KR100968231B1 - Nonwoven Nanofibrous Membranes for Guiding Bone Tissue Regeneration and Their Preparation Method - Google Patents

Abstract

The present invention relates to a bone membrane-induced regeneration shielding membrane and a method for manufacturing the same, and more particularly, consisting of nanofibers comprising a biodegradable polymer and a biocompatible inorganic material, and a bone tissue-induced regeneration shielding membrane comprising a non-woven porous structure and It relates to a manufacturing method thereof, the shielding membrane has a certain strength, biocompatibility and biodegradability, excellent bone regeneration effect can be used suitably in the field of bone tissue engineering.

Bone regeneration, shielding membrane, tissue engineering, nanofiber

Description

Nonwoven Nanofibrous Membranes for Guiding Bone Tissue Regeneration and Their Preparation Method}

The present invention relates to a bone membrane-induced regeneration shielding membrane having a certain strength, biocompatibility and biodegradability, and excellent in bone regeneration effect, which can be suitably used in the field of bone tissue engineering, and a method of manufacturing the same.

In order to be implanted, the periodontal bone must be maintained intact to be firmly fixed. In this case, the periodontal bone can be regenerated using a shielding membrane, and implantation is possible after the periodontal bone is damaged.

As a material of the shielding film, a polymer such as polytetrafluoroethylene was initially used, but a problem such as having to go through the removal of the shielding film after the periodontal bone was formed also occurred.

Accordingly, various attempts have been made to manufacture shielding membranes using biodegradable polymers. The use of biodegradable barriers has been reported to eliminate the need for reoperations to remove the barriers and to show no significant difference in tissue regeneration compared to shields made of non-degradable materials.

Korean Patent Laid-Open No. 2003-2224 discloses a membrane for inducing tissue regeneration in which a porous biodegradable polymer membrane in which micropores are formed between chitosan nonwoven fabrics and a method of manufacturing the same are disclosed.

Korean Patent Laid-Open No. 2005-48360 discloses one or two natural polymers selected from the group consisting of chitosan, collagen and alginic acid, homopolymers of lactic acid, copolymers of lactic acid and glycoic acid, homopolymers of glycoic acid and these A shielding membrane for tissue regeneration in which a nanofiber made from a mixture of one synthetic polymer selected from the group consisting of a nonwoven fabric is formed.

However, it is made of a membrane made of only a polymer as described above has a disadvantage that is not effective in regenerating bone tissue. In addition, even when a shielding membrane manufactured using a biodegradable material is not clinically applied, it does not have sufficient strength to maintain a certain shape, and does not secure a space for tissue growth, thereby causing secondary inflammation caused by the material. Another problem occurred.

In order to increase the affinity with bone tissue, electrospinning method was used to prepare a scaffold with nanostructures similar to bone tissue [Biological efficacy of silk fibroin nanofiber membranes for GBR, KH Kim et al . Journal of Biotechnology 120 (2005) 327-339)

In addition, Korean Patent No. 10-762928 shows that the bone fibroin solution obtained by removing sericin from the silk fiber is rapidly frozen after dialysis and dried, and the bone obtained by dissolving the dried silk fibroin in an electrospinning solvent and then electrospinning. A membrane for bone tissue-induced regeneration of blast cells was proposed.

Korean Patent Publication No. 2003-0022425 discloses an organic-inorganic complex by reacting a biodegradable polymer and a coupling agent at a predetermined ratio to react a ceramic precursor with a coupled biodegradable polymer, and the complex rapidly forms bones after implantation. It is said to be able to form chemical bonds and to effectively suppress bone resorption.

Republic of Korea Patent No. 10-0650453 proposes a composite material for the bone replacement, while using the cyanoacrylate as the material to give the adhesive force and shape retention power, using bone conductive inorganic material as an inorganic material to secure new bone conduction ability It refers to bone substitutes that blend these compositions to make it possible.

