KR100953366B1 - Nano fiber for tissue regeneration and fabrication method thereof - Google Patents

Abstract

The present invention relates to a nanofibrous support for tissue regeneration and a method for manufacturing the same, and more particularly, to a tissue regeneration nanofiber support and a method for producing the same, including a lactide / ε-caprolactone copolymer, and a crosslinked natural polymer. It is about.

The support has excellent mechanical properties of polylactide and flexibility of polycaprolactone to obtain excellent flexibility and elasticity of mechanical properties, and has elasticity and biological functionality of natural polymers to effectively treat patients suffering from skin tissue. It is possible to apply to the treatment.

Tissue regeneration, nanofiber, skin damage

Description

Nanofiber support for tissue regeneration and its manufacturing method {NANO FIBER FOR TISSUE REGENERATION AND FABRICATION METHOD THEREOF}

The present invention relates to a nanofibrous support for tissue regeneration and a method of manufacturing the same, and more particularly, excellent mechanical properties of polylactide and flexibility of polycaprolactone are mutually complemented to obtain excellent mechanical properties of flexibility and elasticity, The present invention relates to a nanofibrous support for tissue regeneration and a method of manufacturing the same, which can be applied to effectively treat a patient suffering from damage to skin tissue by having elasticity and biological functionality of natural polymers.

Biodegradable synthetic polymers, which are widely used to develop scaffolds for tissue regeneration, are polyalphahydroxyester-based polymers, and polylactide, polyglycolide, and copolymers thereof are widely used.

Since these polymers provide excellent mechanical properties and decompose in vivo, they have the advantage of eliminating the need for a secondary procedure for removing a support.

The biodegradable synthetic polymers have been used in the development of a support for tissue regeneration by showing excellent mechanical strength and biodegradability [YZ Zhang et al, Characterization of the surface biocompatibility of the electrospun PCL-Collagen nanofibers using fibroblasts, Biomacromolecules 6, 2583- 2589, 2005; Mengyan Li et al , Co-electrospun poly (lactide-co-glycolide), gelatin, and elastin blends for tissue engineering scaffolds, J Biomed Mater Res 79A: 963 ?? 973, 2006; Yanzhong Zhang et al , Electrospinning of Gelatin Fibers and Gelatin / PCL Composite Fibrous Scaffolds, J. Biomed. Mater. Res. Part B: Appl Biomater 72B: 156-165, 2005; Eva Schnell et al , Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-e-caprolactone and a collagen / poly-e-caprolactone blend, Biomaterials 28 3012-3025 2007; J. Venugopal et al , In vitro study of smooth muscle cells on polycaprolactone and collagen nanofibrous matrices, Cell Biology International 29 861-867 2005].

The support for tissue regeneration using the biodegradable synthetic polymer is ideally considered as a material for bone tissue regeneration that requires excellent mechanical strength by showing excellent strength through hard properties.

However, tissues such as muscles, blood vessels, and skin are elastic tissues, and the tissues are composed of repeated contraction and relaxation. Thus, the properties of the biodegradable synthetic polymers are limited to withstand repeated contraction / relaxation of muscles. It is known.

In addition, polylactide, polyglycolide, and their copolymers have been used to develop scaffolds for tissue regeneration by showing excellent mechanical strength and biodegradability, but these polymers have very low hydrophilicity, making it difficult for cells to adhere to the scaffold and survive. In addition, there is a disadvantage in that it does not promote the growth of cells in the support due to the absence of biological functional groups that can promote the interaction with the cells.

On the other hand, artificial skin made of biocompatible, biodegradable polymers has been commercialized and used in the treatment of skin regeneration using such artificial skin. In cases of skin damage due to accidents or burns, a coating may be used to induce the skin to regenerate.

Recently, in order to increase affinity with skin tissue, a method of manufacturing the support with nanofibers morphologically similar to skin tissue has been proposed. [Korean Patent Publication No. 2005-40186, Korean Patent Publication No. 2006-8084] , Korean Patent Publication No. 2006-70641, Korean Patent Publication No. 2006-71890, Korean Patent Publication No. 2006-87663, Korean Patent Publication No. 2007-6551, Korean Patent Publication No. 2007-99926]. Nanofiber-type supports have a higher surface area than conventional porous supports, so that more cells can be attached, and high porosity ensures perfect interconnection between the pores, thereby providing an excellent supply of nutrients.

