KR100621569B1 - Nano-microfibrous scaffold for enhanced tissue regeneration and method for preparing the same - Google Patents

Abstract

The present invention relates to a biomimetic porous composite support composed of nanofibers and microfibers for effectively regenerating damaged tissue and a method of manufacturing the same.

The porous composite support of the present invention is a micro-fiber as a basic skeleton, the nanofibers obtained from the physiologically active polymer is composed of a two-dimensional or three-dimensional network form, by using a microfiber has a certain shape and strength biological tissue By allowing nano-fibers to be regenerated in three dimensions, the porosity is improved to increase the surface area associated with cells and provide a structure in which cells adhere and proliferate well. In particular, nanofibers have a feature of increasing tissue regeneration ability by using a polymer that gives physiological activity or a polymer that can increase biocompatibility to existing polymers.

Nanofibers, tissue regeneration, complexes.

Description

Bio-mimetic nanofiber and microfiber composite support for inducing tissue regeneration and its manufacturing method {NANO-MICROFIBROUS SCAFFOLD FOR ENHANCED TISSUE REGENERATION AND METHOD FOR PREPARING THE SAME}             

1 is a view showing a schematic diagram of the electrospinning apparatus of the present invention,

Figure 2 is a differential scanning micrograph (3500 times) of the collagen nanofibers prepared in Preparation Example 1,

3 is a differential scanning micrograph (3500 times) of nanofibers prepared from a mixture of chitosan and polylactic acid according to Preparation Example 3,

4 is a differential scanning micrograph (2000 times) of nanofibers prepared from a collagen and polylactic acid mixture according to Preparation Example 4;

Figure 5 is a differential scanning microscope picture (750 times) of the nanofiber and microfiber composite support prepared according to Example 1,

Figure 6 is a graph showing the XRD analysis of the collagen, collagen nanofibers, polylactic acid nanofibers and polylactic acid / collagen nanofibers of the present invention,

Figure 7 is a differential scanning micrograph (500 times) showing the cartilage adhesion of the nanofibers and microfiber composite support prepared in Example 1.

Explanation of symbols on the main parts of the drawings

1: spinning liquid reservoir, 2: nozzle

3: voltage transfer cord, 4: collector

5: voltage generator, 6: micro fiber

The present invention relates to a porous composite support for regenerating damaged tissue and a method of manufacturing the same.

Tissue regeneration is effective to regenerate tissues by providing cells, drug supports, etc., when organs or tissues in the human body lose function or induction, and at this time, the support for tissue regeneration may be physically stable at the implant site and control regenerative efficacy. It must have bioavailability, and must be degraded in vivo after formation of new tissue, and the degradation product must not be toxic.

The support for tissue regeneration has conventionally been prepared with a porous support using a polymer having a certain strength and form, and has been produced and used as a sponge or fibrous matrix or gel-type cell culture support using such a polymer.

Of these, the support of the fibrous matrix has open pores and has a structure in which the pores are large and cells adhere well and proliferate. However, it is not used much at present. This is because when the support is made of natural polymer, the strength in the water phase is so weak that it does not maintain its shape due to disintegration or shrinkage, and even if a synthetic polymer is used, it is difficult to secure a certain space by the fiber form alone. It has a problem of being formed in a two-dimensional structure. For reference, in regenerating tissues, the three-dimensional structure is a very important structure for cell activity and regeneration ability.

In addition, such a support alone has a limitation in encapsulating the drug to control and release all of the natural polymers having physiological activity.

On the other hand, the extracellular matrix in vivo maintains the network structure of the collagen nanofibers and basic substances such as glycosaminoglycans, and the cells attach and proliferate therebetween to form tissues. In the present invention, the structure that mimics the shape of the extracellular matrix is designed to have a network similarity, and at the same time, to form a network structure including a factor capable of inducing tissue regeneration or enhancing biocompatibility, to form a nanofiber and a microfiber. To prepare a composite support comprising a.

Conventional methods for preparing scaffolds for regenerated tissues are generally methods of preparing scaffold-type scaffolds, including particulate leaching, emulsion freeze-drying, high pressure gas expansion, and Phase separation and the like are used.

