KR101731865B1

KR101731865B1 - Tissue engineering biodegradable polymer scaffold and a method for manufacturing it

Info

Publication number: KR101731865B1
Application number: KR1020150038437A
Authority: KR
Inventors: 강길선; 김혜민; 김수민; 김은영
Original assignee: 전북대학교산학협력단
Priority date: 2015-03-19
Filing date: 2015-03-19
Publication date: 2017-05-02
Also published as: KR20160112541A

Abstract

The present invention relates to a biodegradable polymer scaffold for tissue engineering, and more particularly, to a biodegradable polymeric scaffold for tissue engineering, and more particularly to a biodegradable polymeric scaffold for biodegradable flavonoid, The present invention relates to a biodegradable polymer scaffold for biotechnology that is biocompatible and has a reduced biodegradability and biocompatibility when a polymer scaffold is formed using a dispersion medium, which has increased solubility, increased bioavailability, and antioxidant and anti-inflammatory properties.

Description

TECHNICAL FIELD [0001] The present invention relates to a biodegradable polymer scaffold and a method for manufacturing the biodegradable polymer scaffold,

In modern society, it is necessary to replace damaged or weakened tissues or organs due to aging due to aging of human life, industrial accidents caused by industrialization, danger of accidents due to traffic development, etc. However, The problem is that there is a risk of immune rejection between the organs of the donor and the patient after organ transplantation. In addition, treatment with animal organs is exposed to many risks such as immune rejection due to xenotransplantation and animal virus infection not existing in humans. For this reason, studies on methods and materials that can replace organs and tissues became necessary, and these studies are called 'tissue engineering'.

Tissue engineering can be said to be a study in which the fusion of biotechnology and engineering is combined with the study of damaged organs. For such studies, it is necessary to provide a support capable of attaching and growing in a three-dimensional space by specific binding between a cell and an extracellular matrix to maintain and express the inherent properties of the cell.

In tissue engineering, scaffolds are elements that support the proliferative environment of cells for tissue and organ regeneration. Cells sown on the scaffold should not die in the body after implantation and should maintain their function well, It must be naturally degraded and replaced by a growing tissue. Such a support material is divided into ceramic, metal, and polymer. Polymers that have relatively good processability and are capable of easily imparting necessary properties to a site where biomaterials are used are widely used as biomaterials. However, when a polymer is used as a biomaterial, there are disadvantages such as low mechanical strength, time-dependent deformation, and inflammation due to biodegradation.

Among them, lactide glycolide copolymer (PLGA) is a biomaterial polymer approved by the US Food and Drug Administration, which has excellent biocompatibility, biodegradability and processability, and has an advantage of controlling the decomposition period because it controls the monomer . It is also the most widely used polymer to carry out long clinical trials because of its rigid physical properties, its high mechanical strength and its potential for continuous drug delivery.

However, since it is a hydrophobic material that interferes with the adherence of early cells, appropriate studies are needed to improve the cell adhesion properties by modifying the surface through immobilization of ligands and adsorption of proteins. In addition, when transplanted into the body, it causes an immune response due to damage of the surrounding tissues, resulting in an acute inflammatory reaction caused by the inflammatory cells located on the tissue and the graft surface, and also as an acidic substance, resulting in a continuous inflammatory reaction. It is known that the cell growth rate is decreased due to toxicity and the acidic liquid is secreted by the reaction on the surface between PLGA and foreign giant cells, so that the local site tissue reaction occurs continuously and the pH is further lowered. Therefore, in order to take advantage of the excellent biomaterial properties of PLGA, it is necessary to study the modification of PLGA surface and the inflammation reaction.

Meanwhile, Korean Patent No. 10-1109668 discloses a porous support for tissue engineering including silica and PLGA. Here, it is disclosed that it has excellent mechanical stiffness in inducing bone differentiation, is not toxic, and is effective for bone regeneration. However, And anti-inflammatory effect, and thus it is difficult to achieve satisfactory results in inducing cell proliferation and growth due to inflammatory reaction.

