited thereto.

Next, the method for producing a hydrogel according to the present invention is dried by cross-linking cartilage-derived tissue prepared in step (b) to powder again, and then dispersed in a buffer solution to prepare a hydrogel [(c) Step].

Before the dispersion, it may be further mixed and crosslinked with a fluorescent substance-containing solution having a carboxyl group. The fluorescent material has a carboxyl group as a functional group, and thus has an advantage of chemically reacting with an amine group of collagen. At this time, IR 783, IR 780, FITC (Fluorescein isothiocyanate), rhodamine, and PI (Propidium iodide) may be used as the fluorescent material before the functional group is introduced.

The dispersion may be dispersed in 5% to 30% by weight of the cross-linked cartilage-derived tissue powder in a buffer solution, preferably 15% to 30% by weight, but is not limited thereto. As the content of cross-linked cartilage-derived tissue powder in the buffer solution increases, the storage modulus, loss modulus, and composite viscosity tend to increase.

In addition, the present invention provides a hydrogel prepared by the above method.

For example, in the frequency range of 0.1 Hz to 10 Hz of the hydrogel, the storage modulus (G ') and loss modulus (G ”) measured using a rheometer are 5.0 × 10 ² Pa to 1.0 × 10 ⁴ Pa, respectively. And 1.0 Х 10 ¹ Pa to 5.0 Х 10 ² Pa, but is not limited thereto. In addition, the complex viscosity at 37 ° C of the hydrogel may be 5.0 Х 10 ¹ Pa · s to 1.0 Х 10 ³ Pa · s, but is not limited thereto. Such rheological properties can be regarded as depending on the content of the crosslinking agent and the content of the cross-linked cartilage-derived tissue powder in the buffer solution.

In addition, the present invention provides an injectable formulation comprising the hydrogel.

Since the above-described hydrogel has been described through a method of manufacturing the hydrogel, a duplicate description will be omitted.

The injectable preparation means a form injectable into a syringe. For example, the injectable preparation is preferably injectable into a syringe having a needle inner diameter of 10G to 32G, and injected into a syringe having a diameter of 20G to 30G. What is possible is more preferable, but is not limited thereto. At this time, if the inner diameter of the needle of the syringe becomes too small, the compressive strength per compression distance is continuously increased, and thus there is a problem that it is impossible to inject.

The injectable preparation may be for tissue repair, drug delivery, or cell delivery.

Specifically, the hydrogel may be used as a carrier for sustained release of the drug or bioactive factor through injection into the body by carrying the drug or bioactive factor therein. In addition, the hydrogel can be used as a support for bone differentiation when injected into the body by supporting stem cells therein.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Manufacturing example  One: In pigs  Separation of cartilage-derived tissue and preparation of water-soluble porcine cartilage-derived tissue powder

Under sterile conditions, articular cartilage was separated from the forelimbs and hind legs of a pig weighing about 100 kg, 6 months old. At the time of separation, the cartilage part was not separated from the cartilage part and was cut with a blade so that the separation was performed. The isolated articular cartilage tissue was treated with a storage buffer of 10 mM Tris-HCl at pH 8.0 for 12 hours, and then centrifuged using a centrifuge to remove the supernatant. After the precipitated porcine cartilage-derived tissue powder was transferred to a beaker, SDS buffer was added and stirred at 4 ° C for 2 hours. Then, sterile tertiary water was added, and the supernatant was removed by centrifugation at 2,000 rpm for 10 minutes using a centrifuge, and then the precipitated porcine cartilage joint was transferred to a beaker, and DNase solution was added and stirred at 100 rpm for 12 hours. After centrifugation at 2,000 rpm for 10 minutes, the supernatant was removed, and then freeze-crushed in liquid nitrogen at 3 cycle conditions with pre-cooling 50 seconds / grinding (60 Hz) 240 seconds / cooling 50 seconds to obtain porcine cartilage-derived tissue powder. Obtained. The obtained powder was put in a beaker and DNase solution was added and stirred at 100 rpm for 12 hours. Thereafter, the solution was centrifuged at 2,000 rpm for 10 minutes using a centrifuge, and after removing the supernatant, a small amount of sterilized tertiary water was added to suspend the tissue powder derived from pig cartilage, and the conditions were -70 ° C and 5 mTorr in a cryogenic freezer. And freeze-dried for 48 hours. The solution was prepared by stirring the lyophilized pork cartilage-derived tissue powder by adding 25 ml of 800 unit / ml pepsin aqueous solution per 1 g. After adding the NaOH solution to the prepared solution to neutralize it to pH 7.4, fix only one end of dialysis, pour the neutralized solution into the dialysis membrane, fix the other end, put it in 2 L of sterile tertiary water and add 4 ℃ It was stirred for 24 hours. After the dialysis, the aqueous solution of pepsin was lyophilized in a container to be lyophilized in a cryogenic freezer at -70 ° C and 0 mTorr for 48 hours to prepare water-soluble porcine cartilage derived tissue powder.