US Patent Publication No. 2006-88569 discloses a method for inducing regeneration of mesenchymal precursor tissue by immobilizing mesenchymal precursor cells and extracellular matrix on the surface of a porous sheet of bioabsorbable polymer containing medium. It is mentioned.

U.S. Patent Publication No. 2005-226904 proposes a fiber composite for tissue engineering, suggesting that the composite can be prepared in a fiber form including oxides and biodegradable polymers such as SiO ₂ and TiO ₂ having nano-level pores. have.

US Pat. No. 6,863,694 relates to osteogenic bone implant sheets, which disclose that bone-derived particles can be used to produce bone implants having a pore volume of up to 32%.

However, even if the above-mentioned materials are used, it is difficult to sufficiently secure the physical strength and effectively induce bone tissue, and in terms of the support structure, the production method is complicated and the porosity is difficult to control.

An object of the present invention for solving the above problems has a certain strength, biocompatibility and biodegradability, easy to control the pore size of micropores, induction of bone tissue in the form of organic / inorganic mixed non-woven fabric that can be manufactured by a simple manufacturing process To provide a shielding film for regeneration and a method of manufacturing the same.

In order to achieve the above object, the present invention

Provided is a bone membrane-induced regeneration membrane comprising a nanofiber comprising a biodegradable polymer and a biocompatible inorganic material, and including a porous structure in the form of a nonwoven fabric.

Also,

S1) mixing a solution containing a biodegradable polymer and a biocompatible inorganic material;

S2) inserting the obtained mixture into a spinning needle (needle) and then performing a spinning process; And

S3) injecting the nanofibers obtained after spinning into a mold and freeze drying;

It provides a method for producing a bone tissue-induced regeneration shielding membrane comprising a.

Bone tissue-induced regeneration shielding membrane according to the present invention is a bone-induced material is mixed with biodegradable polymer and biocompatible inorganic material and spun into nanofibers by electrospinning method, it is produced as a shielding membrane having a porosity to maximize the surface area by volume in three dimensions Nanostructured support.

The shielding membrane has a certain strength, biocompatibility and biodegradability, and excellent bone regeneration effect can be used suitably in the field of bone tissue engineering.

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

Bone tissue-induced regeneration shielding membrane according to the present invention comprises a biodegradable polymer and a biocompatible inorganic material.

Bone tissue-induced regeneration shielding membrane including the above composition is formed of a biodegradable polymer and a biocompatible polymer into a nanofiber state to have a nonwoven fabric having a porous structure.

The shielding film can control the size of the fiber from the nanostructure to the micro, and preferably has a form in which the nanofibers having a nano level diameter are formed into a nonwoven fabric.

The shielding membrane is easy to use without special training because it maintains the mechanical strength that is easy for the procedure, and the high porosity made of three-dimensionally has high air permeability and excellent properties to supply nutrients when the material is inserted into the human body. .

By controlling the structure of the shielding membrane, the interaction with tissues and osteogenic cells can be controlled to effectively promote cell adhesion, migration, differentiation, and growth.

The biodegradable polymer is not limited in the present invention, but important considerations in the selection of the biodegradable polymer material that can be used in the present invention is that adhesion and proliferation of tissue cells should be good, and the function of differentiated cells It should be preserved, and after being implanted into the body, it should be well compatible with the surrounding tissues so that there is no inflammatory response, and after a certain period of time, it should not decompose itself and remain as a foreign substance.

Preferably, the biodegradable polymer may be a natural polymer and a synthetic polymer as known in the art. The natural polymer may be one selected from the group consisting of fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, and combinations thereof, and the synthetic polymer may include polylactic acid and polyglycolic acid (poly (glycolic acid). ), PGA), poly (lactic-co-glycolic acid) (PLGA), poly-ε- (caprolactone), polyanhydride, polyorthoester, polyvinyl alcohol, 1 selected from the group consisting of polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) copolymer, copolymers and combinations thereof Paper is possible. In this case, the natural polymer and the synthetic polymer may be mixed and used to have an appropriate strength and biodegradability for using the bone tissue-induced regeneration shielding membrane, and the mixed use degree may be selected by a person skilled in the art. More preferably, polylactide, polyglycolide, polycaprolactone and copolymers thereof are used.