However, it is not yet satisfactory to satisfy the two aspects, such as proper mechanical properties and affinity with skin tissue, to serve as a support. In this regard, various studies have been conducted in terms of materials and methods of the support, but the effects are not satisfactory.

An object of the present invention relates to a nanofibrous support for tissue regeneration and a method for producing the same, which are excellent in mechanical properties such as elasticity and flexibility, and have improved cellular suitability and can be used for tissue regeneration treatment.

In order to achieve the above object, the present invention provides a nanofiber support for tissue regeneration comprising a lactide / ε-caprolactone copolymer, and a crosslinked natural polymer.

Also,

The lactide / ε-caprolactone copolymer and the natural polymer are dissolved in a solvent and then mixed,

After the obtained mixed liquid is thrown into the spinning nozzle, the spinning process is performed.

It provides a method for producing a nanofiber support for tissue regeneration, including the step of crosslinking the natural polymer in the spun nanofibers.

In the nanofiber support for tissue regeneration according to the present invention, the lactide / ε-caprolactone copolymer copolymerized by appropriately adjusting the composition of lactide and ε-caprolactone has excellent mechanical properties of polylactide and flexibility of polycaprolactone. Complementary to obtain excellent mechanical properties of flexibility and elasticity, and has the elasticity and biological functionality of natural polymers.

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

The nanofiber support for tissue regeneration according to the present invention includes a lactide / ε-caprolactone copolymer, and a crosslinked natural polymer.

Wherein the lactide / ε-caprolactone copolymer has a repeating unit of Formula 1 below:

[Formula 1]

(Wherein x and y are molar ratios of each monomer, x is 0.2 to 0.8, y is 0.8 to 0.2, x + y = 1 and n is an integer of 25 or more).

Preferably, in said formula, x is 0.4-0.6, y is 0.4-0.6, n is an integer of 25-50, More preferably, x and y are each 0.5, n is an integer of 25-30.

At this time, the molar ratio of each monomer of the lactide / ε-caprolactone copolymer x, y is to maximize the effect of the respective physical properties, to ensure the proper decomposition time by polylactide, poly ε-caprolactone It provides the elasticity and flexibility that is suitable for manufacturing a nanofiber support for tissue regeneration. Thus, the lactide / ε-caprolactone copolymer of Chemical Formula 1 is complementary to the excellent mechanical properties of the polylactide and the flexibility of the polycaprolactone. It has biodegradability that decomposes itself after a certain period of time after the procedure.

The lactide / ε-caprolactone copolymer can be a conventional ring-opening polymerization method, and is not particularly limited in the present invention.

For example, the lactide / ε-caprolactone monomer may be prepared by subjecting to ring-opening polymerization by heating under reduced pressure in the presence of a polymerization catalyst. Hereinafter, the manufacturing process of the lactide / ε-caprolactone copolymer of the present invention will be described in detail.

The ring-opening polymerization reaction is carried out by a heating and decompression method using a conventional ring-opening polymerization reaction catalyst. In this case, the reaction temperature is about 100 to 230 ° C, preferably about 130 to 200 ° C. The reaction time is at least about 5 hours, preferably about 5 to 50 hours.

In this case, as the catalyst used in general, all known catalysts used in the ring-opening polymerization reaction of lactide and ε-caprolactone can be used. Typically, catalysts used in the ring-opening polymerization reaction include tetraphenyltin, tin powder, tin octosan, stannous chloride, stannous chloride, tin oxide, zinc powder, diethylzinc, zinc octoate, and zinc chloride. And zinc oxide.

The amount of the catalyst added is about 0.0001 to 5 mol%, preferably about 0.001 to 0.5 mol%, based on the monomer of lactide and ε-caprolactone.

Once the polymerization is initiated, the molecular weight of the polymer is gradually increased, where the structural analysis and molecular weight measurement of the polymer are carried out using hydrogen nuclear magnetic resonance spectroscopy ( ¹ H-NMR), differential scanning calorimetry (DSC) and gel permeation chromatography. The molecular weight is adjusted to have an appropriate molecular weight using (GPC) or the like.

The crosslinked natural polymer not only increases the elasticity of the nanofiber support for tissue regeneration but also increases hydrophilicity. In addition, by imparting biological functionality to compensate for the shortcomings of the lactide / ε-caprolactone copolymer improves the cell compatibility of the nanofiber support for tissue regeneration.