Particle leaching is a method of mixing a particle such as salt that is insoluble in an organic solvent solution of a biodegradable polymer to remove the solvent, and then eluting and removing the salt particles with water. A porous structure having pore size and porosity can be obtained, but there is a problem that cells are damaged by remaining salt salts or rough shapes (AG Mikos, G. Sarakinos, SM Leite, JP Vacant i, and R. Langer, Biomaterials (1993) 14, 5, 323-330; AG Mikos, A. J. Thorsen, LA Czerwonka, Y. Bao, R, Langer, DN Winslow, and JP Vacan ti, Polymer (1994) 35, 5, 1068- 1077).

In addition, the emulsion freeze drying method is a method of removing the organic solvent and water by freeze-drying the organic solvent solution / water emulsion of the polymer, and the high pressure gas expansion method is to put the biodegradable polymer in the mold without using the organic solvent to apply a pressure pellet After injecting a high-pressure carbon dioxide gas into the biodegradable polymer at an appropriate temperature, the pressure is gradually lowered to release the carbon dioxide gas in the matrix to form pores. However, these methods have limitations in making open cellular pores difficult (see K. Whang, CH Thomas, KE Healy, G. Nuber, Polymer (1995) 36, 4, 837-842; DJ Mooney, DF Baldwin, NP Suh, JP Vacanti, R. Langer, Biomat erials (1996) 17, 1417-1422).

Recently, a phase separation method is used in which a porous support is prepared by adding a sublimable substance or a solvent having different solubility to the organic solvent solution of a polymer and phase separation of the solution according to sublimation or temperature change. Culture is difficult (see H. Lo, MS Ponticiello, KW Leong, Tissue Eng. (1995) 1, 15-28; H. Lo, S. Kadiyala, SE Guggino, KW Leong, J. Biomed). Mater.Res . (1996) 30, 475-484; Ch. Sc hugens, V. Maguet, Ch. Grandfils, R. Jerome, Ph. Teyssie, J. Biomed.Mate.Res . (1996) 30, 449- 461).

The above methods are for producing a three-dimensional polymer support that can induce cell adhesion and differentiation, but there are still many problems in the method for making a three-dimensional tissue regeneration support from biodegradable polymers.

An object of the present invention is to solve the above problems, to provide a porous composite support and a method of manufacturing a network structure that has a structural stability and at the same time can induce tissue regeneration as well as increase the biocompatibility. .

In order to achieve the above object, the present invention provides a porous composite support made of microfibers and nanofibers and a method of manufacturing the same.

Hereinafter, the present invention will be described in detail.

The present invention provides a porous composite support made of microfibers and nanofibers. Specifically, a microfiber as a backbone, and nanofibers obtained from a physiologically active polymer provides a porous composite support having a two-dimensional or three-dimensional network form.

Figure 5 shows an example of the porous composite support of the present invention, the coarse and black fibers are microfibers. Microfibers have a diameter of 30 to 50 µm and are supports having a basic skeleton of a porous composite support. Such a support exhibits a certain form and strength to the porous composite support of the present invention to allow tissue to be three-dimensionally regenerated.

In the present invention, the microfiber is a mixture of synthetic polymer and natural polymer, and the synthetic polymer is polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, L polyesters such as -lactide-co-glycolide (PLGA) and poly (caprolactone), poly (valerolactone), poly (hydroxybutyrate) and poly (hydroxy valerate), and the natural polymer is collagen gelatin, hyaluronic acid Uses are obtained from the group consisting of lonic acid, chitin and chitosan, preferably those obtained from poly (D, L-lactic acid- vegetable-glycolic acid) However, in the present invention, the microfibers can be used for tissue regeneration. The microfiber may be a compound obtained by mixing synthetic polymer and natural polymer in a ratio of 2: 1 to 1: 2, preferably 1: 1. What was obtained by mixing is used.

Also, as shown in FIG. 5, the thin and white fibers are nanofibers. Nanofibers have a diameter of 0.2 μm or less, and have a two-dimensional or three-dimensional network form on the basic skeleton of the microfibers. These nanofibers increase the porosity to increase the surface area associated with the cells and provide a structure in which cells adhere well and proliferate. In particular, it is possible to improve the tissue regeneration ability by manufacturing using a polymer that gives a biological activity or a polymer that can increase the synthesis in vivo to the existing polymer.

