KR101750984B1

KR101750984B1 - Manufacturing method of silk fibroin bone fixation device and silk fibroin bone fixation device manufactured by the same

Info

Publication number: KR101750984B1
Application number: KR1020160014280A
Authority: KR
Inventors: 박찬흠; 김수현; 김동욱; 문보미; 박현정; 박예리; 이민채; 이옥주; 이정민; 정주연; 주형우
Original assignee: 한림대학교 산학협력단
Priority date: 2016-02-04
Filing date: 2016-02-04
Publication date: 2017-06-26

Abstract

The present invention provides a method for manufacturing a bone fixation device for a bone fixation using a centrifugal separation method and a bone fixation plate made of a silk fibroin aqueous solution and a bone fixation screw, and a silk fibroin bone fixation device manufactured by the method . INDUSTRIAL APPLICABILITY According to the method of manufacturing a silk fibroin bone anchoring device of the present invention, it is possible to provide a bone anchoring device of a dense structure having a smooth surface and no bubbles.

Description

Technical Field [0001] The present invention relates to a method of manufacturing a silk fibroin bone fixation device and a silk fibroin bone fixation device manufactured by the method,

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for manufacturing a silk fibroin bone anchoring apparatus and a silk fibroin bone anchoring apparatus manufactured by the method, and more particularly, to a method for manufacturing a silk fibroin bone anchoring apparatus manufactured from an aqueous solution of silk fibroin And a silk fibroin bone anchoring device manufactured by the method.

Fracture refers to the state of fracture or fracture of bone tissue due to external forces. In order to bond fractured bone, the fracture site must be fixed for a long time.

For the treatment of these fractures, commonly used plates for bone fixation and screws for bone fixation are used together to fix, support and union the bones. The bone fixing plate and the bone fixing screw are collectively referred to as a bone fixing device.

The material of the bone fixation device may be classified into a metallic material and an absorbent polymer material. First, titanium, stainless steel, cobalt alloy, magnesium alloy, or the like can be used as the metallic material, and can be processed into a bone fixing device through a process such as casting and plasma coating. Examples of the absorbent polymer material include polylactic acid (PLA), polyglycolic acid (PGA), poly-L-lactic acid (PLLA), poly-D-lactic acid lactic-co-glycolic acid (PLGA), and the like can be used, and they can be processed into a bone anchoring device through a processing method such as extrusion molding and injection molding .

Since these bone anchors are implants inserted into the human body, their biocompatibility and chemical, physical and mechanical properties should be excellent. However, the bone fixation plate and screws of the metallic material can not be decomposed in the body, resulting in an inflammatory reaction. In addition, after the second surgery, there may be traces of surgery on the site where the screw for bone fixation is inserted.

In the case of the absorbent polymer material, which is developed to compensate for the disadvantages of the metallic bone fixation plate and the screw, it is possible to replace the metallic material in a hard part, to eliminate the secondary operation for removal, It can be minimized and there is no sign of surgery. Therefore, the range of use is increasing. However, the bone-anchoring device of the absorbent polymer material is disadvantageous in that the mechanical strength thereof is lower than that of the metallic product, and the cost is high.

As a method for solving such a problem, research on a method using silk fibroin has been carried out for a long time.

Unlike other polymers, the silk fibroin is highly biocompatible because it does not cause an immune response in the living body and is a natural polymer approved by the Food and Drug Administration (FDA). In addition, silk fibroin is excellent in strength and durability and can control the rate of biodegradation according to the degree of crystallization, and has an advantage that it can be easily obtained and is inexpensive.

However, there have been various problems in the process of manufacturing a support such as a bone anchoring device using silk fibroin. The biggest problem is that there is a limit to increase the concentration of silk fibroin aqueous solution. In particular, when the aqueous solution of silk fibroin having a low concentration was used, it was difficult to produce a support due to a phenomenon such as warping and cracking after drying. Therefore, conventionally, porous or sponge-like supports have been mainly studied as a method of applying to silk fibroin using a damaged or missing portion of a bone.

