KR20110040388A - Silk/hydroxyapatite hybrid nanofiber scaffold for bone regeneration - Google Patents

Abstract

PURPOSE: A silk/hydroxyapatite complex nanofiber supporter for osteanagenesis is provided to promote a cell growth promotion effect and to remove cytotoxin. CONSTITUTION: A silk/hydroxyapatite complex nanofiber supporter for osteanagenesis constituted as follows. In the nanofiber supporter, hydroxyapatite is coated on the surface thereof and silk nanofiber having the diameter of tens or hundreds nanometers is three-dimensionally arranged. The silk nanofiber maintains the form of a strand of fiber without conglomeration or entanglement thereof.

Description

Silk / hydroxyapatite hybrid nanofiber scaffold for bone regeneration}

The present invention relates to a silk / hydroxyapatite composite nanofiber support for bone regeneration.

Bone grafts are most common after blood transfusions, and are being performed in various areas to treat bone defects that are commonly seen in orthopedic surgery. According to reports, more than 500,000 bone grafts are performed annually in the United States, and around 2.2 million are performed annually worldwide. Bone grafts play a role in stimulating bone growth in areas where bone is missing due to pathological or physiological causes.

The action of bone grafts can be classified into osteoconduction, osteoinduction, and osteoogenesis depending on the mechanism. Bone conduction refers to the formation of bone by migrating osteoblasts from surrounding bone tissue to the bone graft site and mineral deposition, which only occurs when there is bone tissue or differentiated mesenchymal cells around. If the bone conduction material is implanted in other sites, such as subcutaneous tissue, it will not initiate bone growth. When implanted into bone or soft tissue, the bone conduction material is absorbed and replaced with bone tissue through a process similar to the creeping substitution process. Osteoinduction is a process in which bone graft material induces undifferentiated mesenchymal cells and differentiates into bone precursor cells to form new bone tissue. Bone formation is also possible when transplanted into other tissues, such as subcutaneous tissue. Osteoinductive grafts also have a great impact on the process of remodeling. Osteoblasts refer to the generation of bone tissue by living cells in the graft and autologous bone is the only bone forming material.

Materials used for bone grafts include autologous bone, allogeneic bone, and xenograft. Recently, various artificial bone substitutes have been researched, developed, and used. While bone grafts should ideally meet the requirements of inducing bone formation, biocompatibility with the host, ease of collection and manipulation, and cost reduction, the bone graft materials currently in use have some limitations. have.

Autologous bone graft is the best material that has all three characteristics of bone formation, bone induction and bone conduction for bone union and shows better stability and transplantation rate than all other bone graft materials. However, there are drawbacks that can result in other defects and infections in the donor involved in collecting grafts, require additional surgery time, and often fail to obtain sufficient bone. In order to overcome this drawback, extensive research has been conducted to find a bone graft material to replace it.

Currently, the most commonly used allogeneic bone in place of autologous bone is tissue obtained from the same species as the host, and is provided in various forms such as particles, gels, putties, and the like by freezing, lyophilization, and decalcification drying. Allogeneic bone has a relatively large amount and has the advantage of controlling the shape or bone density by using a specific part of the skeleton or by processing. Allogeneic bone as an implant has excellent bone conduction, but bone cells do not survive, resulting in no bone formation and extremely limited bone induction. In addition, the supply is limited, there are restrictions such as ethical problems, there is a disadvantage that there is a risk of transmission or infection of contaminants, toxins and the like. In addition, allogeneic bones, despite the thorough investigation of donors and various tests, may have the possibility of transmission of infectious diseases caused by viruses, and various processing treatments on allogeneic bones may alter the original biological and epidemiological properties in order to reduce this risk. And there is a problem that can weaken the mechanical strength or adversely affect bone conductivity and bone induction.

Xenograft transplantation is limited to the processing of animal bones such as cows or pigs and implanted into the human body, but there is a problem such as the immune response and transmission of infectious diseases, the use is gradually decreasing.

Recently, due to the problems related to autologous bone, allogeneic bone, and xenograft, there is increasing interest in artificial bone substitutes that can provide biocompatibility and safety for bone graft, and many studies have been actively conducted.