In the present invention, the nanofibers are obtained from natural polymers and synthetic polymers, and the natural polymers use chitosan or collagen as having excellent biocompatibility, and specifically, an acid-soluble type 1 collagen or a number average molecular weight of 80,000 to 150,000 chitosan. use. Synthetic polymers have crystallinity in the structure to maintain a stable form in the aqueous phase of 37 ℃ aqueous solution when manufacturing nanofibers, selected from the group consisting of polylactic acid, polylactic acid-glyconic acid copolymer and polyglycolic acid And polylactic acid having a number average molecular weight of 100,000 to 350,000 is preferably used. In this case, one of the natural polymer and the synthetic polymer may be selected and used alone, or a mixture of the natural polymer and the synthetic polymer may be used. This is because it has crystallinity in the structure, not only to maintain a stable form in vivo conditions when producing nanofibers, but also to show the effect of showing biocompatibility, in consideration of these effects, the mixing ratio of natural polymer and synthetic polymer is weight ratio. 0.25: 1-0.5: 1 are preferable.

As described above, the porous composite support of the present invention can improve the tissue reproducibility while maintaining a certain form and strength by using a combination of microfibers and nanofibers, considering the amount of nanofibers The amount of the microfibers is adjusted to 80 to 90% by weight based on the composite support. When the amount of the nanofibers is less than the range, the tissue regeneration ability is insufficient to obtain a sufficient effect, when the amount exceeds the range is difficult to maintain a certain form and strength, there is a disadvantage in that the characteristics as a support falls.

In addition, the present invention includes a drug or growth factor that can induce tissue regeneration in addition to the porous composite support.

An example of a drug capable of inducing tissue regeneration is DHEA and the like, and the growth factor is selected from the group consisting of platelet-derived growth factor, insulin-like growth factor, epidermal growth factor, and transforming growth factor. However, in the present invention, any drug or growth factor can be used as long as it can induce tissue regeneration, without being limited thereto. These drugs or growth factors can be located in the nanofibers to maximize tissue regeneration, using 100 to 5000 ng for the nanofibers.

In addition, the present invention provides a method for producing a porous composite support.

Specifically, preparing a microfiber (step 1),

Preparing a spinning solution by dissolving the natural polymer and the synthetic polymer alone or in an organic solvent (step 2),

After filling the spinning solution in the electrospinner reservoir to prepare a nanofiber using an electrospinner (step 3), and

It provides a method for producing a porous composite support comprising a step (step 4) of producing a porous composite support by spinning the nanofibers of the step 3 on the microfiber prepared in step 1.

In addition, in the preparation of the spinning solution of step 2, natural polymers and synthetic polymers may be dissolved alone or in a mixed organic solvent, and the drug may be further dissolved to prepare a spinning solution.

Step 1 prepares microfibers. Specifically, synthetic polymers and natural polymers are dissolved in a solvent, respectively, and their mixed solution is prepared using an emulsifier. Thereafter, the mixed solution is solution spun to prepare microfibers, and then compressed and cross-linked to complete the microfibers.

First, synthetic polymers and natural polymers are dissolved in a solvent, where synthetic polymers are dissolved in an organic solvent, and natural polymers are dissolved in an acid solvent.

The organic solvent for dissolving the synthetic polymer is selected from acetone, chloroform and methylene chloride, and methylene chloride is preferably used. However, the present invention is not limited thereto, and an organic solvent may be appropriately selected according to circumstances. In addition, the synthetic polymer is used 10 to 20% by weight based on the synthetic polymer solution.

The acid solvent for dissolving the chitosan is selected from acetic acid, lactic acid and hydrochloric acid, and preferably acetic acid is used. However, the present invention is not limited thereto, and an acid solvent may be appropriately selected depending on the situation. In addition, the natural polymer is used 4 to 8% by weight based on the natural polymer solution.

It uses an emulsifier to mix the respective solutions, preferably Tween 80. At this time, the mixing ratio of the mixed solution is preferably synthetic polymer: natural polymer = 0.5: 1 to 2: 1, it can be appropriately adjusted according to the desired properties.