The biocompatible inorganic material can be any inorganic material (or ceramic) known to be biocompatible. In the present invention, in the selection of an in vitro matrix component and / or an inorganic material added to improve mechanical strength and bone tissue regeneration effect, the adhesion and stability with surrounding bones or tissues when implanted in the human body are preserved. After implantation in the body, it should be well compatible with the surrounding tissues so that there is no inflammatory response.

Preferably, the biocompatible inorganic material is selected from the group consisting of hydroxyapatite, fluorinated apatite, oxy apatite, tricalcium phosphate, monocalcium phosphate, tetracalcium phosphate, calcium metaphosphate, demineralized animal bone, and mixtures thereof. One kind is possible. More preferably, one selected from the group consisting of hydroxyapatite, tricalcium phosphate, and mixtures thereof is possible.

In this case, the biodegradable polymer and the biocompatible inorganic material are adjusted in consideration of the physical properties of the nanofibers constituting the shielding membrane, the size of the pores, the thickness of the shielding membrane, the in vitro matrix component and / or mechanical strength, and the effect of bone tissue regeneration.

Preferably, in the bone tissue-induced regeneration membrane according to the present invention, the biodegradable polymer and the biocompatible inorganic material are mixed in a weight ratio of 1: 9 to 9: 1, more preferably in a weight ratio of 3: 7 to 7: 3. If the content of the biodegradable polymer is excessive, the effect of using the biocompatible inorganic material is not obtained, the bone tissue regeneration effect is not sufficient. On the contrary, when the content of the biocompatible inorganic material is excessive, it is impossible to form a shielding film made of nanofibers, and thus it is impossible to apply the shielding film for bone tissue induction.

In addition, the bone tissue-induced regeneration membrane further includes various additives such as hormones, drugs, growth factors, regeneration enzymes, and the like, if necessary.

As described above, the membrane for bone tissue induction regeneration

S1) mixing a solution containing a biodegradable polymer and a biocompatible inorganic material;

S2) inserting the obtained mixture into a spinning needle (needle) and then performing a spinning process; And

S3) The nanofibers obtained after the spinning are injected into a mold, and then manufactured through freeze drying.

First, in step S1), a solution containing a biodegradable polymer and a biocompatible inorganic material are mixed.

The solution containing the biodegradable polymer is prepared by mixing the biodegradable polymer with a solvent to perform spinning.

The solvent is not particularly limited in the present invention, and any solvent can be used as long as it can dissolve the biodegradable 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 are possible.

The concentration of the solution containing the biodegradable polymer is used in a suitable range for performing electrospinning, preferably in a concentration of 1 to 20% by weight. At this time, it is possible to control the thickness of the nanofibers by adjusting the concentration of the solution, it is used within the above range to have a bone tissue regeneration effect.

In this case, the mixing ratio of the solution containing the biodegradable polymer and the biocompatible inorganic material is appropriately selected in consideration of the content of the biodegradable polymer and the biocompatible inorganic material in the final membrane.

Next, in step S2) the mixture obtained in the step is put into a spinning needle (needle) and then the spinning process is performed.

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. Do this.

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 10 to 20 kV, and preferably 18 to 24 gauge needles are used.

At this time, the needle is periodically shaken during electrospinning so that the biodegradable polymer and the biocompatible inorganic material in the nanofibers are uniformly mixed.

Next, in step S3), the nanofibers obtained after the spinning are injected into a mold, and then freeze-dried to prepare a nonwoven fabric-type bone tissue induction regeneration shielding membrane.