Such natural polymers include functional groups capable of crosslinking in the molecular structure, for example, functional groups of at least one of a hydroxyl group, an amine group, or a carboxyl group. Preferably, the natural polymer may be one selected from the group consisting of collagen, gelatin, chitosan, cellulose, alginic acid, hyaluronic acid, and mixtures (blends) thereof, and more preferably collagen, gelatin, or the like. Mixtures, most preferably gelatin. Such collagen or gelatin is a polymer material whose molecular structure is very close to biological tissue and has a large physiological activity, and its potential is already known in the field of tissue regeneration treatment. However, collagen or gelatin alone is not suitable for use as a support for tissue regeneration due to brittleness due to external force when the support is prepared and used (see Experimental Example 4).

The nanofibrous support for tissue regeneration according to the present invention is present in a form in which the lactide / ε-caprolactone copolymer and the crosslinked natural polymer are uniformly mixed, in consideration of the role of each composition 1: 9 to 9: The content is adjusted to be present in a weight ratio of 1, preferably in a weight ratio of 3: 7 to 7: 3. If the content of the copolymer is excessive, there is a problem that the biocompatibility is greatly reduced. On the contrary, when the content of the natural polymer is excessive, the moisture content and cellular affinity of the support increase, but the flexibility and elasticity obtained by using the copolymer cannot be secured, and the thickness of the fiber decreases, making it impossible to apply the nanofiber support for tissue regeneration. .

The nanofiber support for tissue regeneration is mixed with a lactide / ε-caprolactone copolymer and a natural polymer, the resulting mixture is spun into a fiber form, and then subjected to a crosslinking reaction of the natural polymer in the spun fiber It is prepared by working up.

First, the lactide / ε-caprolactone copolymer of Formula 1 and the natural polymer are dissolved in a solvent, and then uniformly mixed by adjusting the content ratio of the composition.

Preferably, the copolymer and the natural polymer are used by dissolving in a solvent for uniform mixing and subsequent spinning process, wherein each concentration is 5 to 20% by weight using a volume ratio of 1: 9 to 9: 1. Preferably, the volume ratio is 3: 7 to 7: 3. If the content ratio is out of the above range, the phase separation between the copolymer and the natural polymer occurs, and thus the copolymer and the natural polymer in the finally prepared nanofiber do not exist uniformly, are present as agglomerates into some regions, or precipitated on the surface. Application as a fiber support is difficult.

The solvent is not particularly limited in the present invention, and any solvent can be used as long as it can dissolve the lactide / ε-caprolactone copolymer and the natural polymer, and is not particularly limited in the present invention. For example, the solvent may include water; Lower alcohols such as methanol, ethanol, propanol and butanol; Acetone; Trifluoroethanol; Tetrahydrofuran; Dichloromethane; And mixed solvents thereof.

Next, the mixed solution obtained above is spun through a spinning needle and processed into a fiber form.

The spinning is possible using a method known in the art, preferably through electrospinning.

Methods for producing nanofibers include drawing, template synthesis, phase separation, self assembly, electrospinning, and the like. To do it.

Electrospinning is a process for producing nanofiber nonwovens by releasing millimeter diameter liquid jets through needles (capillary tubes, or nozzles) in a way that can produce nanofibers continuously and in large quantities. The electrospinning can produce nanofibers having various diameters ranging from several nm to several thousand nm by adjusting process conditions, and have a high porosity of the prepared membrane as well as a high surface area to volume ratio. Specific process conditions for such electrospinning is not particularly limited in the present invention, it may be appropriately adopted by those skilled in the art.

In the present invention, the inner diameter of the needle is 0.3 to 0.7 mm, and the electrospinning is performed by operating the voltage between the needle and the collector at 15 to 25 kV, and preferably a needle of 18 to 24 gauge is used.

At this time, the needle is regularly shaken during the electrospinning so that the lactide / ε-caprolactone copolymer and the natural polymer are uniformly present in the nanofiber support.

Next, after the cross-linking the electrospun nanofibers obtained above to prepare a nanofiber support for tissue regeneration.

The nanofibers made in the foil in the previous step are dried for 12 hours at room temperature and then immersed in a solution in which the crosslinking agent is dissolved to crosslink the natural polymer present in the nanofibers.

The crosslinking agent may be any known crosslinking agent capable of crosslinking a natural polymer, for example, dimethylaminopropyl-ethylcarbodiimide hydrochloride, hydroxybenzotriazole, O 0-benzotriazol-1-yl-N, N, N'N'-tetramethyluronium tetrafluoroborate and their One selected from the group consisting of mixtures is possible. In addition, the solvent is water; Lower alcohols such as methanol, ethanol, propanol and butanol; Acetone; Tetrahydrofuran; dichloromethane; And mixed solvents thereof.