For example, Patent Document 1 (Korean Patent Laid-Open Publication No. 10-2014-0140204) discloses a process for producing silk fibroin by lithium bromide, calcium chloride, lithium chloride, zinc chloride, To obtain a silk fibroin aqueous solution; and dissolving the silk fibroin aqueous solution in water containing at least one salt selected from the group consisting of: And b) performing a dialysis process using a cellulose dialysis membrane to remove the salts present in the aqueous solution of silk fibroin to prepare a silk fibroin aqueous solution, and using the same to prepare a silk fibroin porous support and using it as a bone graft material .

However, since such a type of silk fibroin scaffold has a low mechanical strength, it has been difficult to develop a bone fixation device composed of a bone fixing plate and a bone fixing screw, which are usually used for fracture treatment.

Therefore, there is a need for establishing an effective method for producing a non-porous support using a silk fibroin aqueous solution and studying a method for manufacturing a bone anchoring device using such a method.

KR 1020140140204 A

The present invention provides a method for producing a silk fibroin bone anchoring device from a solution of silk fibroin using centrifugal force.

The present invention also provides a silk fibroin bone anchoring device having a dense internal structure without bubbles produced by the above-described production method.

SUMMARY OF THE INVENTION In order to solve the above problems, the present invention provides a method of manufacturing a bone fixation device for a bone fixation device comprising a bone fixation plate and a screw for bone fixation, which are manufactured from a solution of silk fibroin using a centrifugal separation method.

Preferably, the manufacturing method includes the steps of: (i) mounting the mold of the bone fixing plate and the bone fixing screw into a centrifuge separator; (Ii) introducing the aqueous solution of silk fibroin into the centrifugal separator through the step (i); (Iii) rotating the centrifugal separator through the step (ii), separating and drying the mold in the centrifugal separator; (Iv) removing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw from the mold dried according to the step (iii), and then crystallizing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw ; And (v) washing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw crystallized in accordance with the step (iv).

The centrifugal separator may be a custom centrifugal separator manufactured by a 3D printer.

The molding material of the mold may include any one or two or more selected from the group consisting of polydimethylsiloxane, silicon, urethane, and resin.

The concentration of the silk fibroin aqueous solution may be 18 to 30 wt%.

In the step (iii), the rotating speed of the centrifugal separator may be 2000 to 4000 rpm and the rotating time may be 3 to 7 hours. In the step (iii), the drying temperature of the mold may be 50 to 70 ° C.

In the step (iv), the crystallization may be performed by immersing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw into any one or two or more mixed solutions selected from the group consisting of methanol, ethanol and propanol.

In addition, the present invention provides a silk fibroin bone anchoring apparatus manufactured by the above-described manufacturing method.

INDUSTRIAL APPLICABILITY According to the present invention, since a centrifugal separation method is used in manufacturing a bone anchoring device using an aqueous solution of silk fibroin, it is possible to provide a bone augmenting device having a smooth surface and a bubble free structure.

Fig. 1 schematically shows a step of manufacturing a customized centrifuge tube for making a bone anchoring device with a 3d printer.
Fig. 2 schematically shows the second concentration step of the aqueous solution of silk fibroin according to the embodiment.
Fig. 3 schematically shows a process of manufacturing a bone anchor mold according to an embodiment and a process of mounting the mold in a centrifuge cage.
FIG. 4 is a schematic view showing a process of preparing a bone fixation after injecting a silk fibroin aqueous solution into a centrifugal separator through the process of FIG.
5 is a microscope image of a silk fibroin bone fixing plate and a screw manufactured according to the embodiment.
Fig. 6 is a SEM image of the surface of a screw for fixing a bone prepared according to the example (right) and the comparative example (left).
Fig. 7 shows a scanning electron microscope image of a bone screw thread section prepared according to the example (right) and the comparative example (left).
8 is a graph showing FT-IR analysis results of a bone fixing plate and a screw manufactured according to the embodiment.
FIG. 9 is a photograph showing a procedure of implanting the bone anchoring device manufactured according to the embodiment into an SD rat.
FIG. 10 is a micro-CT image of the femur of an SD rat taken at 2 weeks after the bone fixation device is implanted according to FIG.
FIG. 11 is a microscopic image of a femur of an SD rat observed histologically through H & E staining at 2 weeks after the bone fixation device was implanted according to FIG.
Fig. 12 is an enlarged microscope image of the portion (a) of Fig.
13 is an enlarged micrograph image of the portion (b) of Fig.
FIG. 14 is an enlarged microscope image of the portion (c) of FIG.
Fig. 15 is an enlarged microscope image of the portion (d) in Fig.
Fig. 16 is an enlarged microscope image of the portion (e) of Fig.