Artificial bone substitutes are required to meet the following requirements. First, it should not cause infection and no or no antigen response, second, it should be easy to handle without shattering, and third, have good hygroscopicity in vivo, and fourth, sufficient mechanical strength to withstand the pressure of surrounding tissue after surgery. Fifth, to maintain a constant volume until bone tissue is sufficiently formed and mature in the support, sixth, to have a proper surface roughness to adhere well to the existing tissue, seventh, to adhere to the growth and differentiation of cells The nutrients or excreta must be porous enough to diffuse well. Eighth, additional products by decomposition of artificial bone substitutes should be biocompatible.

Currently, calcium phosphate-based compounds are in the spotlight as artificial bone materials. The most commonly used hydroxyapatite has the same structure as the main component of bones and teeth of the human body, and is used in the medical field by coating it on powder form, dense body or metal, and has excellent biocompatibility and biocompatibility. Since the bone conduction of the surrounding bone causes the development of artificial bone material, research and clinical applications are actively being conducted. However, in the case of the hydroxyapatite powder preparation, since the strength is low and it is difficult to use it as it is, research on hybrid materials with organic materials has been made to solve this problem.

Organic materials to hybridize with hydroxyapatite include biodegradable synthetic polymers such as polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), collagen, which is an organic component of bone, etc. Is used. However, biodegradable synthetic polymers such as polyglycolic acid, polylactic acid-glycolic acid copolymers, and polylactic acid have excellent structural stability and tensile strength, but are caused by the fact that the biodegradation period is longer than 2 years and the decomposition products are acidic substances. There are problems such as inflammatory reactions. In addition, since collagen has to be extracted from animals, it is difficult to manufacture and store, and it is not suitable for mass production, and thus the manufacturing cost is high, so it is limited for clinical use, the dissolution rate in the body is too fast, immune response and infectious disease It may cause, there is a problem that the tension is weak in vivo.

On the other hand, as the hydroxyapatite coating method, the plasma spraying method is most used, which is a method of melting the hydroxyapatite powder in the high temperature plasma region of 20,000 to 30,000 degrees and then fusion to the metal target. The coating layer coated by the plasma spraying method has a higher adhesion strength than the coating layer coated by chemical vapor deposition, sputtering, etc., but there is still a problem in that the destruction of the coating layer occurs on the metal surface and the surface of the coating layer. Recently, metal implants are deposited in a solution containing calcium and phosphate ions to precipitate hydroxyapatite on the metal surface, or the surface is modified and deposited in a simulated body fluid (SBF) to hydroxyapatite on the surface. A method of forming a layer and a method of coating apatite fine particles using electrophoresis have been developed, but when coating with hydroxyapatite using a human-like solution, deposition takes a long time. Although there are examples of coating hydroxyapatite on fibers and the like, examples of application to silk nanofibers have not been reported so far, and particularly, even coatings of nanofiber surfaces are not reported.

The inventors of the present invention, while studying to develop a bone graft substitute to replace the existing artificial bone substitute, hydroxyapatite is coated on the surface, silk nanofibers having a diameter of tens to hundreds of nanometers in three dimensions Sponge-formed silk / hydroxyapatite composite nanofiber support is not only biocompatible but does not show cytotoxicity, does not inhibit cell proliferation, promotes cell proliferation, and shows excellent bone regeneration. The present invention was found to be very useful as a material for bone regeneration in place of the existing artificial bone substitute, and completed the present invention.

An object of the present invention to provide a silk / hydroxyapatite composite nanofiber support for bone regeneration.

In order to achieve the above object, the present invention is coated with hydroxyapatite on the surface, silk nanofibers for bone regeneration in the form of sponges nano-fibers having a diameter of several tens to hundreds of nanometers three-dimensionally arranged It provides a nanofiber support.

The silk / hydroxyapatite composite nanofiber support according to the present invention is a sponge in which hydroxyapatite is coated and silk nanofibers having a diameter of several tens to hundreds of nanometers are three-dimensionally arranged. It does not show toxicity, does not inhibit cell proliferation, has an excellent effect on promoting cell proliferation, and exhibits excellent bone regeneration effect, and thus may be very useful as a material for bone regeneration in place of an existing artificial bone substitute.

The present invention provides a silk / hydroxyapatite composite nanofiber support for bone regeneration.

The silk / hydroxyapatite composite nanofiber support according to the present invention is coated with hydroxyapatite on its surface, and is provided in a sponge form in which three-dimensionally arranged silk nanofibers having a diameter of several tens to several hundred nanometers are provided. The natural bone extracellular matrix consists of 25% water, 30% collagen protein, and 45% minerals. Natural bone extracellular matrix consists of hydroxyapatite on the surface of collagen type I protein consisting of nanofibers, and calcium phosphate is attached along the nanofiber surface of collagen protein. The silk / hydroxyapatite composite nanofiber support of the present invention mimics the structure of this natural bone extracellular matrix.