In order to solve the inflammatory reaction caused by decomposition of polymer such as PLGA, the necessity of nature-friendly materials having anti-inflammation and antioxidant action is being raised.

Among them, flavonoids are collectively referred to as yellow pigment components of plants, and polyphenols are included therein. Flavonoids are classified into flavonols, isoflavones, and catechins depending on the difference in structure. Flavonoids have antibacterial, anticancer, antiviral, antiallergic and anti-inflammatory activities, and toxicity is reported to be almost absent. In addition, as the fact that it inhibits in vivo oxidative action is known, there is a growing interest in the development and utilization of flavonoid materials.

However, a polymer scaffold for tissue engineering utilizing such a flavonoid-based material has not yet been proposed.

It should be understood that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention and is not to be construed as adhering to the prior art already known to those skilled in the art.

Korean Patent No. 10-1109668

The present invention addresses the development of a biocompatible and bioavailable scaffold while applying the merits of conventional PLGA and compensating for its disadvantages.

Particularly, in the present invention, since the flavonoid series is low in the value of materials and excellent in antioxidative and antiinflammatory effects, it has a low solubility, and thus a solution to effectively increase the solubility of such flavonoids is another problem.

As a result of efforts to solve the above problems, the present invention provides a pretreatment method for effectively improving the low solubility of flavonoids in order to overcome disadvantages of PLGA, and a solubility thereof is further improved by preparing a solid dispersion using a water-soluble polymer The present invention has been completed.

Accordingly, it is an object of the present invention to provide a biodegradable flavonoid solid dispersion having increased solubility and bioavailability.

Another object of the present invention is to provide a biodegradable polymer scaffold for tissue engineering comprising flavonoids and biopolymers.

It is still another object of the present invention to provide a method for producing the biodegradable polymer scaffold for tissue engineering.

In order to solve the above problems, the present invention provides a biodegradable polymer scaffold for tissue engineering comprising a biodegradable flavonoid solid dispersion in which flavonoids are dispersed in a water-soluble polymer and a biodegradable biopolymer.

The present invention also provides a method for producing a water-soluble polymer, comprising: (a) dissolving a water-soluble polymer and a flavonoid in an organic solvent to prepare a solution; (b) preparing a dissolution liquid as a solid dispersion; (c) adding a solid dispersion to a biodegradable biopolymer dissolved in an organic solvent to prepare a support preparation solution; And (d) preparing a support preparation solution as a film or a three-dimensional support. The biodegradable polymer scaffold of the present invention can be used as a biodegradable polymer scaffold.

The present invention also provides a biodegradable flavonoid solid dispersion wherein the flavonoid is dispersed in a water-soluble polymer.

The biodegradable polymer scaffold comprising the flavonoid according to the present invention has biocompatibility and, particularly, a solid dispersion is prepared by using a water-soluble polymer, thereby effectively increasing the dissolution rate of the poorly soluble flavonoid, Flavonoids are useful as biomaterials for reducing the inflammatory reaction caused by synthetic polymers in vivo and inducing cell growth and proliferation.

1 is a DSC graph comparing a thermodynamic change of a biodegradable PVA / Hesperidin solid dispersion prepared according to an embodiment of the present invention with a comparative example.
2 is an XRD graph comparing the crystallographic change of the biodegradable PVA / Hesperidin solid dispersion prepared according to an embodiment of the present invention with the comparative example.
FIG. 3 is a graph comparing the results of HPLC of the biodegradable hesperidin solid dispersion / PLGA supporter prepared according to one embodiment of the present invention over time with a comparative example.
4 is an MTT graph comparing the toxicity of the biodegradable hesperidin solid dispersion / PLGA scaffold prepared according to an embodiment of the present invention with the comparative example.
FIG. 5 is a graph showing the results of mRNA expression of the biodegradable hesperidin solid dispersion / PLGA scaffold prepared according to an embodiment of the present invention by comparing the inflammation relieving time with a comparative example.
6 is a photograph of an actual product of a biodegradable hesperidin solid dispersion / PLGA support prepared according to an embodiment of the present invention.