The mold is not particularly limited in the present invention, any material may be used as long as it is a material for producing a suitable structure used in the field of tissue engineering for regenerating bone tissue, and the structure form is also appropriately in three-dimensional form according to the bone tissue to be reproduced. Manufacturing is possible.

The nanofibers obtained in the previous step are transformed into a suitable form applicable to regenerating bone tissue by injecting into a mold, which is immersed in distilled water to remove the solvent and remaining impurities such as various salts by dialysis.

Next, freeze-drying at -100 to 0 ° C to produce a bone tissue-induced regeneration shielding membrane. In case of high temperature drying, shrinkage of the biopolymer may occur. In the present invention, freeze drying is performed to maintain the shape of the nanofibers present in the mold, thereby preserving its properties.

Bone tissue-induced regeneration shielding membrane according to the present invention prepared through such a step, as described above, the biopolymer and the biocompatible inorganic material is mixed to form a nanofiber, the nanofiber has a non-woven fabric having a porosity.

Bone tissue-induced regeneration shielding membrane according to the present invention can control the thickness of the membrane and the size of the pores of the porous structure by controlling the thickness of the nanofibers and the degree of accumulation of nanofibers at the time of forming the nonwoven fabric. Preferably, the diameter of the nanofibers is 0.1-0.3 μm, the pore size is 4-10 μm, and the thickness of the shielding film is 0.2-0.3 μm.

In addition, the bone tissue-induced regeneration shielding membrane has excellent tensile strength of 4 to 5 MPa, elongation of 60 to 110%, and Young's modulus of 25 to 40 MPa.

The bone tissue-induced regeneration shielding membrane according to the present invention can be used for regeneration of bone tissues of various damaged bones, in other words, osteoblasts, osteoclasts, and proliferation of osteocytes.

Specifically, bone tissue is regenerated by implanting the damaged portion in the shielding membrane.

In particular, it can be used to basically promote the formation of periodontal bone derived from periodontal disease, it can bring about an osteoinductive effect so that the periodontal bone is formed into the space formed in the portion and periodontal bone implanted in the shielding membrane. Furthermore, it may serve to effectively block the formation of externally formed inflammation and fibrous tissue.

Furthermore, the bone tissue-induced regeneration shielding membrane controls the diameter, pore size, porosity, and thickness of nanofibers to control interaction with various cells for bone tissue regeneration, thereby effectively controlling cell attachment, migration, differentiation, and growth. I can promote it.

This bone-induced regenerative shield maintains the mechanical strength that is easy for the procedure for bone tissue regeneration, and can be easily used without special training. The high porosity in three dimensions provides air permeability and nutrients when materials are inserted into the human body. It may have excellent properties.

According to Experimental Example 6 of the present invention, stem cells collected from human jaw bone were attached to the shielding membrane and grown in culture for 3 weeks. As a result, the stem cells are well attached to the shielding membrane, and the number of the stem cells was increased.

In addition, the measurement of calcium content confirmed that the mineral process essential for bone regeneration was promoted.

According to another preferred Experimental Example 7 of the present invention, after implanting the shielding membrane in the skull of the rat through in vitro experiments, after measuring the degree of bone regeneration after 3 months, showed excellent regeneration rate of about 90% or more, Fusion with bone confirmed that new bone continued over most of the damage zone.

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

≪ Example 1 >

After mixing 0.5 g of PLA solution in which polylactide (PLA) was dissolved in ethanol at a concentration of 5% by weight and 0.025 g of demineralized bone powder (DBP, demineralized bone powder, particle size: 0.01 to 0.05 μm), It was injected into a 23G stainless steel syringe and spun.

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 15 kV, causing the solution to be sprayed onto the collector running for 24 hours.

The nanofibers sprayed on the collector were immersed in distilled water at 25 ° C. for 2 minutes to remove the remaining chemicals, and then lyophilized at 0 ° C. to produce a bone tissue-induced regeneration shielding membrane.