Thereafter, a sodium chloride solution is dissolved in a solution of 1: 1 mixing ethylene glycol and methanol to prepare a coagulation bath. Alkaline methanol solution is used as a coagulation bath in the mixed solution prepared above, followed by solution spinning, followed by washing and drying until neutral to prepare microfibers. The ratio of the coagulation bath is most preferably 25% ethylene glycol, 70% methanol, 5% by weight sodium chloride. The natural polymer of the present invention is dissolved in an acid solution and coagulated in an alkaline solution, the synthetic polymer of the present invention is well dissolved in methylene chloride and coagulated in methanol, and the properties of such a solvent can be used to prepare a desired coagulation bath. .

The alkaline methanol coagulant is a mixed solution of ethylene glycol and methanol containing a strong base selected from sodium hydroxide and potassium hydroxide, wherein the base is 4-20%, and the methanol is preferably 30-60%.

Finally, the microfibers prepared above can be filled and compressed into a mold suitable for the shape and size of cartilage defects, equilibrated in an aqueous phase such as the body for at least 1 hour, and then freeze-dried to dissolve polymers such as methylene chloride. Crosslinking is carried out for 2-8 hours in the presence of steam to produce a support suitable for the application site. The pressure is preferably 5000kg ~ 2ton, more preferably 1 ~ 2ton.

In step 2, a spinning solution in which natural polymer and synthetic polymer are dissolved is prepared, and the spinning solution is further prepared by dissolving the drug together.

The natural polymer and the synthetic polymer are raw materials for producing nanofibers, and the natural polymer is one selected from the group consisting of chitosan and collagen with excellent biocompatibility. Preferably, the type 1 collagen or the number average molecular weight is 80,000. Use chitosan of ˜150,000.

In addition, the synthetic polymer has a crystallinity in the structure to maintain a stable form in vivo in the aqueous phase of 37 ℃ aqueous nanofibers are selected from the group consisting of polylactic acid, polylactic acid-glyconic acid copolymer and polyglycolic acid Synthetic polymers are used, and polylactic acid having a number average molecular weight of 100,000 to 350,000 is preferably used. In this case, one of the natural polymer and the synthetic polymer may be selected and used alone, or a mixture of the natural polymer and the synthetic polymer may be used. This is because it has a crystallinity in the structure to maintain a stable form in vivo conditions at 37 ℃ aqueous solution when manufacturing nanofibers, as well as to show the characteristics showing biocompatibility.

The amount of the polymer used is 5 to 10% by weight of natural polymer and 10 to 20% by weight of synthetic polymer, respectively, based on the spinning solution.

The solvent used in step 1 is one that can easily dissolve synthetic and natural polymers, selected from the group consisting of 1,1,1,3,3,3-hexafluoropropanol, methylene chloride and acetone, or The above liquid mixture is preferably a solvent in which 1,1,1,3,3,3-hexafluoropropanol and methylene chloride are mixed in a ratio of 1: 1 to 5: 1 by volume.

In addition, step 1 further dissolves a drug selected from the group consisting of platelet-derived growth factor, insulin-like growth factor, epidermal growth factor and transforming growth factor in the spinning solution, and the amount added is preferably 100 to 5000 ng based on the polymer. . The drug is included in the porous composite support to be added to maximize tissue regeneration. The addition method is emulsified in the spinning solution using a surfactant such as Span 80 to prepare a water-in-oil emulsion.

In step 3 to prepare a nanofiber using the spinning solution electrospinning machine.

The spinning procedure using the electrospinner is described below with reference to FIG. 1. A constant current flows in the voltage generator 5 to form an electric field between the nozzle 2 and the collector 4. The polymer solution filled in the spinning liquid reservoir 1 is spun onto the collector 4 by the force and gravity of this electric field. The radiation conditions can be varied by adjusting the distance of the electric field between the nozzle 2 and the collector 4 and the nozzle size of the voltage emissive reservoir 1. At this time, it is possible to adjust the shape to be produced according to the concentration and the spinning distance of the spinning solution, in this step to produce nanofibers by optimally adjusting the conditions of the electrospinning machine. The condition of the electrospinner is a radiation distance of 10 to 20 cm, and a voltage of 15 to 25 V is preferable. In the present invention, the electrospinning apparatus may use a DH High Voltage Generator (model name: CPS-40K03VIT) manufactured by Chungpa EMT (Korea).

Finally, in step 4, the porous composite support is manufactured by spinning the nanofibers of step 3 on the prepared microfibers. Specifically, the nanofibers are spun into a moderately thick microfiber matrix in a thinly laid state, stacked and compressed in a suitable thickness, and then molded to fit the defects to produce a porous composite support.