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

According to one aspect of the present invention, there is provided a biodegradable biodegradable polymer for tissue engineering, which comprises a flavonoid and a water-soluble polymer, which is biodegradable and biodegradable over time, and which has excellent solubility and a biodegradable flavonoid solid dispersion Thereby providing a support.

Recently, many studies have been conducted to find natural materials with anti-inflammatory properties to solve the inflammatory reaction caused by decomposition of synthetic polymers. Of the materials derived from nature and used in biomaterials, the components of plant extracts have been studied such as alkaloids, phenol compounds, and flavonoids which are antioxidant.

The flavonoids used in the present invention are non-naturally occurring pigments. In most cases, they are present as glycosides bonded with saccharides such as rhamnose, glycose and lutinose, and are known to have antioxidative and antimicrobial properties.

In particular, flavonoids have antioxidant, antiinflammatory, antioxidant and antioxidant effects by inhibiting active oxygen, thereby inhibiting cell, tissue and organ damage by forming antioxidant, antiinflammatory, and metal ion and complex salt. However, the flavonoid is a poorly soluble material, and when applied without a series of pretreatment steps, its solubility is very low, making it difficult to apply it as a biomaterial.

According to a preferred embodiment of the present invention, a biodegradable flavonoid solid dispersion in which flavonoids are dispersed in a water-soluble polymer is prepared in order to utilize flavonoids which are difficult to utilize as described above. Such a solid dispersion is preferably prepared from a polymer scaffold and applied as a tissue engineering material.

According to a preferred embodiment of the present invention, the water-soluble polymer used in the biodegradable flavonoid solid dispersion is selected from the group consisting of PEG, polyvinyl alcohol (PVA), PVP, cellulose derivative, polyacrylate and polymethacrylate, urea, May be used. More preferably, PVA can be used.

According to a preferred embodiment of the present invention, a surfactant may be further added to the biodegradable flavonoid solid dispersion. Examples of the surfactant include polyethylene glycol fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid monoesters , Alkylene glycol fatty acid monoesters, polyoxyethylene polypropylene glycols, and polyoxyethylene sorbitan mono fatty acid ethers. The surfactant may be used in an amount of 0 to 15% by weight, more preferably 0.01 to 5% by weight based on the total weight of the solid dispersion, and the stability of the solid dispersion is further improved according to the use of the surfactant, The bioavailability of the drug can be maintained longer.

According to a preferred embodiment of the present invention, as the flavonoid, at least one selected from genistein, quercetin, red wine and grape seed extract, proanthocyanidin, catechins, rutin and hesperidin can be used .

According to a preferred embodiment of the present invention, the final content ratio of flavonoid in the solid dispersion is 5 to 25 parts by weight, more preferably 10 to 20 parts by weight, and most preferably 15 parts by weight per 100 parts by weight of the solid dispersion to be. If the content of the water-soluble polymer is too small, the action and effect of the solid dispersion can not be expected. Therefore, the availability of the flavonoid such as improvement in solubility can not be expected. If the content of the water-soluble polymer is too high, There is a problem that the bioavailability of the flavonoid may be reduced due to the reduced content.

According to a preferred embodiment of the present invention, the present invention provides a method for producing a water-soluble polymer comprising adding a flavonoid to at least one water-soluble polymer selected from the group consisting of PEG, polyvinyl alcohol (PVA), PVP, cellulose derivative, polyacrylate and polymethacrylate, urea, 5 to 25% by weight of a biodegradable flavonoid solid dispersion.

