KR20190005593A - Porous sponge for hemostasis and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a porous sponge for hemostasis and a manufacturing method thereof. More specifically, the present invention relates to a porous sponge for hemostasis, which is manufactured by mixing collagen derived from duck feet with silk and exhibits excellent hemostatic properties and coagulability. The present invention also relates to a manufacturing method thereof. To this end, the porous sponge for hemostasis includes a collagen-silk hybrid structure.

Description

TECHNICAL FIELD [0001] The present invention relates to a porous sponge for hemostasis and a method of manufacturing the sponge,

The present invention relates to a porous sponge for hemostasis and a method of manufacturing the same, and more particularly, to a porous sponge for hemostasis which can be produced by mixing collagen and silk derived from a duck foot, ≪ / RTI >

Hemostasis plays an important role in all surgical procedures. The basis of good surgical techniques is minimization of bleeding, and modern surgeons have a variety of medications and tools to help with these efforts. Topical hemostatic agents are effective in reducing bleeding and reducing surgical mortality. Thus, the development of these hemostatic agents is an important topic of medical history.

Over the past several decades, the number of various hemostatic agents has increased significantly. The type of bleeding, the precise mechanism of action, cost, and availability are considerations for selecting the most appropriate agent. