KR101409312B1 - Biocompatible small intestinal submucosa sheet with adjustable degradation time in vivo, and method for preparing the same - Google Patents

Abstract

The present invention provides a biocompatible intestinal submucosal tissue sheet capable of regulating the decomposition period in vivo in which crosslinks are formed by reacting small intestinal submucosa of an animal other than a human with a cross-linking agent, and a method for producing the same. The biocompatible intestinal submucosal tissue sheet according to the present invention can delay the decomposition period in vivo by cross-linking of the cross-linking agent and can control the decomposition period according to the type of cross-linking agent and the degree of cross- It can be very useful as an engineering support, a drug delivery system, a wound dressing or a hemostatic agent.

Description

TECHNICAL FIELD The present invention relates to a biocompatible intestinal submucosal tissue sheet capable of controlling the decomposition period in vivo, and a method for preparing the biocompatible small intestinal submucosal tissue sheet,

The present invention relates to a biocompatible intestinal submucosal tissue sheet capable of controlling the decomposition period in vivo, and a method for producing the same.

Natural materials are derived from natural materials, animals, and humans and have excellent biocompatibility. A typical example is the extracellular matrix (ECM), which is a biomaterial extracted from complex human and animal tissues, and is capable of controlling cell function. The support made of natural materials is evaluated as an ideal material for a tissue engineering support since it has less inflammatory reaction after transplantation into living body and can provide excellent biocompatibility and biodegradability.

Of these natural materials, small intestinal submucosa of pigs is a cell-free tissue with almost no immune response. More than 90% of the constituents are composed of type I and type II collagen in the skin, and others It is composed of small amounts of type V and type VI collagen. Also, the small intestinal submucosa of the pig is composed of an extracellular matrix (ECM) containing glycosaminoglycan and fibronectin, chondroitin sulfate, heparin, heparin sulfate, hyaluronic acid and basic fibroblast growth factor-2 (FGF- 2), nerve growth factor (NGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor endothelial growth factor (VEGF), and insulin-like growth factor-1 (IGF-1), which are involved in cell adhesion, growth, migration, and differentiation It can be applied to many fields besides tissue regeneration by helping.

In order to heal organs damaged by various diseases, it is sometimes necessary to receive treatment for bone marrow transplantation or organ donation such as heart, kidney, eyeball, etc. However, since supply is insufficient compared to demand, Development is needed. Such tissue engineering is a field that requires mutual cooperation between scholars applied to the recovery of biological substitutes for the purpose of restoring or preserving the function of the lost organ using the principles of life sciences and engineering. It can be applied as a tissue engineering support for the development of artificial organs to regenerate damaged tissue.

The study of the small intestinal submucosa was initiated by Badylak in the "Purdue University" in the United States and has been actively studied by a number of leading pharmaceutical companies such as "COOK". As a result of these studies, small intestinal submucosa is currently commercialized as a vein or an artery vessel, a graft of a dermis, an epithelium, a bone, a bile duct, and a graft for the bladder of an incontinent patient. Therefore, the small intestinal submucosa is expected to be a biocompatible material that can be applied to various biologic alternatives.

However, since a small intestine submucosa of a pig having high biocompatibility as a natural material has poor physical properties such as a short decomposition period, there is a limit to its application, so it is necessary to develop a method of enhancing physical properties through physical or chemical treatment thereof . Particularly, a biocompatible crosslinking agent and a method for enhancing the physical properties by using the crosslinking agent are not known at all, and there is no research on this.

Therefore, there is a desperate need to develop biocompatible intestinal submucosal tissue sheet capable of regulating the decomposition period in vivo by reacting small intestinal submucosa with a cross-linking agent to form cross-linking.

As a result of studying biocompatible materials, the present inventors have found that when a small intestinal submucosa in which immune reaction hardly occurs is reacted with a cross-linking agent to form a cross-linking agent, the mechanical properties are improved in proportion to the presence or absence of cross- And the present invention has been completed.

Accordingly, the present invention provides a biocompatible intestinal submucosal tissue sheet capable of regulating the decomposition period in vivo in which crosslinks are formed by reacting small intestinal submucosa of an animal other than a human with a cross-linking agent.

The present invention also provides a method for producing a biocompatible intestinal submucosal tissue sheet in which the in vivo decomposition period can be controlled.