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for manufacturing a bone fixation device for a bone fixation using a centrifugal separation method and a bone fixation plate made of an aqueous solution of a silk fibroin and a bone fixation screw, and a silk fibroin bone fixation device manufactured by the method .

Specifically, the method for manufacturing the silk fibroin bone fixation apparatus includes the steps of: (i) mounting the mold of the bone fixing plate and the bone fixing screw into a centrifuge separator; (Ii) introducing the aqueous solution of silk fibroin into the centrifugal separator through the step (i); (Iii) rotating the centrifugal separator through the step (ii), separating and drying the mold in the centrifugal separator; (Iv) removing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw from the mold dried according to the step (iii), and then crystallizing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw ; And (v) washing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw crystallized in accordance with the step (iv).

Hereinafter, a method of manufacturing a silk fibroin bone anchoring device according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

(I) mounting step of the bone anchorage mold

This step is a step of mounting the plate of the bone fixing plate and the mold of the bone fixing screw into the centrifuge separating barrel.

The centrifugal separator may be a custom centrifugal separator manufactured by a 3D printer, and one embodiment of the centrifugal separator manufacturing process is schematically shown in FIG. 1, the centrifugal separator has a portion to which a thread fixing screw mold can be attached on both sides of the lower side of the centrifuge separator, and a plate mold for fixing a bone can be mounted in the center of the centrifuge separator. .

Meanwhile, FIG. 3 schematically shows an embodiment of the process of manufacturing the mold and the mode in which the mold is mounted in the centrifugal separator. Referring to FIG. 3, the molds 13 and 23 are inserted into molds 12 and 22 for molding a bone, respectively, and a bone fixing screw model 21 is inserted into the molds 12 and 22, And molding the molding material into the molds 12 and 22. The screw mold 23 for fixing the cogs among the molds 13 and 23 manufactured as described above is mounted on both the lower ends of the circular separator 3 and the plate mold 13 for fixing the corrugations, .

(Ii) Step of injecting aqueous solution of silk fibroin

This step is a step of injecting a silk fibroin aqueous solution into the centrifugal separator equipped with the mold for the bone fixing plate and the bone fixing screw according to the step (i), which is shown in (1) of FIG.

The concentration of the silk fibroin aqueous solution may be 18 to 30 wt%, and more preferably 28 to 30 wt%. If the concentration of the aqueous solution of silk fibroin is less than 18 wt%, it may be difficult to manufacture a bone anchoring device due to a phenomenon such as twisting or cracking after drying. On the other hand, in order to produce an aqueous solution of silk fibroin having a concentration exceeding 30 wt% May fall.

The aqueous solution of silk fibroin may be prepared by a conventional method. Preferably, the method comprises dissolving silk fibroin in an aqueous solution containing a salt to obtain an aqueous mixed solution (step 1); Removing the salt in the mixed aqueous solution to obtain a silk fibroin aqueous solution (step 2); And a first concentration step (step 3) of concentrating the aqueous solution of silk fibroin using polyethylene glycol (PEG). In this case, a silk fibroin aqueous solution having a concentration of 18 to 20 wt% can be obtained.

The silk fibroin solution is prepared by mixing an aqueous solution of silk fibroin having been subjected to the first concentration step with an aqueous solution of polyethylene oxide (PEO), separating the mixture into an upper layer solution and a lower layer solution, Concentration step (step 4). In this case, it is more preferable to obtain a high concentration silk fibroin aqueous solution of 28 to 30 wt%.

The process for preparing the silk fibroin aqueous solution will be described step by step.

Stage 1 : silk Step of obtaining fibroin-salt mixed aqueous solution

First, silk fibroin can be obtained by removing sericin protein and impurities from the cocoon through a refining process, and the refining process can be carried out through various methods known in the art. For example, the refining process of the silk fibroin is a cocoon Marseille soap (Marseilles soap), sodium bicarbonate (Sodium bicarbonate; NaHCO _3), sodium carbonate _{(Sodium carbonate; Na 2 CO 3} ), sodium hydroxide (Sodium Hydroxide; NaOH), silicate Sodium selenate (Na ₂ SiO ₃ ) and papain enzymes, at 90 to 100 ° C for 30 minutes to 2 hours, washing with water and drying .