<Example 2>

A bone tissue-induced regeneration shield was prepared in the same manner as in Example 1, except that 0.1 g of DBP was used as the inorganic material.

Comparative Example 1

In the same manner as in Example 1, using the PLA alone without using an inorganic material to prepare a bone tissue-induced regeneration shielding membrane.

Experimental Example 1 Scanning Electron Microscope and Porosity Analysis

In order to determine the surface state of the porous shielding membranes prepared in Examples 1 and 2 and Comparative Example 1 were analyzed by scanning electron microscopy (Scanning electron microscopy), the obtained results are shown in Figure 1 below.

Figure 1 (a) is a photograph of the porous shielding membrane prepared in Examples 1, 2 and Comparative Example 1, (b) is a scanning electron micrograph of them, (c) is an enlarged photograph of (b).

Referring to Figure 1, compared to the case of Comparative Example 1 using PLA alone, the porous shield membrane of Examples 1 and 2 prepared according to the present invention vary in thickness in the outer and inner cross-section of the support, In addition to being very uniform in shape, it can be seen that the shielding film is made of nanofibers having excellent porosity.

In addition, the pore size of the support was analyzed by using a porosity analyzer (Porosimetry analyzer) of the porous shielding membrane prepared in Examples 1, 2 and Comparative Example 1. As a result, the pore size of the porous structure was found to be 4 ~ 10 ㎛, it can be seen that the pores are formed very uniform over the entire shielding film.

And as shown in the portion indicated by the arrow of Figure 1 (c), in the case of the shielding film of Example 2 it can be seen that the DBP protrudes on the surface of the nanofiber. Since the DBP is friendly to bone cells in bone, it can be seen that the rate of progression can be faster when the shielding membrane is used for regeneration of bone tissue.

Experimental Example 2 Surface Condition Analysis by AFM

The surface morphology of the bone tissue-induced regeneration shielding membranes prepared in Example 1 and Comparative Example 1 was measured by an atomic force microscope.

Figure 2 (a) is a view showing the atomic force microscopic analysis of the surface form of the shielding film prepared in Comparative Example 1, (b) is an atomic force microscopic analysis of the surface shape of the shielding film prepared in Example 1 Figure showing.

It can be seen that the shielding film of Comparative Example 1 shown in (a) of FIG. 2 has a very smooth surface by using PLA alone. In contrast, it can be seen that the shielding film of Example 1 of (b) has a very rough surface, and this roughness is due to the inorganic material contained in the shielding film.

Experimental Example 3 Measurement of Physical Properties

Physical properties of the bone tissue-induced regeneration shielding membranes prepared in Examples 1 and 2 and Comparative Example 1 were measured and shown in FIGS. 3 to 5.

Figure 3 is a graph showing the tensile strength (tensile strength) of the bone tissue-induced regeneration shielding membrane prepared in Examples 1 and 2 and Comparative Example 1, Figure 4 is a nanofiber support of Examples 1 and 1 and Comparative Example 1 Elongation, FIG. 5 is a graph showing the young's modulus of the nanofiber supports prepared in Examples 1 and 2 and Comparative Example 1. FIG.

Referring to FIG. 3, in the case of the shielding film of Example 1, tensile strength was slightly reduced (not significant) as compared with the shielding film of Comparative Example 1 using PLA alone (Example) as the content of DBP, which is an inorganic material, increased (Example 2) It was confirmed that the tensile strength was increased.

Referring to FIG. 4, the elongation was increased as the content of PLA in the shielding film increased, and it decreased as the content of DBP, which is an inorganic material, increased (Example 2).

Referring to FIG. 5, in the case of Young's modulus, Examples 1 and 2 are much superior to Comparative Example 1, and the elasticity is improved as compared with the case of using PLA alone.