The present invention provides a biocompatible intestinal submucosal tissue sheet capable of regulating the decomposition period in vivo in which crosslinks are formed by reacting small intestinal submucosa of an animal other than a human with a cross-linking agent.

The present invention also provides a method for preparing a biocompatible intestinal submucosal tissue sheet in which the in vivo decomposition period can be controlled.

The biocompatible small intestine submucosal tissue sheet according to the present invention can delay the decomposition period in vivo by forming a crosslinking with the crosslinking agent and can control the decomposition period according to the type of crosslinking agent and the degree of crosslinking according to the crosslinking time, It can be very useful as a support for tissue engineering, a drug delivery system, a wound dressing or a hemostatic agent.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing the chemical structure of a crosslinking agent usable as a crosslinking agent in a small intestine submucosal tissue sheet of the present invention. FIG.
FIG. 2 is a diagram showing a chemical structure of a crosslinking agent prepared by introducing a carboxyl group at the PEG end according to Example 2-1 of the present invention and reacting with EDC-NHS, and NMR spectrum thereof.
FIG. 3 is a diagram showing the chemical structure of a cross-linking agent prepared by introducing a carboxyl group into the terminal and / or side chain of the PCLA polymer according to Example 2-2 of the present invention and reacting with DCC, and NMR spectrum thereof.
FIG. 4 is a schematic view showing the decomposition behavior experiment of biocompatible small intestinal submucosal tissue sheet through cross-linking of the present invention.
FIG. 5 is a view showing the degradation behavior of biocompatible small intestinal submucosal tissue sheet according to Example 1 of the present invention. FIG.
FIG. 6 is a graph showing changes in weight with time of the biocompatible intestinal submucosal tissue sheet prepared in Example 1 according to the type of cross-linking agent according to the present invention. FIG.
FIG. 7 is a graph showing degradation behavior of biocompatible small intestinal submucosal tissue sheet prepared in Example 2 according to the crosslinking reaction time. FIG.
FIG. 8 is a graph showing changes in weight of biocompatible intestinal submucosal tissue sheet prepared according to Example 2 of the present invention over time according to types of cross-linking agents. FIG.

 The present invention provides a biocompatible intestinal submucosal tissue sheet capable of regulating the decomposition period in vivo in which crosslinks are formed by reacting small intestinal submucosa of an animal other than a human with a cross-linking agent.

The present invention also provides a method for preparing a biocompatible intestinal submucosal tissue sheet in which the in vivo decomposition period can be controlled.

In addition, the present invention provides a tissue engineering support, drug delivery system, wound dressing, and hemostatic agent containing the small intestine submucosal tissue sheet.

Hereinafter, the present invention will be described in more detail.

The intestinal submucosal tissue sheet according to the present invention is characterized in that a small intestinal submucosa of an animal other than a human, preferably a mammal other than a human, is separated and reacted with a cross-linking agent to form cross-linking.

The crosslinking agent usable in the present invention may be at least one selected from the group consisting of an aldehyde compound, a water-soluble carboimide compound, an organic solvent-soluble carboimide compound, an epoxy compound and a diisocyanate, more preferably a water- Ethyl-3- (3-dimethylaminopropylcarbamido), 1-ethyl-3- (2-morpholinyl-4-ethyl) carbomide as an organic solvent-soluble carbomide compound, dicyclohexyl Carboimides, and aldehyde-based compounds such as formaldehyde, glutaraldehyde, and dextrin aldehyde, but are not limited thereto.

The crosslinking agent that can be used in the present invention may be a biocompatible polymer having a carboxyl group at a terminal or side chain. The biocompatible polymer may be selected from the group consisting of polyethylene glycol (PEG), polycaprolactone (PCL), glycolide But are not limited to, at least one selected from the group consisting of GA, glycolide, lactide (LA), and co-polymers thereof. In the present invention, the biocompatible polymer having a carboxyl group at the terminal or side chain may be a crosslinking agent represented by the following general formula (1) or (2).

[Chemical Formula 1]

In the above formula (1), n is an integer representing the repeating unit of polyethylene glycol, preferably an integer of 1 to 20.

(2)

In the general formula (2), n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20, and m is an integer of 1 to 10 representing a segment constituting the polyester.

Such animals include, but are not limited to, pigs, cows, rabbits, mice, and the like.