In addition, the individual silk nanofibers constituting the sponge-like support according to the present invention are provided to maintain the shape of the fiber strands without agglomeration or entanglement with each other. In this case, when the hydroxyapatite is coated too much, the silk / hydroxyapatite composite nanofiber support loses the shape of the nanofibers and becomes planar, and thus cannot be used as the nanofiber support.

Furthermore, the silk / hydroxyapatite composite nanofiber support according to the present invention preferably has a pore size between 50 and 1000 μm between nanofibers. When the pore size is less than 50 μm, a problem may occur that cells do not easily enter the support while proliferating, and when the pore size exceeds 1000 μm, the cells do not adhere well and may escape between the pores. This can happen.

In addition, the silk / hydroxyapatite composite nanofiber support according to the present invention preferably has a porosity of 90% or more. If the porosity is less than 90%, there is a problem that the exchange of materials that supply sufficient air and nutrients and discharge waste products may not be performed well.

Silk / hydroxyapatite composite nanofiber support according to the present invention can be prepared by performing the following steps 1 to 6.

Step 1: silk  Preparing the spinning stock solution

The silk spinning stock solution comprises the steps of removing sericin from the silk fibers (step a); Washing, drying and dialysis the silk fibers from which sericin has been removed to prepare an aqueous silk fibroin solution (step b); Freeze-drying the aqueous solution of silk fibroin to prepare a silk fibroin sponge (step c); And it can be prepared through the step of dissolving the silk fibroin sponge in a predetermined solvent and filtering (step d).

The silk fiber refers to a fiber made of yarn extracted from silkworm cocoons that grow and eat mulberry, and has been used as a high quality fiber material for a long time due to its high tensile strength, inherent gloss, and excellent dyeability. In addition, silk fibers have a structure in which two strands of fibroin are wrapped in a sericin outer membrane, and since fibroin has excellent biocompatibility and does not affect any surrounding tissue, it is manufactured in various forms such as powder, aqueous gel solution, and the like. It can be used in various ways. When preparing the spinning stock solution using such silk fibers, it is preferable to remove sericin from the silk fibers in order to maximize biocompatibility. Sericin may be removed by immersing the silk fibers in a sodium oleate and sodium bicarbonate solution followed by heating.

Silk fibers from which the sericin is removed can be washed repeatedly with distilled water and dried, dissolved in a mixed solution of calcium chloride / water / ethanol, and then dialyzed in distilled water to prepare a silk fibroin aqueous solution. The dialysis membrane is preferably a dialysis membrane having a MWCO of 12 to 14 kDa.

The silk fibroin solution may be freeze-dried to prepare a silk fibroin sponge, and then dissolved in formic acid, followed by filtration to prepare a silk spinning stock solution.

The silk spinning stock solution is preferably prepared in a fiber spinning stock solution at a concentration of 10 to 18% to smoothly perform the spinning process and to obtain a uniform fiber. Increasing the concentration within the concentration range of 10 to 18% increases the thickness of the fiber. If the concentration of the spinning stock solution is less than the above range, the viscosity of the spinning stock solution is too low to form fibers, and if it exceeds the range, the spinning stock solution may have high viscosity, which makes it difficult to form fibers by electric force, thereby stably electrospinning. No problem occurs.

Step 2: Prepared in Step 1 silk  Electrospinning the spinning stock solution

Electrospinning can be performed using a predetermined radiating device while applying a predetermined voltage. The electrospinning apparatus according to the present invention comprises a radiating portion, a direct current power supply portion and a dispersion portion. The spinning unit receives the spinning stock solution and serves to spin the received spinning stock solution into the nanofibers. When spinning the spinning solution from the spinning unit, it is preferable to keep the spinning speed constant, and therefore, it is preferable to use a syringe pump suitable for keeping the spinning speed constant. The DC power supply unit serves to apply a voltage during radiation, and is connected to the radiation unit. The dispersion part serves to disperse and solidify the spun fibers, and consists of a coagulation bath filled with coagulant liquid. The coagulation bath is made of a metal container and is grounded by a predetermined wire.