Experimental Example 4 TGA Analysis

Thermal gravimetric analysis of the shielding membranes prepared in Examples 1 and 2 and Comparative Example 1 and DBP, which is an inorganic material, were performed, and the obtained results are shown in FIG. 6. In this case, DBP alone was also measured for comparison.

6 is a thermogravimetric analysis spectrum of the shielding membrane and inorganic DBP prepared in Examples 1,2 and Comparative Example 1.

Referring to FIG. 6, both of the shielding films of Comparative Examples 1 and 1 began to exhibit pyrolysis at 300 ° C. or higher and completely decomposed at 400 ° C. FIG. However, in Examples 1 and 2, 4 to 13% of the remaining inorganic components were found even at 400 ° C. or more due to the inorganic materials present in the shielding film.

Experimental Example 5 ATR-FTIR Analysis

The functional groups of the bone tissue-induced regeneration shielding membrane and DBP powder prepared in Example 2 and Comparative Example 1 were confirmed by Attenuated total reflectance-Fourier transform infreared (ATR-FTIR) and Raman analysis.

7 is an ATR-FTIR spectrum of a shielding film prepared in Example 2 and Comparative Example 1 and DBP alone.

7, was born the receive characteristics such as the peak of 1237 cm ^-1, 1030 cm ^-1, and 604cm ^-1 in the case of phosphate DBP, this peak appeared in the spectrum of Example 2; In addition, C = O (1759 cm ^-1 ), which is a characteristic peak of PLA, can also be confirmed in the spectrum of Example 2.

These results show that the shielding film of Example 2 coexists with PLA-DBP.

Experimental Example 6 Bone Tissue Induction Regeneration Effect Characteristics

In order to determine the regeneration of bone tissue for the shielding membrane prepared in Example 2 and Comparative Example 1 was performed as follows.

Cell growth

Stem cells harvested from human jaw bone were cultured and then attached to the respective shielding membranes prepared in Example 2 and Comparative Example 1 at 2 × 10 ⁴ cells / cm ² . Subsequently, the cells were grown in osteogenic cultures for 3 weeks, and then the degree of cell growth was confirmed by DNA measurement. The results obtained are shown in FIG. 8.

8 is a graph measuring the DNA content of the stem cells attached to the shielding membrane of Example 2 and Comparative Example 1.

Referring to FIG. 8, it can be seen that the shielding membranes of Example 2 and Comparative Example 1 are well adhered to cells for 3 weeks, and their number increases with time. These results indicate that both the shielding membranes of Example 2 and Comparative Example 1 had no harm to the cells.

ALP activity

In order to determine the degree of differentiation of the stem cell cultured stem cells in the above three weeks, bone differentiation marker ALP (alkaline phosphatase) activity was measured, and the results are shown in FIG. 9.

9 is a graph measuring the ALP activity of the stem cells attached to the shielding membrane of Example 2 and Comparative Example 1.

Referring to FIG. 9, the activity of both Example 2 and Comparative Example 1 was increased, and after 3 weeks, at the end of differentiation, the value tended to decrease, but in general, Example 2 and Comparative Example 1 showed similar trends. Seemed.

Calcium content

In order to infer the degree of bone regeneration during the three weeks of the membrane cultured stem cells, the calcium content was measured and shown in FIG. 10.

 10 is a graph measuring the content of calcium present in the shielding film of Example 2 and Comparative Example 1.

Referring to FIG. 10, in the second week, calcium in the shielding film of Example 2 was detected at least about twice as much as the calcium content in the shielding film of Comparative Example 1, and the calcium content of the shielding film of Example 2 was excellent even in the third week. It was. These results indicate that DBP, a biosynthetic mineral contained in the shield, promotes the mineral processes essential for bone regeneration.

Mineralization

After stem cell attachment, the surface of the shielding membranes of Example 2 and Comparative Example 1 was measured by scanning electron microscopy, and the results are shown in FIG. 11.