According to a preferred embodiment of the present invention, the manufacturing method used in the production of the biodegradable flavonoid solid dispersion can be produced by one or more methods selected from the group consisting of dry granulation and wet granulation.

According to a preferred embodiment of the present invention, in the biodegradable polymer scaffold for tissue engineering according to the present invention, the content of flavonoid in the whole polymer scaffold is preferably 0.15 to 2.25% by weight, more preferably 1.0 to 1.5% by weight. If the content is less than 0.15% by weight, the effect of the flavonoid in the polymer scaffold is negligible in vivo, and the effect can not be expected. When the content exceeds 2.25% by weight, the peroxidation of the flavonoid causes death of peripheral cells, Inflammation, and the like.

According to a preferred embodiment of the present invention, the biodegradable biopolymer used for mixing the solid dispersion into a polymer scaffold may be a polyester, polyglycolic acid, polylactic acid, polycaprolactone, polypropylene fumarate, At least one selected from the group consisting of hydroxybutyric acid, polyoxyethylene, polyoxyethylene, hydroxybutyric acid, polyoxyethylene, polyoxyethylene, polyoxyethylene, polyoxyethylene, hydroxybutyric acid, polyanhydride, polyalkylcyanoacrylate, polylactide-glycolide copolymer, chitin, chitosan and alginic acid. Preferably, a polylactide-co-glycolide (PLGA) may be used as the polylactide-glycolide copolymer.

The PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA), and the degree of degradation can be controlled according to the mixing ratio and the molecular weight. The mixing ratio of polylactic acid and polyglycolic acid is 40:60 to 60:40 Mixing ratio may preferably be used.

According to another aspect of the present invention, there is provided a method for producing a water-soluble polymer, comprising: (a) dissolving a water-soluble polymer and a flavonoid in an organic solvent; (b) preparing a dissolution liquid as a solid dispersion; (c) adding a solid dispersion to a biodegradable biopolymer dissolved in an organic solvent to prepare a support preparation solution; And (d) preparing a support preparation solution as a film or a three-dimensional support. The biodegradable polymer scaffold for tissue engineering may be prepared by a method comprising:

In step (a), the water-soluble polymer and the flavonoid are dissolved in an organic solvent. According to a preferred embodiment of the present invention, the mixing ratio of the water-soluble polymer and the flavonoid in the solid dispersion preparation is (30 to 90) ) Weight ratio, more preferably (60 to 80): (40 to 20), most preferably 70: 30 weight ratio. According to a preferred embodiment of the present invention, at least one selected from the group consisting of methanol, ethanol, MC, ethylacetate, butanol and methylene chloride may be used as the organic solvent, more preferably ethanol.

According to a preferred embodiment of the present invention, at least one selected from the group consisting of a vacuum melting method, a spray drying method, and a solution evaporation method may be used in step (b) It is more preferable to use the spray drying method in view of the yield, the manufacturing cost, the manufacturing time, the complexity of the manufacturing process, the decrease in the content during the manufacturing process, and the like.

In the step (c), the biopolymer is dissolved in an organic solvent. According to a preferred embodiment of the present invention, the organic solvent used herein is dichloromethane, methylene chloride, dimethylformamide, chloroform, acetone, dioxane, tetrahydrofuran At least one selected from the group consisting of furan, trifluoroethane, and hexafluoroisopropane may be used, more preferably methylene chloride may be used.

In the step (c), the biodegradable biopolymer is dissolved in the organic solvent in an amount of 15 to 40% by weight, more preferably 20 to 30% by weight.

The content of the flavonoid solid dispersion to be added to the biodegradable biopolymer in the step (c) is preferably 1 to 15 parts by weight, more preferably 3 to 15 parts by weight, per 100 parts by weight of the biodegradable biopolymer, To 7 parts by weight, and most preferably 5 parts by weight. If the content of the solid dispersion is less than 1 part by weight, there is a problem in that the antioxidant and anti-inflammatory effects are insufficient. When the content is more than 15 parts by weight, physical stability of the support is insufficient.