The voltage applied during the electrospinning is preferably set in the range of 10 to 15 kV because it can perform a stable spinning process. When the voltage increases within the voltage range of 10 to 15 kV, the spinning speed increases.

Step 3: The radiation emitted in step 2 silk  Dispersing Nanofibers

It can be made by dispersing the silk nanofibers spun in step 2 in a coagulation bath filled with a predetermined coagulation solution. Methanol may be used as the coagulating solution, but is not necessarily limited thereto.

Step 4: dispersed in step 3 above silk  Freeze-dried nanofibers in a predetermined container silk  Steps to prepare nanofibers

In step 3, the fibers dispersed in the coagulation bath are nano-sized to have a gel-like state. Before performing freeze-drying, it is preferable to perform a process of removing the coagulation liquid present in the dispersed fibers. The coagulating solution can be removed by washing the fibers dispersed in the step 3 in distilled water. Freeze-drying is carried out in a state in which the fiber from which the coagulant has been removed is contained in a predetermined container, so that nanofibers in the form of sponges three-dimensionally dispersed in various shapes according to the shape of the container can be made.

Step 5: the three-dimensional dispersed sponge form prepared in step 4 silk  Nanofibers at a concentration of 5 to 15 times Human body  solution( simulated body fluid , SBF Immersed in) With hydroxyapatite  Complex coating step

The calcium / phosphorus (Ca / P) molar ratio of hydroxyapatite having Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ structure is 10/6, or 1.67, similar to human bone. The human bone is a structure in which the PO ₄ and OH groups are partially substituted with CO ₃ groups in the above structure, rather than pure hydroxyapatite.

Since the human-like solution is similar to the ionic composition of human plasma, when the porous biodegradable polymer support in which hydroxyapatite is formed is provided using the same, the formation of hydroxyapatite in the porous biodegradable polymer support is provided by providing more environmental conditions. Can promote. In this case, the human body similar to the solution at a concentration of 10 times to 15 times is in 1ℓ of distilled water _{^{Ca 2 + 2.5 mM, H 2}} PO 4 2 - including 1.0 mM, and Na ^+, K ^+, Mg ^{2 +,} Cl ^-, HCO ^{3 -,} SO ₄ ^{2 -,} based on the human body similar solution containing a chemical species, and ^{(1 × SBF), Ca 2} + and H ^₂ PO _{4 2} for the standard solution ^- the concentration of five times to increase 15-fold the I mean.

The three-dimensionally dispersed sponge-shaped silk nanofibers prepared in step 4 were immersed in hydroxyapatite by immersing in a solution of human-like solution having a temperature of 37 ± 0.5 ° C. and a pH range of 7.2 to 7.4 for 30 minutes to 90 minutes. Can be coated. After the reaction is terminated, the remaining hydroxyapatite, which is not coated on the silk nanofibers, may be further washed with distilled water. If the washing is not done properly, unnecessary salt may remain, so washing with water is preferably performed three or more times.

Step 6: In Step 5 above With hydroxyapatite  Composite coated silk  Forming voids in the nanofibers

The pores are immersed in the dispersion medium of the silk nanofibers coated with the hydroxyapatite complex (step a); Adding particles for forming pores to the mixed solution (step b); Preparing a silk / hydroxyapatite composite nanofiber foam including the pore-forming particles using the mixture comprising the pore-forming particles obtained in step b (step c); And forming a pore-formed silk / hydroxyapatite composite nanofiber support by removing the pore-forming particles of the nanofiber foam (step d).

Step a is a step of immersing the silk nanofiber complex coated with hydroxyapatite in a dispersion medium. The type of the dispersion medium is not particularly limited as long as it does not dissolve the pore-forming particles and does not chemically and physically change the hydroxyapatite on the silk and the surface of the silk, but dioxane, carbon tetrachloride, trichloroethane, benzene, isopropanol , Cyclohexane, or a mixed solvent thereof, and most preferably dioxane. The dioxane is an organic solvent suitable for freeze drying because it is frozen at 10 ° C. or lower and sublimation occurs at room temperature in a vacuum condition of 10 mTorr. It also does not dissolve silk fibroin and pore-forming particles, making it well suited for shaping and freeze-drying while maintaining the silk / hydroxyapatite composite nanofiber form.