Example  One: Glutaraldehyde  Preparation of biochemical crosslinking reaction and powder using

0.2 g of water-soluble porcine cartilage derived tissue powder prepared in Preparation Example 1 was added to a 30 mL vial, dispersed in 15 mL of sterilized tertiary distilled water, and stirred at room temperature for 24 hours. After stirring, the solution was poured into Teflon mold (75 mm × 75 mm) and 1.5 mL of a crosslinking agent glutaraldehyde solution was evenly added dropwise with a glass pipette, followed by reaction in a shaker at 37 ° C. at a rate of 100 rpm for 12 hours and It was dried. At this time, compared to the total content of water-soluble pig cartilage derived tissue powder and glutaraldehyde, the content of glutaraldehyde is 0.1% by weight, 0.15% by weight, 0.2% by weight, 0.3% by weight, 0.5% by weight, 1% by weight, 3% by weight And 5% by weight. The dried solid form of cross-linked porcine cartilage-derived tissue powder is washed alternately for 30 minutes with 3 times of distilled water and phosphate buffer solution sterilized in a shaker at 37 ° C at 100 rpm, and freeze-dried for 48 hours to crosslink biochemically. Pork cartilage derived tissue powder was prepared (FIGS. 1A and B).

Example  2: Butanediol diglycidyl ether (BDDE)  Biochemical crosslinking reaction and Powder of  Produce

0.2 g of water-soluble porcine cartilage derived tissue powder prepared in Preparation Example 1 was added to a 30 mL vial, dissolved in 1% NaOH aqueous solution and stirred for 24 hours. After stirring, the solution was poured into a Teflon mold (75 mm × 75 mm) and 1.5 mL of a crosslinking agent butanediol diglycidyl ether (BDDE) solution was evenly added dropwise with a glass pipette, followed by 12 at 100 rpm at 37 ° C. Reaction and drying in a shaker for an hour. At this time, compared to the total content of water-soluble pig cartilage-derived tissue powder and butanediol diglycidyl ether (BDDE), the content of butanediol diglycidyl ether (BDDE) was adjusted to 0.3% by weight. The dried solid form of cross-linked porcine cartilage-derived tissue powder is washed alternately for 30 minutes with 3 times of distilled water and phosphate buffer solution sterilized in a shaker at 37 ° C at 100 rpm, and freeze-dried for 48 hours to crosslink biochemically. Pork cartilage derived tissue powder was prepared.

Example  3: Poly ( Caprolactone -co- Lactide -co- Glycolide )- NHS / Polyethylene glycol / Poly (caprolactone-co-lactide-co-glycolide) -NHS biochemical crosslinking reaction and preparation of powder

First, to prepare poly (caprolactone-co-lactide-co-glycolide) -NHS / polyethylene glycol / poly (caprolactone-co-lactide-co-glycolide) -NHS crosslinker, poly (caprolactone) 1-ethyl-3- (3-dimethylaminopropylcarboimide) (EDC) to 1 g of -co-lactide-co-glycolide) / polyethylene glycol / poly (caprolactone-co-lactide-co-glycolide) ) Was prepared by dissolving 0.01 g of 0.01 g and N-hydroxysuccinimide (NHS) in a methylene chloride solution, stirring for 12 hours, removing the solid with a filter, and drying the remaining solution at 60 ° C. for 24 hours.