Post-treatment at this time includes conventional washing and drying. For example, after washing three times in distilled water to remove unreacted substances or impurities remaining on the surface or inside of the crosslinked nanofibers, lyophilization is performed.

The nanofiber support for tissue regeneration produced by this method has a structure in which the pores of the three-dimensional structure formed between the nano-unit fibers are interconnected. In addition, the support shows a high frequency in the average diameter of 200 to 250 nm in the nanofiber average diameter size of 50 to 500 nm. At this time, the support has a thickness of 50 to 150 μm, preferably 70 to 110 μm.

According to Experimental Example 2 of the present invention, the nanofibrous support for tissue regeneration is appropriately controlled by the lactide / ε-caprolactone copolymer to control hydrophobicity due to hydrophobicity and gelatin, thereby easily controlling water absorption. have.

In addition, according to Experimental Example 4, the nanofiber support for tissue regeneration compensates for the shortcomings of brittle gelatin due to elasticity and flexibility by the lactide / ε-caprolactone copolymer, thereby improving tensile strength and deformation of the support. The recovery rate and recovery rate can be appropriately controlled.

In particular, according to Experimental Example 5, the lactide / ε-caprolactone copolymer and gelatin both have biodegradability, it was confirmed that the decomposition time of the nanofiber support by controlling the mixing ratio thereof.

In addition, referring to Experimental Example 6, after culturing human skin fibroblasts and keratinocytes on the support, as a result of measuring the cell proliferation, the problem of the conventional lactide / ε-caprolactone copolymer was increased by increasing the content of gelatin. It can be seen that can be solved.

In addition, as a result of measuring the regenerative effect on the skin tissue through Experimental Example 7, it can be seen that the regenerative effect of the skin tissue can be effectively controlled by controlling the content of collagen / gelatin added.

The nanofiber support of the present invention is useful for damaged tissue regeneration because it is easily attached to cells and facilitates growth, and has a characteristic of being naturally biodegraded and absorbed by the human body afterwards. Such supports may be applied in the form of maskpack materials, wound dressings, artificial skin, drug delivery materials or ion exchange membranes, and if necessary, include absorbing a therapeutically effective amount of the drug or nanocoating the drug-containing particles. It may further include.

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.

Production Example 1 Preparation of Lactide / ε-caprolactone Copolymer

14.46 g of lactide and 26.64 g of epsilon -caprolactone and 0.0182 g of octosan tin were diluted with 0.128 ml of toluene as a catalyst. Teflon-coated magnetic bars were placed in the ampoules containing the reactants, and the ampoules were kept in a vacuum at 0.005 mmHg for about 2 hours to remove toluene and water.

Subsequently, dry nitrogen injection and vacuum maintenance were repeated 10 times, and the ampoule was heat-sealed. The sealed ampoules were stirred using a magnetic plate while raising the temperature to 170 ° C at 1 ° C / min in a 105 ° C oil bath.

Thereafter, the solid phase polymerization was performed at 170 ° C. for 24 hours. As the polymerization proceeded, the viscosity of the polymerization system increased and stirring was impossible. The polymerization system was initially a clear gel but turned to a brown solid as the reaction proceeded.

After the reaction was completed, the ampoule was sufficiently cooled using liquid nitrogen, and then the ampoule was destroyed to recover the copolymer. The recovered sample was dissolved in chloroform and precipitated in methanol to remove unreacted monomer to recover the copolymer.

The molar ratio of lactide / ε-caprolactone copolymer (Mn = 271,000, Mw = 348,000) measured by hydrogen nuclear magnetic resonance analysis was 5.0 / 5.0.

Production Example 2 Preparation of Gelatin Solution

Gelatin was dissolved in trifluoroethanol at a concentration of 10% by weight in a mixer to prepare a gelatin solution.

Production Example 3 Preparation of Collagen / Gelatin Mixed Solution

The solution was prepared by dissolving collagen and gelatin in trifluoroethanol at a concentration of 10% by weight in a mixer.

Production Example 4 Preparation of Hyaluronic Acid Solution

A solution was prepared by dissolving hyaluronic acid in trifluoroethanol at a concentration of 10% by weight in a mixer.