The step b is a step of adding the pore-forming particles to the mixed solution, the pore-forming particles are insoluble in the dispersion medium, and are removed by the steps described below, the kind is not particularly limited, sodium chloride, potassium chloride, calcium chloride , Sucrose, aluminum chloride and the like, and most preferably sodium chloride. The pore size of the pore-forming particles is preferably 100 to 200 μm, and it is preferable to use 0.01 to 0.02 g of pore-forming particles per 2 mg of the silk nanofibers coated with hydroxyapatite.

Step c is a step of preparing a silk / hydroxyapatite composite nanofiber foam (foam) containing the pore-forming particles using the mixture containing the pore-forming particles obtained in step b. The silk / hydroxyapatite composite nanofiber foam may be prepared by freeze-drying the mixture including the pore-forming particles.

Step d is a step of forming the pores formed silk / hydroxyapatite composite nanofiber support by removing the particles for forming the pores of the nanofiber foam. The silk / hydroxyapatite composite nanofiber support is a solvent that solubilizes the pore-forming particles after vapor cross-linking the silk / hydroxyapatite composite nanofiber foam of step c using steam. It can be formed by immersing in, and performing the process of freeze drying the immersed silk / hydroxyapatite composite nanofiber foam. The use of the vapor crosslinking has the advantage that it is not necessary to wet the material to be crosslinked in the aqueous solution, so that the material vulnerable to moisture can be crosslinked. Here, the vapor is not particularly limited because it may vary depending on the nanofiber foam to be crosslinked, glutaraldehyde, carbodiimide, diphenylphosphoryl azide, ethyldimethylaminopropylcarbodiimide, toylene diisocyanate, hexamethylene It is preferable to use diisocyanate or the like. Subsequently, the vapor crosslinked nanofiber foam may be dipped into a solution in which the pore forming particles are soluble. At this time, it is preferable that the residual toxicity of the steam used in the steam crosslinking is removed. Subsequently, the immersed silk / hydroxyapatite composite nanofiber foam may be washed with a predetermined solvent and then lyophilized to prepare a nanofiber support according to the present invention.

Hereinafter, the present invention will be described in detail by way of examples, with the following examples being merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

< Example  1> silk Of Hydroxyapatite  Preparation of composite nanofiber supports 1

Step 1: silk  Preparation of Spinning Solution

The dried gamma cocoons were immersed in a bath ratio of 1:25 in 0.3% soap and 0.2% sodium bicarbonate solution and treated at 100 ° C. for 1 hour to remove sericin protein. The cocoons from which sericin was removed were repeatedly washed with distilled water and dried, and then dissolved in a solution of calcium chloride / water / ethanol in a molar ratio of 1: 8: 2 at 85 ° C. for 3 minutes in a bath ratio of 1:25. The dissolved solution was dialyzed for 3 days using a dialysis membrane to prepare an aqueous silk fibroin solution. The silk fibroin aqueous solution was freeze-dried to prepare a silk fibroin sponge, which was dissolved in 98% formic acid at a concentration of 12% and the insolubles were filtered to prepare a silk spinning stock solution.

Step 2: silk  Electrospinning of Radiant Solution

The silk spinning stock solution prepared in step 1 was subjected to an electrospinning process using the electrospinning apparatus described above. In more detail with respect to the electrospinning apparatus, a syringe pump including a 22 G injection needle was used as the spinning unit, and the distance between the spinning unit and the coagulation bath was set to about 15 cm, Electrospinning was performed with an applied voltage.

Step 3: radiated silk  Dispersion of Nanofibers

As a coagulation bath of the nanofibers prepared in step 2, methanol was used. After performing the electrospinning process, the nanofibers dispersed in the coagulation bath were placed in a dialysis membrane and dialyzed with distilled water for 2 days to remove methanol used as the coagulation bath.

Step 4: Lyophilization

Silk fibroin nanofibers from which methanol was removed in step 3 was lyophilized in an appropriate container to obtain silk nanofibers in the form of sponge.

Step 5: With hydroxyapatite  Composite coated silk  Preparation of Nanofibers

NaCl 58.430 g, KCl 0.373 g, CaCl ₂ 3.675 g, MgCl ₂ 1.016 g, NaH ₂ PO ₄ 1.420 g, NaHCO ₃ 0.840 g in 1 L distilled water in 10-fold concentration (10 × SBF solution) ) Was prepared. Here, the silk nanofibers were immersed at a rate of 50 ml of the human-like solution having a 10-fold concentration per 10 mg of the silk nanofibers obtained in step 4, and reacted at 37 ° C. for 30 minutes to coat with hydroxyapatite. After the reaction, the silk nanofibers were washed three times with distilled water and then lyophilized to obtain silk nanofibers in the form of sponges coated with hydroxyapatite.