0.2 g of water-soluble porcine cartilage derived tissue powder prepared in Preparation Example 1 was added to a 30 mL vial, dispersed in 15 mL of sterilized tertiary distilled water, and stirred at room temperature for 24 hours. After stirring, the solution was poured into Teflon mold (75 mm × 75 mm) and crosslinking agent poly (caprolactone-co-lactide-co-glycolide) -NHS / polyethylene glycol / poly (caprolactone-co-lactide- 1.5 mL of a co-glycolide) -NHS solution was evenly added dropwise with a glass pipette, and then reacted and dried in a shaker at 37 ° C at a rate of 100 rpm for 12 hours. At this time, the total content of water-soluble porcine cartilage-derived tissue powder and poly (caprolactone-co-lactide-co-glycolide) -NHS / polyethylene glycol / poly (caprolactone-co-lactide-co-glycolide) -NHS In contrast, the content of poly (caprolactone-co-lactide-co-glycolide) -NHS / polyethylene glycol / poly (caprolactone-co-lactide-co-glycolide) -NHS was adjusted to 0.3% by weight. The dried solid form of cross-linked porcine cartilage-derived tissue powder is washed alternately for 30 minutes with 3 times of distilled water and phosphate buffer solution sterilized in a shaker at 37 ° C at 100 rpm, and freeze-dried for 48 hours to crosslink biochemically. Pork cartilage derived tissue powder was prepared

Example  4-6: Near infrared Fluorescence imaging  for Fluorescent material  Preparation of containing powder

In order to introduce a fluorescent substance into the amine group (-NH ₂ ) of the pig cartilage derived tissue powders prepared in Examples 1 to 3, IR783 (80 mg, 0.11 mmol) and 4-mercaptobenzoic acid ( 51 mg, 0.33 mmol) was dissolved in DMF (3 mL) and stirred at room temperature for 24 hours. After the reaction, in order to remove unreacted substances and impurities from the reacted solution, precipitated in 30 mL of a mixed solvent of ethanol and diethyl ether (v / v = 1/19) and filtered to dry the green solid obtained by vacuum to IR783-COOH. It was prepared (Fig. 2a).

Next, IR783-COOH (1.4 mg, 1.6 μμmol) and DMTMM (0.66 mg, 2.4 μmol) were dissolved in sterilized tertiary distilled water (5 mL) in a 20 mL vial, and pork cartilage derived tissue prepared in Example 1 0.1 g of powder was added to the IR783-COOH solution, and the mixed solution was stirred at room temperature for 24 hours. After the reaction, the reaction solution was poured into a dialysis membrane (molecular weight cut-off: 2.0 kDa, Spectrum Laboratories, CA, USA), and then dialyzed in tertiary distilled water for 3 days to remove unreacted material and lyophilized by lyophilization for 4 days. The material was introduced, and biochemically cross-linked porcine cartilage derived tissue powder was prepared (FIG. 2B).

Example  7 ~ 9: Hydrogel  Produce

Hydrogels were prepared by uniformly dispersing the biochemically cross-linked porcine cartilage-derived tissue powders prepared in Examples 1 to 3 in a phosphate buffer solution in 5 mL vials at 10 wt%, 15 wt% and 20 wt%, respectively. It was confirmed that a hydrogel usable as a mold was formed (FIG. 3).

Experimental Example  1: Animal cartilage-derived tissue Hydrogel  Measurement and evaluation of mechanical properties

(One) Hydrogel Rheological  Characteristic evaluation

To measure the rheological properties of the tissue cartilage derived from pig cartilage prepared in Example 7, using a rheometer (MCR 102, Anton Paar, Ostfildern, Germany) storage modulus and storage modulus Was measured, and complex viscosity was measured at 37 ° C., and the results are shown in FIGS. 4A to C.

Figures 4a and b is a graph showing the results of measuring the rheological properties (storage modulus, loss modulus and composite viscosity) of the tissue cartilage derived from porcine cartilage prepared in Example 7.