In this step, the refined silk fibroin is dissolved in an aqueous solution containing a salt to prepare a mixed aqueous solution.

The salt may be any one or two or more selected from the group consisting of lithium bromide (LiBr), lithium chloride (LiCl ₂ ), zinc chloride (ZnCl ₂ ) and calcium chloride (CaCl ₂ ) For about 30 minutes to 4 hours.

Step 2: silk Step of obtaining fibroin aqueous solution

This step is a step for obtaining a silk fibroin aqueous solution by removing the salt from the mixed aqueous solution obtained from the step 1 above.

Specifically, the salt removal may be performed through a dialysis process using a cellulose dialysis membrane having a weight-average molecular weight cut-off of 12,000 to 14,000. For example, in the dialysis process, the mixed aqueous solution is placed in the dialysis membrane Sealed and then immersed in distilled water.

The dialysis process may be performed for 36 to 72 hours. Through this process, a pure aqueous solution of silk fibroin in which salt is removed from the mixed aqueous solution and only silk fibroin is left can be obtained. The concentration of the silk fibroin aqueous solution thus obtained may be 8 to 10 wt%.

Step 3: Primary enrichment step

This step is a step of primarily concentrating the aqueous solution of silk fibroin obtained in the above step 2 by using polyethylene glycol (PEG).

Specifically, the concentration may be performed through a dialysis process performed using a cellulose dialysis membrane having a weight-average molecular weight cut-off of 6,000 to 8,000. For example, in the dialysis step, the silk fibroin aqueous solution obtained in step 2 may be placed in the dialysis membrane, sealed, and then PEG may be sprayed on the outer surface. In this case, distilled water in the dialysis membrane And the aqueous solution of silk fibroin inside the dialysis membrane is concentrated. The concentration of the aqueous solution of silk fibroin (hereinafter referred to as "primary concentrate") that has undergone the primary concentration step may be 18 to 20 wt%.

Step 4: Second enrichment step

This step is a step of mixing the primary concentrate with an aqueous solution of polyethylene oxide (PEO) to separate the supernatant into a supernatant and a lower supernatant, and then obtaining the supernatant. The procedure is schematically shown in Fig. 2 . In Fig. 2, "silk" represents the primary concentrate, and "PEO" represents the PEO aqueous solution. The lower layer of the layered mixture solution is obtained in a test tube.

At this time, it is preferable that the primary condensate and the aqueous solution of PEO are mixed at a weight ratio of 3: 1 to 5: 1, and when the mixing ratio of the aqueous solution of PEO is out of the above range and mixed excessively, There is a possibility that the concentration of the silk fibroin in the third concentrate may be mixed with PEO or the loss of the silk fibroin may be increased.

In addition, the concentration of the PEO aqueous solution is preferably 25 to 35 wt%, and when the concentration is less than 25 wt%, layer separation may not be induced properly in the second concentration method of inducing layer separation due to density difference, If it is more than 35 wt%, there is a possibility that PEO remains in the lower layer after the layer separation.

This step is a step for producing an aqueous solution of high concentration of silk fibroin that can be more preferably used for manufacturing a bone anchoring device by secondarily concentrating the primary concentrated liquid. The concentration of the aqueous solution of silk fibroin obtained by this step is 28 ~ 30 wt%.

(Iii) Mold drying step

This step is a step of rotating the centrifugal separator into which the aqueous solution of silk fibroin is injected in accordance with the step (ii), and then separating and drying the mold in the centrifugal separator. In steps (2) and One embodiment of the step is shown sequentially.

The rotation speed of the centrifugal separator is preferably 2000 to 4000 rpm, and the rotation time is preferably 3 to 7 hours. When the rotation speed and the rotation time are less than the lower limit value of the above range, the silk fibroin aqueous solution is not sufficiently filled in the mold, which may cause difficulty in forming the osseous fixation. On the other hand, .