Next, in step (d), the support solution prepared in step (c) is prepared as a film or a three-dimensional support. According to a preferred embodiment of the present invention, In order to prepare the polymer scaffold by effectively improving the dissolution rate by complementing the characteristics of the insoluble flavonoid, a biodegradable flavonoid solid dispersion obtained through the steps (a) and (b) is prepared and then the polymer scaffold is prepared It is necessary.

According to a preferred embodiment of the present invention, the film-form support of step (d) may be a solvent evaporation method which is prepared by pouring the solution prepared in step (c) on a plate.

According to a preferred embodiment of the present invention, in the step (d), a solution prepared by adding 80 to 95% by weight of NaCl having an average diameter of 180 to 600 탆 to the solution prepared in step (c) After pouring and drying, it is immersed in deionized water for 2 to 3 days to extract salt and form pores.

According to a preferred embodiment of the present invention, in the case of the biodegradable flavonoid solid dispersion as described above, the flavonoid is encapsulated into the water-soluble polymer to lose its original crystallinity and be dispersed evenly in an amorphous state. . According to a preferred embodiment of the present invention, the powder of the solid dispersion is prepared to have a particle size of 1 to 300 mu m, more preferably 10 to 200 mu m. In preparing the polymer scaffold, the flavonoid dissolves and the cell attachment And it is preferable in terms of helping growth.

The biodegradable polymer scaffold for tissue engineering according to the present invention prepared by such a manufacturing method effectively improves the antioxidant and anti-inflammatory effects, and thus is useful as a biomaterial for reducing the inflammatory reaction caused by the in vivo synthesis of the polymer and inducing cell growth and proliferation. Can be widely applied.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Preparation of biodegradable PVA / Hesperidin solid dispersion

The solid dispersion was prepared by solvent evaporation. To dissolve PVA, a mixed solution of 70/30 (weight ratio) of acetic acid / water mixture was used to dissolve anhydrous methanol and hesperidin. 70 g of PVA was dissolved in 500 ml of anhydrous methanol and completely dissolved at room temperature. 30 g of hesperidin was dissolved in 500 ml of acetic acid / water mixture solution while heating for 1 hour, and ultrasonication was performed for 20 minutes until the mixed solution was completely dissolved . Once the PVA and the hesperidin were completely dissolved, the two solutions were mixed and sonicated for 15 minutes without heat. Then, the PVP / Hesperidin film was obtained by completely evaporating the solvent using a rotary evaporator under reduced pressure at 60 ° C to completely remove the solvent. Thereafter, the powder was pulverized using a freeze grinder, and a solid dispersion powder having a size of 180 탆 or less was used.

1 mg of the obtained solid dispersion powder was sufficiently dissolved in 1 ml of DMSO, and the solution was filtered with a 0.45 μm syringe filter. The content of hesperidin was measured by HPLC and it was confirmed that 15% of the sample was contained in the water-soluble polymer.

Example 2 Production of Biodegradable Hesperidin Solid Dispersion / PLGA Film

PLGA (lactide / glycolide molar ratio 75/25, Resomer RG756, Boehringer Ingelheim Chem. Co., Germany) was used with an average molecular weight of 90,000 g / mole. PLGA / hesperidin films were prepared by solvent evaporation methods. Specifically, 300 mg of PLGA was dissolved in 5 mL of methylene chloride (MC, Tedia Co. Inc., USA) and various amounts of biodegradable hesperidin solid dispersion (3 wt%, 5 wt%, 10 wt%) were added. The above solution was mixed, slowly poured into a glass dish (diameter 30 mm), and dried at room temperature until the film was cast. This dried film was sterilized with 70% ethanol to prepare a final biodegradable hesperidin solid dispersion / PLGA hybrid film.