The porous composite support of the present invention is for effectively regenerating tissue when the organs or tissues in the human body read or perform a function, and is a porous composite support useful for attaching cells. The porous composite support may be applied to a variety of chondrocytes or bone cells, and preferably applied to chondrocytes.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Preparation Example 1 Collagen Nanofiber Preparation

1 g of acid-soluble collagen 1 obtained from calf skin of young cows at concentrations of 5%, 8% and 10%, respectively, 1,1,1,3,3,3, -hexafluoropropanol (1,1 , 1,3,3,3 hexafluoropropanol) solution to prepare a spinning solution. Nanofibers were prepared using the spinning solution electrospinning method (see FIG. 1 ).

The electrospinning machine used DH High Voltage Generator (Model: CPS-40K03VIT) manufactured by Chungpa EMT (Korea).

The detailed method of electrospinning was described in conjunction with FIG. 1 attached as follows.

The collagen solution (spinning liquid) having each concentration prepared above was filled into the spinning liquid reservoir 1. The spinning solution reservoir (1) was used as a 10 ml pipette and the inlet of the pipette was heated with a fire and adjusted to a diameter of 0.5 mm to serve as a nozzle (2). The rate of emission of the spinning liquid was adjusted to 0.5 ml / min during spinning, the voltage was adjusted between 15 and 25 V, and the distance of the electric field was adjusted to 10 to 20 cm. Collagen nanofibers were prepared.

At this time, it was confirmed that the obtained collagen nanofibers had a thickness of 2 μm or less.

FIG. 2 shows a differential micrograph (3500 times) of collagen nanofibers obtained with 8% collagen staph solution under conditions of 20 V voltage and 20 cm electric field distance.

Preparation Example 2 Preparation of Chitosan Nanofibers

A spinning solution was prepared by dissolving chitosan having a number average molecular weight of 80,000 to 150,000 in an organic solvent at a concentration of 0.5 to 2%. The organic solvent used was a mixed solution of 1,1,1,3,3,3, -hexafluoropropanol (1,1,1,3,3,3 hexafluoropropanol) and methyl chloride. It was 5-40 weight% with respect to the mixed solution.

Chitosan spinning solution of each concentration was spun in the same manner as in Preparation Example to prepare chitosan nanofibers.

Preparation Example 3 Preparation of Nanofibers Composed of Mixture of Collagen and Polyester

A spinning solution was prepared by dissolving 320 mg of acid-soluble collagen type 1 and 640 mg of polylactic acid having a number average molecular weight of 100,000 in 5 ml of 1,1,1,3,3,3, -hexafluoropropanol.

As shown in FIG. 1, the spinning solution was filled in the spinning solution reservoir 1 of the spinning apparatus and spun in the same manner as in Preparation Example 1 to prepare nanofibers.

In the case of spinning at an electric field distance of 15 cm and a voltage of 20 V, the most preferable type of nanofibers could be produced, and the nanofibers thus obtained were observed through a differential microscope. The results are shown in FIG.

Collagen is a structure that does not have its own crystallinity, and when it is made of nanofibers, it is easily contracted in an aqueous solution at 37 ° C., which is the same condition as the body, and the fibers are aggregated together or lose nano form. However, nanofibers prepared by adding polylactic acid having crystallinity to collagen were able to maintain a stable state while maintaining the original form in the aqueous solution.

Preparation Example 4 Preparation of Nanofibers Composed of a Mixture of Chitosan and Polyester

100 mg of chitosan used in Preparation Example 2 and 300 mg of polylactic acid having a number average molecular weight of 100,000 were 1,1,1,3,3,3, -hexafluoropropanol and methyl chloride 2: 1 (w / w). It was dissolved in 10 ml of the mixed solution prepared to prepare a spinning solution.

As shown in FIG. 1, the spinning solution was filled in the spinning solution reservoir 1 of the spinning apparatus and spun in the same manner as in Example 1 to prepare nanofibers.

In the case of spinning at an electric field distance of 15 cm and a voltage of 25 V, nanofibers of the most preferred form could be prepared, and the nanofibers thus obtained were observed through a differential microscope. The results are shown in FIG.