After the centrifugal separation is completed, the mold separated from the centrifuge can be dried at 50-70 ° C. In this process, water in the mold is removed and a bone fixation formed only by silk fibroin can be manufactured.

(Iv) the step of crystallizing the bone fixation device

This step is a step of crystallizing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw after removing the silk fibroin bone fixing plate and the bone fixing screw from the mold dried according to the step (iii) An embodiment of this step is schematically shown in (4) of FIG.

The crystallization process is performed to control the decomposition degree and maintain the physical properties of the silk fibroin bone fixation device in vivo. The silk fibroin bone fixation plate and the screw for fixing the silk fibroin bone, which have undergone the drying process, are composed of methanol, ethanol and propanol , And the immersion may be carried out for 10 to 15 minutes.

(V) washing step

This step is a step of washing the silk fibroin bone fixation plate crystallized according to the step (iv) and the screw for fixing the silk fibroin bone, and an embodiment of this step is schematically shown in (5) of Fig. have. The washing may be performed using distilled water. In this process, the immersion solution such as ethanol used in the crystallization process in step (iv) may be removed from the silk fibroin bone anchoring device. Also, in this process, the PEO that can remain in the silk fibroin during the second concentration of the aqueous solution of silk fibroin may be removed together.

Meanwhile, the present invention provides a silk fibroin bone anchoring device manufactured according to the above-described manufacturing method. Since the internal structure of the bone fixation apparatus is dense without bubbles, the bone fixation apparatus has excellent mechanical strength as compared with conventional porous structures or sponge-like supports. Thus, when the silk fibroin bone anchoring device manufactured according to the present invention is applied to conventional fracture treatment, it can provide sufficient stability during bone regeneration and growth period.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these embodiments.

Example

(1) Preparation of aqueous solution of silk fibroin

The cocoon of Bombyx mori was placed in an aqueous solution of 0.02M sodium carbonate (Na ₂ CO ₃ ), heated to 100 ° C for 1 hour to remove sericin protein and impurities, and then washed with distilled water to obtain refined pure silk fibroin.

The refined silk fibroin was placed in a 9.3 M aqueous solution of lithium bromide (LiBr) and dissolved at 60 캜 for 1 hour to obtain a silk fibroin-LiBr mixed aqueous solution.

The mixed aqueous solution was placed in a cellulose dialysis membrane having a weight molecular weight cut-off of 12,000 and dialyzed in distilled water for 48 hours to remove LiBr in the mixed aqueous solution. The concentration of the silk fibroin aqueous solution thus obtained was 10 wt%.

The aqueous solution of silk fibroin was placed in a cellulose dialysis membrane having a weight molecular weight cut-off of 6,000, and polyethylene glycol (PEG) was sprayed on the outer surface thereof. In this process, distilled water in the dialysis membrane is diffused out of the dialysis membrane by the osmotic phenomenon, whereby the aqueous solution of the silk fibroin is first concentrated. The concentration of the aqueous solution of silk fibroin (hereinafter referred to as "primary concentrate") after the primary concentration step was 18 wt%.

The primary concentrate was mixed with a 30 wt% aqueous solution of polyethylene oxide (PEO) in a weight ratio of 4: 1, followed by layer separation. When the layer separation is performed, the first concentrated liquid is subjected to the second concentration by taking the lower layer liquid as shown in FIG. The final concentration of the aqueous solution of silk fibroin (hereinafter referred to as "second concentrated solution") after the second concentration step was 30 wt%, and the second concentrated solution was used as the aqueous solution of silk fibroin for preparing the bone fixation device of the present invention.

(2) Manufacture of plate for bone fixing and screw for fixing bone

First, as shown in FIG. 1, a customized centrifuge can be manufactured by using a 3D printer (NBR-T, BT-3000) to which a bone anchor fixing mold can be attached, and the centrifuge tube is connected to a centrifuge Industrial Corp., Table Top Centrifuge, PLC-03).

3, a bone fixing plate 11 and a bone fixing screw model 21 are inserted into molds 12 and 22 for mold production, respectively, and the mold 12 22 and 22 are made of polydimethylsiloxane (PDMS) as a mold-forming material to produce a bone-fixing plate mold 13 and a bone-fixing screw mold 23. The bone fixing plate mold 13 and the screw mold 23 (hereinafter referred to as "PDMS mold") made of the PDMS are respectively mounted on the center portion and the lower end portions of the centrifuge tube 3.