Example 3 Production of Biodegradable Hesperidin Solid Dispersion / PLGA Porous Support

Porous scaffolds were prepared by salt extraction. 1 g of PLGA was mixed with various amounts of biodegradable solid dispersions of hesperidin (3 wt%, 5 wt%, and 10 wt%) and dissolved in methylene chloride. 9 g of NaCl having an average diameter size of 180-250 占 퐉 was added to the above mixture as a porous forming material, followed by thorough mixing by mechanical stirring. The mixture was poured into a mold (diameter: 7 mm, height: 3 mm) and dried overnight at room temperature. The dried mixture was removed from the mold, immersed in deionized water for 2 days, and lyophilized for one day at -80 ° C . The resulting support was sterilized with 70% ethanol to prepare a final porous PLGA / hesperidin support.

Comparative Example 1: Preparation of PLGA film

A pure PLGA / hesperidin film was prepared in the same manner as in Example 2 except that pure hesperidin which had no treatment was used.

Comparative Example 2: Preparation of porous PLGA scaffolds

Porous PLGA / hesperidin supporters were prepared in the same manner as in Example 3 except that pure hesperidin which had no treatment was used.

EXPERIMENTAL EXAMPLE 1: Physical property comparison test

Various experiments were conducted to compare the physical properties of the materials prepared in the above examples with those of the comparative examples. As comparative examples, the same components comparable to the examples were compared as examples in which the conditions were different. The results are shown in Figs. 1 to 5.

FIG. 1 is a DSC graph comparing the thermodynamic changes of the biodegradable PVA / hesperidin solid dispersion prepared according to Example 1, and FIG. 2 is a graph showing the DSC curve of the biodegradable PVA / hesperidin solid dispersion prepared according to Example 1 This is an XRD graph comparing and analyzing crystallographic changes.

FIG. 3 is a graph comparing the HPLC of the biodegradable hesperidin solid dispersion / PLGA scaffold prepared according to Example 3 over time with the HPLC of Comparative Example 2. FIG.

4 is an MTT graph comparing the toxicity of the biodegradable hesperidin solid dispersion / PLGA scaffold prepared according to Example 3 with time according to Comparative Example 2 and the like.

FIG. 5 is a graph showing the results of mRNA expression of the biodegradable hesperidin solid dispersion / PLGA scaffold prepared according to Example 3 over time in comparison with the comparative examples and the like.

6 is a photograph of an actual product of the biodegradable hesperidin solid dispersion / PLGA scaffold prepared according to Example 3 above.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

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(a) dissolving a water-soluble polymer and a flavonoid in an organic solvent to prepare a solution;
(b) removing the solvent from the solution to prepare a solid dispersion;
(c) adding a solid dispersion to a biodegradable biopolymer dissolved in an organic solvent to prepare a support preparation solution; And
(d) preparing the support preparation solution as a film or a three-dimensional support
Wherein the biodegradable polymer scaffold is a biodegradable polymer scaffold.

The method of claim 9, wherein the mixing ratio of the water-soluble polymer to the flavonoid is in the range of (30 to 90): (70 to 10) by weight in the step (a).

[12] The method according to claim 9, wherein the content of the flavonoid solid dispersion added to the biodegradable biopolymer in the step (c) is 1 to 15 parts by weight of the flavonoid solid dispersion relative to 100 parts by weight of the biodegradable biopolymer (Preparation method of biodegradable polymer scaffold for engineering use).

[12] The biodegradable polymer scaffold for tissue engineering according to claim 9, wherein the support in the form of a film in step (d) is a solvent evaporation method in which the solution prepared in step (c) .

[12] The method according to claim 9, wherein the solid support in step (d) is prepared by pouring a solution containing 80 to 95% by weight of NaCl having an average diameter of 180 to 600 탆 into the solution prepared in step (c) Wherein the biodegradable polymer scaffold is prepared by a salt extraction method comprising immersing in deionized water for 2 to 3 days to extract NaCl and forming pores.