Figure 11 (a) is a scanning electron micrograph immediately after and 3 weeks after the stem cell adhesion of the shielding membrane of Comparative Example 1, (b) is a scanning electron micrograph immediately after and 3 weeks after the stem cell adhesion of the shielding membrane of Example 2 .

Referring to FIG. 11, it can be seen that the cells and the extracellular matrix are more widely covered on the surface of the shielding membrane of Example 2 according to the present invention than the shielding membrane of Comparative Example 1 and more minerals are deposited. .

< Experimental Example  7> In vivo Rat  exam

In In Experiment 6 above After confirming the enhanced mineral formation in vitro , we examined the extent of bone regeneration through an in vivo rat model.

CT scan

First, bones of 12-week-old Sprag-Dauri rats (SD rats) showed 8 mm defects, and then the sutures of the shielding membranes prepared in Example 1 and Comparative Example 1 were implanted and sutured. The degree of regeneration was measured using a micro CT scan and the results obtained are shown in FIG. 12.

12 is a CT photograph showing the degree of regeneration of bone implanted in the shielding membrane prepared in Example 2 and Comparative Example 1, about 2 months after transplantation in the case of the shielding membrane of Comparative Example 1 in the implanted group about the end of the damaged area The recovery rate was 15%, and the shielding film of Example 2 was 55%.

In addition, in the third month, the shielding film of Comparative Example 1 showed a regeneration rate of about 38%. However, the shielding film of Example 2 according to the present invention showed an excellent regeneration rate of about 90% or more. It can be seen that the use of a mixed biocompatible inorganic material with Mars can enhance the regeneration effect.

Tissue staining

Three months after the implantation of the shielding membrane, bone tissue was stained for regeneration.

13 is a staining picture of bone tissue using the shielding film prepared in Comparative Example 1, Figure 14 is a staining picture of bone tissue using the shielding film prepared in Example 2.

Referring to FIG. 13, in the mice transplanted with the shielding membrane using PLA alone, some bones were formed only at the ends of the damaged area, and the inside of the mice was hardly regenerated.

In contrast, looking at the photo of Figure 14 using the barrier film of Example 2, it can be seen that in the case of a shield film properly mixed PLA and DBP is fused with the existing bone, new bone is continued over most of the damage site.

Bone tissue-induced regeneration shielding membrane according to the present invention is applied to the tissue regeneration of various damaged bones, it can be used in the field of tissue engineering.

Figure 1 (a) is a photograph of the porous shielding membrane prepared in Examples 1, 2 and Comparative Example 1, (b) is a scanning electron micrograph of them, (c) is an enlarged photograph of (b).

Figure 2 (a) is a view showing the atomic force microscopic analysis of the surface form of the shielding film prepared in Comparative Example 1, (b) is an atomic force microscopic analysis of the surface shape of the shielding film prepared in Example 1 Figure showing.

3 is a graph showing the tensile strength (tensile strength) of the bone tissue-induced regeneration shielding membrane prepared in Examples 1 and 2 and Comparative Example 1.

Figure 4 is a graph showing the elongation (elongation) of the nanofiber support prepared in Examples 1,2 and Comparative Example 1.

FIG. 5 is a graph showing young's modulus of the nanofiber supports prepared in Examples 1 and 2 and Comparative Example 1. FIG.

6 is a thermogravimetric analysis spectrum of the shielding membrane and inorganic DBP prepared in Examples 1,2 and Comparative Example 1.

7 is an ATR-FTIR spectrum of a shielding film prepared in Example 2 and Comparative Example 1 and DBP alone.

8 is a graph measuring the DNA content of the stem cells attached to the shielding membrane of Example 2 and Comparative Example 1.

9 is a graph measuring the ALP activity of the stem cells attached to the shielding membrane of Example 2 and Comparative Example 1.

10 is a graph measuring the content of calcium present in the shielding film of Example 2 and Comparative Example 1.