Example 1 Preparation of Nanofiber Support for Tissue Regeneration

The lactide / ε-caprolactone copolymer prepared in Preparation Example 1 was dissolved in trifluoroethanol at a concentration of 10% by weight, and then mixed with the gelatin solution prepared in Preparation Example 2 at a 30:70 volume ratio. The obtained liquid mixture was put into a syringe and spun using a 23G stainless steel needle.

At this time, the rotary collector was wrapped in aluminum foil and placed at a distance of 20 cm from the needle, and the syringe was injected at a rate of 20 ul / min. The voltage between the needle and the collector continued to act at 18-20 kV, causing the solution to be sprayed into the collector running for 24 hours.

The nanofibers made of foil were dried for 12 hours at room temperature, and the dried specimen was 20 ml of acetone and water (9: 1) added with 0.35% of dimethylaminopropyl-ethylcarbodiimide hydrochloride. The gelatin was crosslinked by soaking the solution slowly for one day.

The crosslinked sample was washed three times in distilled water to remove the remaining chemicals and then lyophilized to prepare a nanofiber support for tissue regeneration.

Example 2 Preparation of Nanofiber Support for Tissue Regeneration

The lactide / ε-caprolactone copolymer prepared in Preparation Example 1 was dissolved in trifluoroethanol at a concentration of 10% by weight, and then mixed with the gelatin solution prepared in Preparation Example 2 at a 70:30 volume ratio. , The same procedure as in Example 1 was carried out to prepare a nanofiber support for tissue regeneration.

Example 3 Preparation of Nanofiber Support for Tissue Regeneration

A nanofiber support for tissue regeneration was prepared in the same manner as in Example 1 except that the collagen / gelatin mixed solution of Preparation Example 3 was used instead of the gelatine solution of Preparation Example 2.

Example 4 Preparation of Nanofiber Support for Tissue Regeneration

Except for using the hyaluronic acid solution of Preparation Example 4 instead of the gelatin solution of Preparation Example 2, it was carried out in the same manner as in Example 1 to prepare a nanofiber support for tissue regeneration.

Comparative Example 1 Preparation of Nanofiber Support for Tissue Regeneration

A nanofiber support for tissue regeneration was prepared in the same manner as in Example 1 except that 10 wt% of gelatin was used alone.

Comparative Example 2 Preparation of Nanofiber Support for Tissue Regeneration

A nanofiber support for tissue regeneration was prepared in the same manner as in Example 1 except that 10 wt% of the lactide / ε-caprolactone copolymer was used alone.

At this time, the contents of the lactide / ε-caprolactone copolymer and gelatin used in each Example and Comparative Example are shown in Table 1 below.

division Volume ratio of copolymer / gelatin Concentration (% by weight) Example 1 30/70 10 Example 2 70/30 10 Comparative Example 1 0/100 10 Comparative Example 2 100/0 10

Experimental Example

Experimental Example 1 Nanofiber Support Form According to Copolymer-Gelatin Mixing Ratio

SEM analysis

The results obtained by measuring the supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2 by a scanning electron microscope are shown in FIG. 1. 1 is a scanning electron micrograph of the support prepared in Examples 1-2 and Comparative Examples 1-2.

Referring to Figure 1, it can be seen that the supports of Examples 1 and 2 and Comparative Examples 1 and 2 are interconnected pores of the three-dimensional structure formed between the fibers of the nano-unit.

Nanofiber Diameter Analysis

In order to measure the average diameter of the support, 30 regions were randomly selected from the scanning electron micrograph, and the diameters of the respective fibers were measured, and the obtained results are shown in FIG. 2. 2 is a graph comparing the average diameters of the supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2. FIG.

Referring to FIG. 2, the supports of Examples 1 and 2 and Comparative Examples 1 and 2 showed a high frequency at an average diameter of 200 to 250 nm in an average diameter of 50 to 500 nm. The thickness of the support showed an approximation of 100 μm.

Experimental Example 2 Surface Characteristics of Nanofiber Support According to Mixing Ratio of Copolymer-Gelatin

Contact angle measurement

The contact angles of the supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2 were measured by an image contact angle measuring instrument, and the results obtained are shown in FIG. 3. At this time, Figure 3 shows the change in contact angle according to the content of gelatin in the support.