Step 6: Preparation of Pore-Formed Silk / hydroxyapatite Composite Nanofiber Support

Silk nanofibers coated with the hydroxyapatite obtained in step 5 were coated in dioxane and left for 12 hours to be immersed sufficiently. Thereafter, sodium chloride having an average particle diameter of 100 to 200 µm was added to disperse the mixed well with the silk nanofibers coated with the hydroxyapatite, and then filled into a glass tube having an inner diameter of 6 mm, and then freeze-dried to give silk / A hydroxyapatite composite nanofiber foam was prepared. The silk / hydroxyapatite composite nanofiber foam was placed in a desiccator saturated with glutaaldehyde vapor and treated for 24 hours to allow crosslinking between silk fibroin. After crosslinking treatment, the mixture was immersed in an aqueous 0.1 M glycine solution and stirred while being exchanged at 6 hour intervals for 24 hours. Upon stirring, the desired pores were formed in the support while the sodium chloride particles melted. Then, washed three times with PBS (phosphate buffer saline) again, soaked and stirred for 24 hours, and through the process of freeze-drying to obtain a silk / hydroxyapatite composite nanofiber support having a porous structure.

< Example  2> silk Of Hydroxyapatite  Preparation of Composite Nanofiber Supports 2

Silk / hydroxyapatite composite nanofiber support having a porous structure in the same manner as in Example 1, except that the reaction was performed for 60 minutes by immersing in a human-like solution having a 10-fold concentration in Example 5 of Example 1. Obtained.

< Example  3> silk Of Hydroxyapatite  Preparation of Composite Nanofiber Supports 3

In the same manner as in Example 1, except for reacting for 90 minutes by immersing in a human-like solution of 10 times the concentration in Example 5, the silk / hydroxyapatite composite nanofiber support having a porous structure Obtained. The scanning electron microscope image of Example 3 is shown in FIG.

< Example  4> silk Of Hydroxyapatite  Preparation of Composite Nanofiber Supports 4

In the same manner as in Example 1, except for immersing in a human-like solution of 10 times concentration in step 5 of Example 1 and reacting for 120 minutes, the silk / hydroxyapatite composite nanofiber support having a porous structure Obtained.

< Comparative example  1> silk  Preparation of Nanofiber Supports

A silk nanofiber support was obtained in the same manner as in Example 1, except that Example 1 was not coated with hydroxyapatite. A scanning electron microscope image of Comparative Example 1 is shown in FIG. 1 (b).

< Experimental Example  1> Cell proliferation effect measurement

In order to determine the degree of cell proliferation of the silk / hydroxyapatite composite nanofiber support according to the present invention, the following experiment was performed.

MC3T3-E1 cells, which are osteoblastic cells of rats, were used for cell proliferation experiments, and cell culture was performed with Eagle medium (α-MEM) to which 10% bovine serum was added. The cells in monolayer culture in a cell culture flask were treated with trypsin-EDTA, and the cells were collected. 2.0 × 10 ⁴ cells were incubated for 1, 3, 5 days in an incubator at 37 ° C., 5% CO ₂ . As a control, an artificial bone substitute TCP (control 1) and the silk nanofiber support of Comparative Example 1 (control 2) were used. After incubation, the medium was removed and the support was washed twice with 1 ml of pH 7.0 phosphate buffered saline, and then 1 ml of medium without bovine serum was added, followed by MTT (3- (4,5-dimethyl-). 0.5 g of 2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) reagent (pH 7.0, 5 mg / ml in PBS) was turned off and the lights were turned off for 4 hours at 37 ° C., 5% CO ₂ The culture was carried out in a conditional incubator. After incubation, the supernatant was removed, 400 μl of DMSO was added to each culture flask, mixed well, 200 μl was transferred to a new 48-well culture flask, and the absorbance was measured at 570 nm using a UV reader. The results are shown in FIG.

As shown in Figure 2, it can be seen that the optical density of the silk / hydroxyapatite composite nanofiber support of Example 2 according to the present invention is higher than the control on the 1st, 3rd and 5th day.

From this, it can be seen that the silk / hydroxyapatite composite nanofiber support according to the present invention not only inhibits cell proliferation but also has an effect of promoting cell proliferation.