The small intestinal submucosal tissue sheet according to the present invention can be used for various purposes as a sheet capable of controlling the decomposition period by forming crosslinks using various crosslinking agents. The small intestinal submucosa is abundant in ECM (extracellular matrix) and cytokine, which helps functional cells such as adhesion, growth, migration and differentiation of cells. In addition to tissue engineering regenerative scaffolds, A drug delivery, a wound dressing, or a hemostatic agent when the drug is contained in a tissue sheet.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. Preparation of small intestinal submucosal tissue sheet using crosslinking agent

1-1. Separation and storage of small intestinal submucosa in pigs and manufacture of intestinal submucosal tissue sheet

The fat tissue was removed using a pig's factory (ileum) within 4 hours after death. After cleaning the inside and outside of the plant cleanly with water, the plant was cut to a length of about 10 cm and placed in saline and washed. Physical force was applied to the washed plant to remove the dense layer in the outer layer, and then, in turn, the muscle layer of the mucosa was removed to separate only the small intestinal submucosa. The resultant was washed with saline and stored in a -80 ° C cooler. One side of the small intestinal submucosa stored at -80 DEG C was cut, unfolded and fixed, and then lyophilized to prepare a sheet-like small intestinal submucosa.

1-2. Cross-linking reaction of intestinal submucosal tissue sheet with cross-linking agent

A cross-linking agent capable of causing a cross-linking reaction in a small intestinal submucosal tissue sheet is shown in Fig. (NHS / EDC / dicarboxyl), 1-ethyl-3- (3-dimethyl-3-pyridyl) Aminopropylcarbodiimide (EDC), carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), and formalin (formailn) were selected and dissolved in 20 ml of a phosphate buffer solution at a concentration of 50 mM. -1, and the mixture was stirred at room temperature for 24 hours to effect crosslinking reaction.

Example 2: Preparation of a biocompatible crosslinking agent cross-linked with small intestinal submucosa on a sheet

2-1. Biocompatibility of formula (1) Cross-linking  Produce

The following experiment was conducted to introduce a carboxyl group into the terminal or side chain of polyethylene glycol (PEG).

Concretely, using toluene as a solvent, 1.3 times glutaric anhydride was added to the hydroxyl group (OH) of polyethylene glycol, and the mixture was stirred at 100 ° C for 24 hours using acetic acid as a catalyst. The prepared polyethylene glycol, 1-ethyl-3- (3-dimethylaminopropylcarbamido) (EDC) and N-hydroxysuccinimide (NHS) were dissolved in distilled water at a molar ratio of 1: 2: 2 And stirred for 2 hours to prepare a biocompatible and biodegradable crosslinking agent represented by the following formula (1).

[Chemical Formula 1]

In the above formula (1), n is an integer representing the repeating unit of polyethylene glycol, preferably an integer of 1 to 20.

The NMR analysis results of the cross-linking agent of Formula 1 prepared above are shown in FIG.

2-2. Biocompatibility of Formula 2 Cross-linking  Produce.

In order to introduce a carboxyl group into the terminal or side chain of a polyethylene glycol / poly (caprolactone-co-lactide) (polyethylene glycol / poly (caprolactone-co-lactide)) having excellent biocompatibility as a raw material for a hydrogel, Experiments were performed.

Concretely, 1.3 times glutaric anhydride was added to the hydroxyl group (OH) of polyethylene glycol / poly (caprolactone-co-lactide) using toluene as a solvent, Lt; / RTI > The prepared polyethylene glycol / poly (caprolactone-co-lactide) (PCLA) and dicyclohexylcarbodiimide (DCC) having a carboxyl group were dissolved in distilled water at a molar ratio of 1: 1, 2 < / RTI > was prepared.

(2)

In the general formula (2), n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20, and m is an integer of 1 to 10 representing a segment constituting the polyester.

NMR analysis results of the cross-linking agent of Formula 2 prepared above are shown in FIG.

Example  3. Biocompatibility To crosslinking agent  by Submucosal Crosslinking experiment

The small intestine submucosal tissue sheet prepared in Example 1-1 was added to 20 ml of a phosphate buffer solution prepared by dissolving the biocompatible crosslinking agent prepared in Example 2-1 at a concentration of 50 mM, and the mixture was stirred at room temperature for 24 hours, Crosslinking reaction was carried out.

The above n is an integer representing the repeating unit of polyethylene glycol, and is preferably an integer of 1 to 20.

In addition, the biocompatible crosslinking agent prepared in Example 2-2 was used to carry out the crosslinking reaction as described below in the small intestinal submucosal tissue sheet.