Referring to FIG. 3, Comparative Example 1 using gelatin alone showed high wettability due to a large number of hydrophilic functional groups, and the contact angle gradually decreased as the content of the lactide / ε-caprolactone copolymer was increased. Able to know. For example, the contact angle of Comparative Example 1, which is gelatin alone, was 24 ± 2.7 °, and in Comparative Example 2, which used the lactide / ε-caprolactone copolymer alone, the contact angle was 89 ± 6.7 °, and Example 1 in which these were mixed was used. In the case of the support of 2, the value between them is shown.

Water absorption

The water absorption of the supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2 was measured by mass change after soaking in distilled water, and the obtained results are shown in FIG. 4. At this time, Figure 4 shows the change in water absorption rate according to the content of gelatin in the support.

Referring to FIG. 4, it is supported that the introduction of hydrophilic natural polymers has a good effect on the hydrophilicity and the interaction with cells. In Comparative Example 2 using the lactide / ε-caprolactone copolymer alone, it was almost 0, whereas in Example 1, in which the lactide / ε-caprolactone copolymer and gelatin were mixed, the water absorption rate was close to 87 ± 5.6%. Indicated.

Experimental Example 3 Properties of Nanofiber Supports According to Copolymer-Gelatin Mixing Ratio

ATR-FT-IR Measurement

ATR-FT-IR measurements were performed to investigate the surface properties of the nanofibers of the supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2.

As a result, peaks of amide groups of 1540 cm ^-1 and 1656 cm ^-1 were observed in Comparative Example 1 and Examples 1 and 2 containing gelatin, and Comparative Examples using the lactide / ε-caprolactone copolymer alone In 2, only the peak of the ester bond was observed at 1750 cm ^-1 .

Thermal analysis

Thermal analysis of the supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2 was performed. As a result, the melting point of Comparative Example 1 had a wide and pointed endothermic point at 93.8 ° C. However, in Examples 1 and 2, the endothermic curve was broad and blunt, and this result is understood to be due to the coagulation of the pulled chain in the electrospinning process.

Experimental Example 4 Mechanical Properties of Nanofiber Support According to Copolymer-Gelatin Mixing Ratio

The supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2 were subjected to stress-strain experiments, and the results obtained are shown in the figure.

The tensile strength

5 is a graph showing the tensile strength of the support prepared in Examples 1-2 and Comparative Examples 1-2.

Referring to FIG. 5, the support of Comparative Example 1 using gelatin alone showed brittle results, and the elasticity increased as the content of lactide / ε-caprolactone copolymer increased (Example 1, 2), the support of Comparative Example 2 using the lactide / ε- caprolactone copolymer alone showed about 220% stretch.

Recovery rate

6 is a graph showing the recovery rates of the supports prepared in Examples 1-2 and Comparative Examples 1-2.

Referring to FIG. 6, Comparative Example 1 exhibited the largest deformation such as semi-permanent break even though only 50% of deformation was added, and when 100% of Comparative Example 2 was applied, it showed almost perfect recovery of 90% or more. As the content of lactide / ε-caprolactone copolymer increased, the recovery rate tended to increase. In addition, the support of Example 1 showed 91% recovery in 50% of deformation and 85% recovery in 100% of deformation.

Strain

7 is a graph showing strains of the supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2;

Referring to FIG. 7, as in the results of FIGS. 5 and 6, while the support of Comparative Example 2 maintained an elasticity degree superior to 16 days or more, Comparative Example 1 split after 6 hours of applying periodic deformation. As the content of, lactide / ε-caprolactone copolymer increased, the recovery rate tended to increase.

Experimental Example 5 Degradation of Nanofiber Support According to Copolymer-Gelatin Mixing Ratio

Residual mass

The supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2 were observed for 4 weeks at 37 ° C. in a culture solution containing 10% FBS, and the obtained results are shown in FIG. 8.

8 is a graph showing the change in residual mass according to the decomposition period of the support prepared in Examples 1-2 and Comparative Examples 1-2.

Referring to FIG. 8, the support of Comparative Example 1 was completely decomposed after 4 weeks. In comparison, Example 1 showed a residual mass of 29 ± 3%, and Example 2 showed a residual mass of 61 ± 3%. In addition, the support of Comparative Example 2 showed a degradation rate of 5% water because it was slowly degraded in the culture.