As shown in Figure 4a, when the content of biochemically crosslinked pig cartilage derived tissue powder in a phosphate buffer solution is 20% by weight, the content of the crosslinking agent (0.1% by weight, 0.2% by weight, 0.3% by weight, 0.5% by weight, 1% by weight, 3% by weight, and 5% by weight)), the storage elastic modulus and the composite viscosity were found to show a tendency to increase. As a result, the mechanical properties of the hydrogel can be controlled according to the content of the crosslinking agent. have.

As shown in Figure 4b, when the content of the cross-linking agent is 0.5% by weight, as the content of biochemically cross-linked pig cartilage derived tissue powder (10%, 15% and 20% by weight) in a phosphate buffer solution increases , It was confirmed that the storage elastic modulus, the loss elastic modulus, and the composite viscosity all showed a tendency to increase, and it can be seen that the mechanical properties of the hydrogel can be controlled according to the content of the biochemically crosslinked pig cartilage derived tissue powder.

Figure 4c is a graph comparing the results of measuring the rheological properties (storage modulus, loss modulus and composite viscosity) of the tissue cartilage derived from pig cartilage prepared in Examples 7-9.

As shown in Figure 4c, when the content of the cross-linking agent is 0.5% by weight, and the content of tissue powder derived from pig cartilage biochemically cross-linked in a phosphate buffer solution is 20% by weight, when glutaraldehyde (GA) is used as a cross-linking agent In addition, butanediol diglycidyl ether (BDDE) or poly (caprolactone-co-lactide-co-glycolide) -NHS / polyethylene glycol / poly (caprolactone-co-lactide-co-glycolide)- Even when NHS (NHS-PEG-NHS) was used, it was confirmed that the storage elastic modulus and composite viscosity could be increased, and thus it could be seen that the mechanical properties of the hydrogel can be controlled using various crosslinking agents.

(2) Hydrogel  Compressive strength and Injectability  evaluation

To evaluate the compressive strength and injectability of swine cartilage-derived tissue hydrogel prepared in Example 7, after being loaded in a 2 mL syringe, compressed using UTM (H5KT) at a rate of 10 mm / min, inside the needle Compressive strength was measured using a syringe with diameters of 30G, 26G, 23G and 21G, and the injectability was evaluated (FIG. 5A).

Figure 5b is a graph showing the compressive strength per compression distance of pig cartilage-derived tissue hydrogel prepared in Example 7, Figure 5c shows the maximum compressive strength per compression distance of pig cartilage-derived tissue hydrogel prepared in Example 7 5D is a table evaluating the injectability according to the results of FIGS. 5B and 5C.

As shown in Figs. 5B to D, a curve in which the compressive strength per compression distance is kept constant can be seen as easily injectable, and a curve in which the compressive strength per compression distance is continuously increased can be seen as non-injectable. Therefore, as the content of the crosslinking agent (0.1% by weight, 0.15% by weight, 0.3% by weight, 0.5% by weight, and 1% by weight) increased, injectability tended to decrease, and as the needle inner diameter of the syringe decreased, , Injectability also tended to decrease. In particular, when the content of the crosslinking agent is 1% by weight or more, it is confirmed that injection injection is very difficult.

Experimental Example  2: Near infrared  Neon Imaging Pig through  Cartilage-derived tissue Hydrogel  Biodegradation behavior observation and evaluation

The fluorescent substance prepared in Example 4 was introduced, and biochemically cross-linked tissue powder derived from pig cartilage, 15% by weight of pig cartilage in a phosphate buffer solution having a pH of 7.4 after sterilization using UV and 70% ethanol A derived tissue hydrogel was prepared. The prepared pig cartilage-derived tissue hydrogel was injected 0.15 mL into a subcutaneous nude mouse (male, 8 weeks old) with a 1 mL syringe (needle inner diameter = 21G), and the results of observation of near infrared fluorescence imaging according to each time are shown in FIG. 6. Did.