Polyester is a biodegradable and stable form, and is widely used as a biomaterial under the approval of the US Food and Drug Administration. However, it has a problem of tissue toxicity due to the high concentration of acid due to degradation products. . Thus, by adding chitosan having biocompatibility to polylactic acid, the produced nanofibers could improve biocompatibility.

Example 1 Preparation of Nanofiber and Microfiber Composite Supports 1

(Step 1) Preparation of the microfiber

First, the polylactic acid-glycolic acid copolymer was dissolved in acetone and chitosan was dissolved in 5% acetic acid. After each of the dissolved solutions were mixed, the two solutions were mixed using an emulsifier Tween 80. At this time, the mixing ratio of the mixed solution was a ratio of polyester: chitosan = 0.5: 1 by weight.

Thereafter, a sodium chloride solution was dissolved in a solution of 1: 1 mixing ethylene glycol and methanol to prepare a coagulation bath. The solution was spun using an alkali-axksdhf solution as a coagulation bath in the mixed solution, washed until neutral, and dried to prepare a microfiber. At this time, the ratio of the coagulation bath is 25% by weight ethylene glycol, 70% by weight methanol, 5% by weight sodium chloride.

Thereafter, the prepared microfibers were filled in a mold having a shape suitable for the shape and size of the cartilage defect. It was compressed, equilibrated for 1 hour in the same water phase as the body, lyophilized and crosslinked for 4 hours in methylene chloride vapor to prepare a support suitable for the application site. At this time, the pressure was 2 tons.

(Step 2) spinning solution preparation

(Step 3) Preparation of Nanofibers

Steps 2 and 3 are as described in Preparation Example 3.

(Step 4)

On the microfiber matrix in a thinly laid state, the nanofibers were radiated using an electrospinning device shown in FIG. At this time, it was carried out under the conditions of the electric field distance cm, voltage 20V.

Figure 5 shows a differential scanning micrograph of the prepared nanofiber and microfiber composite support. As shown in FIG. 5, the nanofibers form a network while maintaining a well mixed form between the microfibers. It can be seen that it is possible to induce tissue regeneration by the mixing of physiologically active nanofibers and to increase the adhesion of cells by increasing the surface area.

Example 2 Preparation of Nanofiber and Microfiber Composite Supports 2

A composite support was prepared in the same manner as in Example 1, except that 300 ng of insulin-like growth factor was emulsified using Span 80 to prepare the spinning solution in the spinning solution prepared in Preparation Example 3. .

The nanofibers containing the drug or growth factor that can induce tissue regeneration in the nanofibers thus radiated can be maximized.

Experimental Example 1 Stability Analysis by XRD Analysis

Collagen, collagen nanofibers of Preparation Example 1, polylactic acid nano by the manufacturing method of the present invention, in order to predict the change of stability of the material itself of the collagen, polylactic acid and mixtures thereof and the decrease of crystallinity in the production of nanofibers XRD (X-ray Diffractometer (XRD 3000PTS); Rich. Seifert & Co.) analysis was performed on the fibers and the polylactic acid / collagen nanofibers of Preparation Example 3. The results are shown in FIG.

As shown in FIG. 6, in the case of collagen, the stability of the nanofibers is further reduced, leading to rapid shrinkage in the actual aqueous solution, which results in agglomeration or loss of shape. Thus, it can be seen that 2θ is mixed with polylactic acid having crystallinity around 17 ° to prepare nanofibers to give crystallinity, thereby increasing stability in aqueous solution and maintaining a constant form.

In fact, non-woven fabrics made of nanofibers were used to measure the change in porosity at drying and porosity in 37 ℃ aqueous solution, the nanofibers made by mixing polylactic acid with collagen compared to the reduction of porosity by more than 50% Showed a porosity reduction of about 10%.

Experimental Example 2 Chondrocyte Attachment Experiment of Support

After disintegrating the composite scaffold prepared in Example 1 with 70% ethanol, the cultured cartilage cells passaged after dynamic culture were observed to observe the state of cells attached by differential scanning microscope.

The cells to which no cells were attached were removed and pre-fixed with 2.5% glutaraldehyde solution obtained by diluting 25 (w / w)% glutaraldehyde in 0.1M phosphate buffered saline (PBS pH 7.4). . After preliminary fixation, the cells were washed with 0.1M phosphate buffered saline and then fixed with 0% for 20 minutes with a 1% osmium tetraoxide solution obtained by dissolving osmium tetrachloride in 0.1M phosphate buffered saline. After post-fixation, water was completely removed with ethanol, and after lyophilization, the samples were coated with G-Paradium and observed with a differential scanning microscope. The results are shown in FIG.