< Experimental Example  2> bone regeneration effect measurement

In order to examine the bone regeneration effect of the silk / hydroxyapatite composite nanofiber support according to the present invention was carried out the following experiment.

24 male Sprague Dawley rats (300-350 g) at 10 weeks of age were anesthetized by inhaling isoflurane-oxygen (FORENE ™, Abbot AG, Switzerland). When skin and muscles were incised and the skull exposed, a hole was drilled using a 7.00 mm diameter drill, and the silk / hydroxyapatite composite nanofiber support of Example 3 was implanted. As a negative control, the group without any treatment was used, and as a positive control, the group to which the silk nanofiber support of Comparative Example 1 was implanted (positive control group 1) and the bone filler Bio-OSS (Geistlich biomaterials, Swiss) were used. The transplanted group (positive control group 2) was used. After 4 and 8 weeks, the rats were euthanized with CO 2 gas and the skulls were recovered. The recovered rat skulls were fixed in 10% formalin for 24 hours and photographed with Micro C.T (SKYSCAN 1072-32, Belgium). 359 images were created for each sample, and three-dimensional images of 1024 × 1024 pixels were generated and analyzed using SKYSCAN's CT reconstruction software. The volume of the regenerated bone was used with an image analysis program (Image Pro 6.2, Media Cybernetics Inc, Bethesda, MD). The results are shown in FIGS. 3 and 4. More specifically, the skull images of rats after 4 weeks are shown in FIG. 3, and the skull images of weeks after 8 weeks are shown in FIG. 4.

As shown in Figures 3 and 4, it can be seen that the bone regeneration rate is significantly low in the case of no control negative treatment. In addition, when the silk / hydroxyapatite composite nanofiber support of Example 3 according to the present invention is implanted, Bio-OSS (Geistlich biomaterials, Swiss) that is a positive control and bone filler implanted with the silk nanofiber support of Comparative Example 1 It can be seen that the positive control group implanted with bone regeneration is performed, the bone regeneration is the best bone regeneration after 8 weeks in the case of implanting the silk / hydroxyapatite composite nanofiber support of Example 3 according to the present invention Able to know.

From this, it can be seen that the silk / hydroxyapatite composite nanofiber support according to the present invention has excellent bone regeneration effect.

Therefore, the silk / hydroxyapatite composite nanofiber support according to the present invention is not only excellent in biocompatibility as a sponge in which hydroxyapatite is coated and silk nanofibers having a diameter of several tens to several hundred nanometers are three-dimensionally arranged. It does not show cell cytotoxicity, does not inhibit cell proliferation, has excellent cell growth promoting effect, and shows excellent bone regeneration effect, so it can be very useful as a material for bone regeneration in place of the existing artificial bone substitute. .

1 is a scanning electron microscope image of a silk / hydroxyapatite composite nanofiber support according to the present invention ((a) Example 3. (b) Comparative Example 1);

Figure 2 is a graph showing the cell proliferation effect of the silk / hydroxyapatite composite nanofiber support according to the present invention;

Figure 3 is a graph showing the bone regeneration effect after 4 weeks of the silk / hydroxyapatite composite nanofiber support according to the present invention ((a) negative control, (b) Comparative Example 1 (positive control group 1), (c) Bio -OSS (positive control group 2), (d) Example 3);

Figure 4 is a graph showing the bone regeneration effect after 8 weeks of the silk / hydroxyapatite composite nanofiber support according to the present invention ((a) negative control, (b) Comparative Example 1 (positive control group 1), (c) Bio -OSS (positive control group 2), (d) Example 3).

Claims (4)

Sponge-type silk / hydroxyapatite composite nanofiber support for bone regeneration in which three-dimensionally arranged silk nanofibers having a composite coating of hydroxyapatite on a surface and a diameter of several tens to hundreds of nanometers are provided. According to claim 1, wherein the silk nanofibers are bone / silk hydroxyapatite composite nanofiber support for bone regeneration, characterized in that to maintain the shape of the fiber strands without agglomeration with each other. The silk / hydroxyapatite composite nanofiber support for bone regeneration of claim 1, wherein the pore size between the nanofibers of the silk / hydroxyapatite composite nanofiber support is 50 to 1000 µm. 2. The silk / hydroxyapatite composite nanofiber support for bone regeneration of claim 1, wherein the porosity of the silk / hydroxyapatite composite nanofiber support is 90% or more.

Images