Subsequently, as shown in Fig. 4, the second concentrate prepared in (1) preparation of aqueous solution of silk fibroin is poured into a centrifuge separator equipped with the PDMS mold. Thereafter, the centrifugal separator poured with the secondary concentrate was rotated at a rotation speed of 3000 rpm for 5 hours, and the PDMS mold was separated from the centrifugal separator and dried in an oven at 60 ° C. Thereafter, the silk fibroin bone fixing plate and screws were removed from the dried PDMS mold, and the silk fibroin bone fixing plate and screws were immersed in 100% ethanol to crystallize, followed by washing with distilled water to remove ethanol, Fibroin bone fixation plates and screws were prepared. FIG. 5 shows a microscope image of the prepared silk fibroin bone fixation plate and screw.

Comparative Example

Bone fixation plate and bone fixation screw BioSorb ^TM FX (Bionix Inc. Finland) made of commercially available absorbable polymer (poly L / DL lactide copolymer) were prepared and prepared.

Assessment Methods

1. Surface and Sectional Analysis

In order to compare the surfaces of the bone-fixing screws prepared according to the examples and the comparative examples, the bone-fixing screws were coated with Au-Pb and then examined with a scanning electron microscope (Carl Zeiss, Germany, model name: RUPRA55V VP-FESEM) After cutting the screw, the cross section was observed with the scanning electron microscope. The results are shown in Figs. 6 and 7, respectively. At this time, scanning electron microscope observation was performed at the Basic Science Research Institute (Chuncheon).

Referring to Figs. 6 and 7, it can be seen that the bone fixing screw (left) of the comparative example shows a rough surface, and bubbles are also observed at the inner end face. On the other hand, the silk fibroin screw (right) manufactured according to the embodiment exhibited a smooth surface, and in the case of the internal cross section, it shows that the structure is dense without bubbles, This is because, in the case of the comparative absorbable bone fixing screw, the bone fixing screw manufactured according to the embodiment of the present invention is manufactured by a secondary step of cutting to shape, Because it adopts.

2. Fourier Transform Infrared Spectroscopy (FT-IR) analysis

Qualitative analysis was carried out using an FT-IR measuring device (Perkin Elmer, UK, model name: Frontier) for the structural analysis of the silk fibroin bone fixation plates and screws manufactured according to the examples. The bone fixation plate and the screw are manufactured by taking the lower layer of the mixed solution of the primary concentrate and the PEO aqueous solution layered in the second concentration step as shown in FIG. 2, and the FT-IR analysis is performed in the bone fixation device It is an analysis for confirming whether PEO remains.

To this end, the FT-IR analysis graph of FIG. 8 shows that the polyethylene oxide (PEO) used in the examples, the purified pure silk fibroin (Silk) according to the embodiment, and the silk fibroin- The FT-IR spectrum of the screw (Silk Plate & screw) was compared.

Generally, PEO is known to exhibit the COC stretching the peak (Straching peak) at about 1100cm ^-1, CH ₂ weging peak (Wagging peak) at about 1350cm ^-1, CH ₂ bending peak (Bending peak) at 1450cm ^-1 for a, and FIG. 8 Looking at the PEO used in the examples may be confirmed that the peak indicating at ^{1095cm -1, 1341cm -1, 1466cm -1} .

On the other hand, the silk fibroin are usually 1650cm ^-1 for the AmideⅠ peak, and a peak at 1550cm ^-1 for AmideⅡ, known to exhibit a peak at 1250cm ^-1 for AmideⅢ, also look at the 8-polishing according to an embodiment of pure silk fibroin (Silk) is 1620cm ^-1, 1514cm ^-1, 1229cm ^{-1 and} confirmed that represents the peak, and a bone screw for fixing plates and prepared according to example (Silk & plate screw) is 1618cm ^-1, 1513cm ^{- 1} and 1229 cm ^-1 , respectively.