As shown in FIG. 6, the degree of overall fluorescence expression decreases with time, but the fluorescence retention period tends to increase as the content of the crosslinking agent (0.15 wt%, 0.5 wt% and 1 wt%) increases. As can be seen, it can be seen that the rate of decomposition in vivo can be controlled according to the content of the crosslinking agent.

Experimental Example  3: Pig cartilage-derived tissue Hydrogel  And bioinjection observation and evaluation of cells

(200 ng/mL)] 및 [가교된 돼지 연골 유래 하이드로젤 + 연골 유래 태아줄기세포(5 x 10⁶ cells) + BMP2 (10 μg/mL)]의 하이드로젤 3군을 상온에서 15 중량% 로 PBS에 현탁시켰다. 이를 마취시킨 SD rat(280-300 g, male, 6주령) 피하에 1 mL 주사기(바늘 내부 직경 = 21G)로 0.5 mL 주입한 다음, 조직을 적출하였다. For the biochemically crosslinked porcine cartilage derived tissue powder prepared in Example 7, 15% by weight of porcine cartilage derived tissue hydrogel is prepared in a phosphate buffer solution having a pH of 7.4 after sterilization using UV and 70% ethanol. Did. To observe and evaluate whether the cartilage-derived hydrogel acts as a carrier of cartilage-derived fetal stem cells, [cross-linked pig cartilage-derived hydrogel + cartilage-derived fetal stem cells (5 x 10 ⁶ cells)], [cross-linked pig cartilage Derived hydrogel + cartilage derived fetal stem cells (5 x 10 ⁶ cells) + TGF-

(200 ng / mL)] and three hydrogels of [cross-linked cartilage-derived hydrogel + cartilage-derived fetal stem cells (5 x 10 ⁶ cells) + BMP2 (10 μg / mL)] at 15% by weight at room temperature Suspended in PBS. This was injected into an anesthetized SD rat (280-300 g, male, 6 weeks old) subcutaneously with a 1 mL syringe (needle inner diameter = 21 G), followed by 0.5 mL of tissue.

Hematoxylin and eosin (H & E) staining and ED1 (mouse anti) were used to fix the extracted tissue in 10% formalin, cut the fixed tissue into a paraffin block, cut it to a thickness of 4 μm, fix it on a slide, and evaluate the immunological inflammatory response. rat CD68: Serotec, UK) was stained, and the results are shown in FIGS. 7a and b, respectively.

As shown in Figure 7a, it is confirmed that the cartilage-derived fetal stem cells are well alive in the hydrogel derived from porcine cartilage. As shown in FIG. 7B, the hydrogel derived from porcine cartilage does not show an immune response, and thus is verified as stable.

Claims (8)

(A) preparing a cartilage derived tissue powder by pulverizing the tissue derived from animal cartilage;
(b) preparing a cross-linked cartilage-derived tissue by mixing and crosslinking the aqueous solution containing cartilage-derived tissue powder prepared in step (a) with a solution containing a cross-linking agent; And
(c) drying the cross-linked cartilage-derived tissue prepared in step (b) to powder again, and then dispersing it in a buffer solution to prepare a hydrogel.
Method of manufacturing a hydrogel.
According to claim 1,
In step (a), the animal is one or more selected from the group consisting of humans, pigs, cows, sheep, horses, and cats, the method of producing a hydrogel.
According to claim 1,
In the step (b), the crosslinking agent has at least one functional group selected from the group consisting of an aldehyde group, an epoxy group, an imide group, an amine group and a carboxyl group, a method for producing a hydrogel.
According to claim 1,
With respect to the total content of the cartilage-derived tissue powder and the crosslinking agent in step (b), the content of the crosslinking agent is 0.05% to 10% by weight, a method for producing a hydrogel.
According to claim 1,
The dispersion in step (c) is a method for producing a hydrogel, wherein the cross-linked cartilage-derived tissue powder is dispersed in a buffer solution at 5% to 30% by weight.
Hydrogel prepared by the method according to claim 1.
Injectable preparation comprising the hydrogel according to claim 6.
The method of claim 7,
The injectable preparation is for tissue repair, drug delivery or cell delivery, injectable preparation.

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