Figure 11 (a) is a scanning electron micrograph immediately after and 3 weeks after the stem cell adhesion of the shielding membrane of Comparative Example 1, (b) is a scanning electron micrograph immediately after and 3 weeks after the stem cell adhesion of the shielding membrane of Example 2 .

12 is a CT photograph showing the degree of regeneration of bone implanted in the shielding membrane prepared in Example 2 and Comparative Example 1.

13 is a staining picture of bone tissue using the shielding membrane prepared in Comparative Example 1.

14 is a staining picture of bone tissue using the shielding membrane prepared in Example 2.

Claims (14)

Polylactic acid, poly (glycolic acid), PGA, poly (lactic-co-glycolic acid), PLGA, poly-ε- (caprolactone), One biodegradable polymer selected from the group consisting of copolymers and combinations; And 1 type selected from the group consisting of hydroxyapatite, fluorinated apatite, oxy apatite, tricalcium phosphate, monocalcium phosphate, tetracalcium phosphate, calcium metaphosphate, demineralized animal bone having a particle size of 0.01 to 0.05 μm, and mixtures thereof Contains biocompatible minerals of The biodegradable polymer and the biocompatible inorganic material comprises a nanofiber including a weight ratio of 1: 1 to 1: 4, including a porous structure in the form of a nonwoven fabric and satisfying the following physical properties: Diameter of Nanofibers: 0.1-0.3 μm Pore size: 4-10㎛ Thickness: 0.2 ~ 0.3㎛ Tensile Strength: 4 ~ 5 MPa Elongation: 60 ~ 110% Young's modulus: 25-40 MPa delete delete delete delete delete delete delete The method of claim 1, In addition, the bone tissue induction regeneration shielding membrane is bone tissue induction regeneration shielding membrane comprising an additive. 10. The method of claim 9, The additive is a bone tissue-induced regeneration barrier membrane is selected from the group consisting of hormones, drugs, growth factors, regeneration enzymes and mixtures thereof. S1) polylactic acid, polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA), poly-ε- (caprolactone), One biodegradable polymer selected from the group consisting of copolymers and combinations thereof was dissolved at a concentration of 1 to 20% by weight in one solvent selected from the group consisting of ethanol, trifluoroethanol, or a mixed solvent thereof. Prepare a solution, Particle size of 0.01 ~ 0.05 ㎛, 1 selected from the group consisting of hydroxyapatite, fluorinated apatite, oxy apatite, tricalcium phosphate, monocalcium phosphate, tetracalcium phosphate, calcium meta phosphate, demineralized animal bone, and mixtures thereof Mixing the biocompatible minerals of the species, wherein the biodegradable polymer and the biocompatible minerals are mixed in a content ratio of 1: 1 to 1: 4; S2) putting the obtained mixture into a spinning needle having a needle inner diameter of 0.3 to 0.7 mm, and then performing electrospinning by operating a voltage between the needle and the collector at 10 to 20 kV; And S3) a step of injecting the nanofibers obtained after spinning into a mold and freeze drying; Diameter of Nanofibers: 0.1-0.3 μm Pore size: 4-10㎛ Thickness: 0.2 ~ 0.3㎛ Tensile Strength: 4 ~ 5 MPa Elongation: 60 ~ 110% Young's modulus: 25-40 MPa The method of claim 11, The solution containing the biodegradable polymer is water as a solvent; Lower alcohols of methanol, ethanol, propanol, and butanol; Acetone; Trifluoroethanol; Tetrahydrofuran; Dichloromethane; And a mixture selected from the group consisting of mixed solvents thereof. The method of claim 11, The solution containing the biodegradable polymer is a method for producing a bone tissue-induced regeneration shielding membrane to be used in a concentration of 1 to 20% by weight. The method of claim 11, The spinning is 0.3 ~ 0.7 mm inner diameter of the needle, the method of producing a bone tissue-induced regeneration shielding membrane to perform electrospinning by operating the voltage between the needle and the collector 10 to 20 kV.

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