Experimental Example 6 Evaluation of Cell Affinity of Nanofiber Scaffold According to Copolymer-Gelatin Mixing Ratio

The supports (diameter: 3 cm, round) prepared in Examples 1 and 2 and Comparative Examples 1 and 2 were soaked in ethanol for 30 minutes, sterilized in ultraviolet light for 30 minutes, and then left in a culture solution for 12 hours. Thereafter, 1 × 10 ⁴ cells / cm ² of human dermal fibroblasts and keratinocytes were cultured and cultured in a growth culture medium for 7 days, and then the proliferation and morphology of the cells were observed by MTT assay and scanning electron microscopy. At this time, as a control example, a support prepared by performing spin coating instead of spinning was used to prepare each support.

9 and 10 are graphs showing changes in cell proliferation according to gelatin content of human skin fibroblasts (FIG. 9) and keratinocytes (FIG. 10), respectively.

Referring to Figure 9, the growth of fibroblasts with time occurred in the support (comparative example 2) consisting only of the lactide / ε-caprolactone copolymer, gelatin increased significantly as the amount of gelatin increases In the case of the support consisting of only (Comparative Example 1) it can be seen that the proliferation of fibroblasts has the highest value. These results indicate that tissue regeneration may occur faster due to the addition of natural polymer gelatin in the preparation of the nanofiber scaffold for tissue regeneration according to the present invention.

In particular, compared to the spin-coated scaffolds, fibroblast proliferation was improved by up to about 3 times in the scaffolds spun with nanofibers. have.

This tendency was similar in the results performed using keratinocytes (FIG. 10).

FIG. 11 is a scanning electron micrograph of the result of FIG. 9. Referring to FIG. 11, it can be seen that the supports of Examples 1 and 2 and Comparative Examples 1 and 2 are formed by forming a network structure three-dimensionally so that the nanofibers have pores.

Experimental Example 7 Evaluation of Cell Affinity of Nanofiber Scaffold According to Copolymer-Gelatin Mixing Ratio

The supports prepared in Controls, Examples 1 and 2 and Comparative Example 1 were soaked in ethanol for 30 minutes, sterilized in ultraviolet light for 30 minutes, and lyophilized. Herein, epidermal growth factor (EGF) was diluted in distilled water, and then 1 ng EGF per support was added and lyophilized.

After removing the skin completely in a 6 mm diameter circle on the rat back, the experimental support was attached and observed for 14 days, followed by histological examination through hematoclinin-eosin staining. The histological examination was performed by dividing the group into a support (Comparative Example 1, Example 1, Example 2) and an unsupported support (control group) containing EGF, and the results obtained are shown in FIG. 12. At this time, the cell cultured support is a circular shape each 15 mm in diameter.

12 is an optical micrograph showing the skin regeneration effect of the support prepared in the control, Examples 1 to 2 and Comparative Example 1.

Referring to FIG. 12, the regeneration effect was excellent in the support of Comparative Example 1, which is a support using gelatin alone, and the supports of Examples 1 and 2 also showed high results. In addition, it can be seen that the effect is further improved when the epidermal growth factor (EGF) is used in combination with the control.

As a result of measuring various physical properties of the support through Experimental Examples 1 to 7, it was found that the lactide / ε-caprolactone copolymer or gelatin was formed by controlling the content thereof rather than forming the support. It could have suitable physical properties. In addition, it can be seen that the content of each composition is involved in tissue regeneration (by cell proliferation) as well as physical properties. Thus, by adjusting the content of each composition, it is possible to control the properties of the support and maximize the effect of tissue regeneration.

In addition, the support is produced in the form of nanofibers through the spinning process, the cell proliferation is more smoothly, it is possible to obtain faster tissue regeneration effect.

The nanofiber support for tissue regeneration according to the present invention can effectively treat a patient who has damaged skin tissue for skin regeneration.

1 is a scanning electron micrograph of the support prepared in Examples 1-2 and Comparative Examples 1-2.

Figure 2 is a graph comparing the average diameter of the support prepared in Examples 1-2 and Comparative Examples 1-2.

3 is a graph showing the change in contact angle according to the content of gelatin in the support.

Figure 4 is a graph showing the change in water absorption according to the content of gelatin in the support.

5 is a graph showing the tensile strength of the support prepared in Examples 1-2 and Comparative Examples 1-2.

Figure 6 is a graph showing the recovery rate of the support prepared in Examples 1-2 and Comparative Examples 1-2.

Figure 7 is a graph showing the strain of the support prepared in Examples 1-2 and Comparative Examples 1-2.

8 is a graph showing the change in residual mass according to the decomposition period of the support prepared in Examples 1-2 and Comparative Examples 1-2.

9 is a graph showing changes in cell proliferation according to the gelatin content of human skin fibroblasts.