In the above reaction formula, n is an integer representing the repeating unit of polyethylene glycol, preferably an integer of 1 to 20, and m is an integer of 1 to 10 representing a segment constituting the polyester.

Experimental Example  One. Cross-linking agent  Manufactured using Small intestinal submucosa  Sheet Decomposition behavior

1-1. Experimental study of cross-linked small intestinal submucosal tissue sheet

The small intestinal submucosa of the sheet prepared in Example 1 was cut into 2 × 2 cm ² and placed in a 20 ml vial. Then, 5 ml of a phosphate buffer solution supplemented with collagenase (60 μg / ml) was added thereto, The degradation behavior was confirmed morphologically by decomposition at 37 ℃ and 1000 rpm.

FIG. 4 shows a schematic diagram of the decomposition behavior test of the biocompatible intestinal submucosal tissue sheet of the present invention, and the result of the decomposition behavior observation is shown in FIG.

As shown in FIG. 5, it was confirmed that the crosslinked small intestinal submucosal tissue sheet was not degraded more than the normal intestinal submucosal tissue sheet without cross-linking, and the form was remarkably excellent.

1-2. Measurement of weight change of crosslinked small intestinal submucosal tissue sheet

After the experiment of Experimental Example 1-1, the remaining impurities and phosphate buffer solution were removed. The small intestinal submucosal tissue sheet was simply washed, and then lyophilized to quantify the decomposition behavior with a change in weight with time.

The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the shape and initial weight of the intestinal submucosa sheet were maintained better when the cross-linking agent was used, though it varied depending on the kind of the cross-linking agent.

Experimental Example  2. Biocompatibility Cross-linking agent  Manufactured using Small intestinal submucosa  Degradation Behavior of Sheet

2-1. Degradation of the intestinal submucosal tissue sheet by crosslinking time

The biocompatible crosslinking agent prepared in Example 2 was used for crosslinking for 24 hours (1 day) or 72 hours (3 days) and then lyophilized to prepare a crosslinked small intestinal submucosal tissue sheet. The sheet-like small intestinal submucosa was cut into 2 × 2 cm ² , placed in a 20 ml vial, and 5 ml of phosphate buffer solution supplemented with collagenase (60 μg / ml) was added thereto. And the degree of decomposition was confirmed morphologically.

The results are shown in Fig.

As shown in Fig. 7, the small intestinal submucosal tissue sheet crosslinked with the biocompatible crosslinking agent was not decomposed more than the normal intestinal submucosal tissue sheet not crosslinked, and the form was remarkably excellent. The shape of the small intestinal submucosal tissue sheet was excellent in the case of performing the crosslinking for 72 hours than in the case of performing the crosslinking for 24 hours. Therefore, the longer the crosslinking reaction time, the better the physical properties of the small intestinal submucosal tissue sheet.

2-2. Measurement of weight change of small intestinal submucosal tissue sheet by time of crosslinking

After the experiment of Experimental Example 2-1, the remaining impurities and the phosphate buffer solution were removed, and the small intestinal submucosal tissue sheet was simply washed and then lyophilized to quantify the decomposition behavior with the change of the weight with time.

The results are shown in Fig.

As shown in FIG. 8, although the initial weight of the intestinal submucosal tissue sheet did not vary greatly depending on the type of the cross-linking agent, it was confirmed that the sheet form was preserved very well as in 2-1 above.

Claims (13)

A method for preparing a biocompatible intestinal submucosal tissue sheet capable of controlling in vivo decomposition time, wherein a small intestinal submucosa of an animal other than a human is reacted with a cross-linking agent represented by the following formula (2)
(2)

In Formula 2, n is an integer of 1 to 20, and m is an integer of 1 to 10. delete delete delete delete delete delete The method according to claim 1, wherein the animal is at least one selected from the group consisting of pigs, cows, rabbits, mice, and a method for producing a biocompatible intestinal submucosal tissue sheet capable of controlling the decomposition period in vivo. 9. A biocompatible intestinal submucosal tissue sheet prepared by the method of claim 1 or 8, wherein the biodegradation period is adjustable. 9. A tissue engineering support comprising the small intestine submucosa tissue sheet of claim 9. 9. A drug delivery system comprising the small intestinal submucosa tissue sheet of claim 9. A wound dressing comprising the intestinal submucosa tissue sheet of claim 9. A hemostatic agent comprising the small intestinal submucosa tissue sheet of claim 9.

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