As a result of the above analysis, the bone-fixing plate and screw finally produced exhibit a spectral pattern almost identical to that of pure silk fibroin, but show a completely different pattern from that of PEO. In the lower layer solution obtained in the second concentration step, It can be seen that the PEO does not remain and therefore the plate for fixing the silk fibroin and the screw made of the lower layer solution are composed of only pure silk fibroin.

3. Animal experiments

An animal experiment was conducted to confirm the bone fixation and regeneration effect using the silk fibroin bone anchoring device manufactured according to the example. In this animal experiment, 14-week-old male Sprague-Dawley (SD) rats weighing 250-300 g were used as experimental animals. The temperature inside the animal room was adjusted to 25 ± 1 ℃ and the contrast was automatically adjusted to 12 hours / day. In addition, feed and water were freely ingested, and all experimental animals were adapted to the animal room environment for 2 weeks before being used in the experiment.

Referring to FIG. 9, in an animal experimental procedure, the femur of the SD rats is first incised, and the femur is exposed to induce fracture (A). At this time, the fracture was induced using an electric drill having a thickness of 1 mm, and then holes were drilled at both ends of the fractured bone using a hand drill having a diameter of 1 mm to secure a screw insertion portion. After fracture induction and screw insertion are completed, the fractured bone is fixed using sterile silk fibroin bone plate and screws (B). After fixing the femur as described above, the femoral portion of the SD rats was divided into a muscle layer and a skin layer. Then, the thighs are fixed using a splint and a plaster cast to prevent the broken bone from moving.

FIG. 10 shows a micro-CT image of a femur of an SD rat in which two weeks have elapsed from the insertion of a bone anchoring device. The yellow arrow in FIG. 10 indicates a screw insertion portion, and the red arrow indicates a fracture portion regeneration portion . 10 (C) is an enlarged image of FIG. 10 (A), and FIG. 10 (D) is an enlarged image of the bone. Referring to FIG. 10, It can be confirmed that the fusion and the formation of new bone are well done.

In addition, the femoral tissues of SD rats 2 weeks after the insertion of the bone anchoring device were extracted and subjected to histological observation through hematoxylin & eosin (H & E) staining. Respectively. Figs. 12 to 16 are enlarged views of the portions (a), (b), (c), (d) and (e) As a result of the histological observation, it was confirmed that the foreign body reaction and the inflammation reaction were not observed on the plate and screw for fixing the silk fibroin bone according to the example, and the binding to the surrounding tissue was well performed.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Should be construed as being included in the scope of the present invention.

11: plate for bone fixing
12: Die-casting mold for mold making
13: plate mold for fixing the bone
21: screw for fixing bone
22: Mold for thread mold for threading
23: Screw mold for fixing the bone

Claims

A manufacturing method of a bone fixation device for a bone fixation device, comprising a bone fixation plate and a bone fixation screw, which are manufactured from an aqueous solution of silk fibroin using a centrifugal separation method,
In the above manufacturing method,
(I) attaching the mold of the bone fixing plate and the bone fixing screw to a centrifuge separator;
(Ii) introducing the aqueous solution of silk fibroin into the centrifugal separator through the step (i);
(Iii) rotating the centrifugal separator through the step (ii), separating and drying the mold in the centrifugal separator;
(Iv) removing the silk fibroin bone fixing plate and the kefibroin bone fixing screw from the mold dried according to the step (iii), and then crystallizing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw ; And
(V) washing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw crystallized according to the step (iv)
Wherein the centrifugal separator is a customized centrifuge separator manufactured by a 3D printer.

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The method according to claim 1,
Wherein the molding material of the mold includes one or more selected from the group consisting of polydimethylsiloxane, silicone, urethane, and resin. Gt;

The method according to claim 1,
Wherein the concentration of the aqueous solution of silk fibroin is 18 to 30 wt%.

The method according to claim 1,
Wherein the rotating speed of the centrifugal separator in the step (iii) is 2000 to 4000 rpm, and the rotating time is 3 to 7 hours.

The method according to claim 1,
Wherein the drying temperature of the mold in step (iii) is 50 to 70 ° C.

The method according to claim 1,
In the step (iv), the crystallization is performed by immersing the silk fibroin bone fixing plate and the silk fibroin bone fixing screw into one or two or more mixed solutions selected from the group consisting of methanol, ethanol and propanol. A method for manufacturing a fibroin bone fixation device.

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