10 is a graph showing changes in cell proliferation according to the gelatin content of keratinocytes.

FIG. 11 is a graph showing changes in cell proliferation of supports prepared in Examples 1 and 2 and Comparative Examples 1 and 2. FIG.

12 is an optical micrograph showing the skin regeneration effect of the support prepared in the control, Examples 1 to 2 and Comparative Example 1.

Claims (23)

Lactide / ε-caprolactone copolymer having the structure of Formula 1, and One type of crosslinked natural polymer selected from the group consisting of collagen, gelatin, chitosan, cellulose, alginic acid, hyaluronic acid, and mixtures thereof (blends), A nanofiber support for tissue regeneration, wherein the copolymer and a crosslinked natural polymer are mixed in a weight ratio of 1: 9 to 9: 1: [Formula 1]

(Wherein x and y are molar ratios of each monomer, x is 0.2 to 0.8, y is 0.8 to 0.2, x + y = 1 and n is an integer of 25 or more). delete The method of claim 1, Wherein x is 0.4 to 0.6, y is 0.4 to 0.6, and n is an integer of 25 to 50. The method of claim 1, Wherein x and y are each 0.5 and n is an integer of 25 to 30. delete The method of claim 1, The natural polymer is collagen, gelatin, or a mixture thereof is a nanofiber support for tissue regeneration. delete The method of claim 1, The natural polymer crosslinked with the lactide / ε-caprolactone copolymer is contained in a weight ratio of 3: 7 to 7: 3 in the nanofiber support. The method of claim 1, The tissue regeneration nanofiber support is an average diameter of 50 to 500 nm, the thickness of 50 to 150 ㎛ tissue regeneration nanofiber support. The method of claim 1, The tissue regeneration nanofiber support is an average diameter of 200 to 250 nm, the thickness of the 70 to 110 ㎛ tissue regeneration nanofiber support. Each of one natural polymer selected from the group consisting of lactide / ε-caprolactone copolymer represented by the following Chemical Formula 1 and collagen, gelatin, chitosan, cellulose, alginic acid, hyaluronic acid, and a mixture (blend) thereof is 1: Dissolved in a solvent in a weight ratio of 9 to 9: 1, and then mixed; After the obtained mixed solution is added to a spinning needle (needle), a spinning process is performed. A method of preparing a nanofiber support for tissue regeneration according to claim 1, comprising the step of crosslinking a natural polymer in the spun nanofibers: [Formula 1]

(Wherein x and y are molar ratios of each monomer, x is 0.2 to 0.8, y is 0.8 to 0.2, x + y = 1 and n is an integer of 25 or more). delete The method of claim 11, Wherein x is 0.4 to 0.6, y is 0.4 to 0.6, and n is an integer of 25 to 50. The method of claim 11, Wherein x and y are each 0.5 and n is an integer of 25 to 30. delete The method of claim 11, The natural polymer is collagen, gelatin, or a mixture thereof is a method of producing a nanofiber support for tissue regeneration. The method of claim 11, Lactide / ε- caprolactone copolymer and the natural polymer when the mixture is used in the form of a solution dissolved in a concentration of 5 to 20% by weight in a solvent, respectively. The method of claim 17, The solvent is water; Lower alcohols such as methanol, ethanol, propanol and butanol; Acetone; Trifluoroethanol; Tetrahydrofuran; Dichloromethane; And a method for producing a nanofiber support for tissue regeneration that is one selected from the group consisting of mixed solvents thereof. delete The method of claim 11, The spinning is performed by the electrospinning method of manufacturing a nanofiber support for tissue regeneration. The method of claim 20, The electrospinning method of producing a nanofiber scaffold for tissue regeneration is performed under the condition that the voltage between the spinning needle and the collector to 15 to 25 kV using a spinning needle of 18 to 24 gauge. The method of claim 11, The crosslinking process is a method for producing a nanofiber support for tissue regeneration that is carried out by immersing the spun fibers in a solution containing a coupling agent. The method of claim 22, The coupling agent is dimethylaminopropyl-ethylcarbodiimide hydrochloride, hydroxybenzotriazole, O-benzotriazol-1-yl-N, N, N'N'-tetra Methylruronium tetrafluoroborate (0-benzotriazol-1-yl-N, N, N'N'-tetramethyluronium tetrafluoroborate) and a nanofiber support for tissue regeneration comprising one selected from the group consisting of Manufacturing method.

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