As shown in Figure 7, the strength was maintained in a constant form by the microfiber after one day. In addition, when the tissues were enlarged and the cells were attached to the scaffold, some cartilage cells were attached to the microfibers, but they were closely attached to a large number of cells or nanofibers. It was found that mixing the nanofibers provides a space to which cells can attach and induces tissue regeneration due to the nanofibers having cellular activity.

 As described above, the composite support for tissue regeneration of the present invention consists of a network of microfibers and nanofibers to maintain a stable strength in vivo by maintaining a certain strength and at the same time having a physiological activity in biomimetic form Can effectively induce tissue regeneration. In particular, the nanofibers constituting the composite support can maximize the adhesion and proliferation of cells by increasing the porosity. In addition, it has an effect of enhancing biocompatibility by using collagen having its own physiological activity or by using chitosan which can neutralize an acid.

Claims (13)

Polyesters selected from the group consisting of polylactic acid, polyglycolic acid and poly (D, L-lactic acid-co-glycolic acid); Poly (caprolactone); Poly (valerolactone); Basic skeleton is a microfiber composed of a mixture of synthetic polymer selected from the group consisting of poly (hydroxybutyrate) and poly (hydroxy valerate) and natural polymer selected from the group consisting of collagen, gelatin, hyaluronic acid, chitin and chitosan The two-dimensional structure or nanofibers composed of a single polymer or a mixture of natural polymers selected from the group consisting of chitosan and collagen and synthetic polymers selected from the group consisting of polylactic acid, polylactic acid-glyconic acid copolymer and polyglycolic acid Porous composite support, characterized in that consisting of a three-dimensional network form. The porous composite support of claim 1, wherein the porous composite support further comprises a drug selected from the group consisting of platelet-derived growth factors, insulin-like growth factors, epidermal growth factors, and transforming growth factors. The porous composite support of claim 1, wherein the nanofibers are contained in an amount of 10 to 20% by weight based on the composite support, and the microfibers are contained in an amount of 80 to 90% by weight based on the composite support. delete delete The porous composite support according to claim 1, wherein the mixing ratio of the synthetic polymer and the natural polymer constituting the nanofibers is 0.25: 1 to 0.5: 1 by weight. In the preparation of the porous composite support, Polyesters selected from the group consisting of polylactic acid, polyglycolic acid and poly (D, L-lactic acid-co-glycolic acid); Poly (caprolactone); Poly (valerolactone); To prepare microfibers comprising a mixture of synthetic polymers selected from the group consisting of poly (hydroxybutyrate) and poly (hydroxy valerate) and natural polymers selected from the group consisting of collagen, gelatin, hyaluronic acid, chitin and chitosan Step (step 1), Preparing a spinning solution by dissolving a mixture of a natural polymer selected from the group consisting of chitosan and collagen and a synthetic polymer selected from the group consisting of polylactic acid, polylactic acid-glyconic acid copolymer and polyglycolic acid in an organic solvent ( Step 2), After filling the spinning solution in the electrospinner reservoir to prepare a nanofiber using an electrospinner (step 3), and Method for producing a porous composite support comprising the step (step 4) of producing a porous composite support by electrospinning the nanofiber of the step 3 on the microfiber prepared in step 1. 8. The method of claim 7, wherein in step 1, a drug selected from the group consisting of platelet-derived growth factor, insulin-like growth factor, epidermal growth factor and transforming growth factor is further dissolved. The preparation of the porous composite support according to claim 7, wherein the solvent of step 1 is at least one selected from the group consisting of 1,1,1,3,3,3, -hexafluoropropanol, methyl chloride and acetone. Way. 8. The method of claim 7, wherein the electrospinning is performed under a voltage of 15 to 25 V and a spinning distance of 10 to 20 cm. The porous composite support of claim 1 useful for attaching cells. 12. The porous composite support of claim 11, wherein the cells are chondrocytes. The porous composite support according to claim 1, wherein the diameter of the microfibers is 30 to 50 µm and the diameter of the nanofibers is more than 0 and 0.2 µm or less.

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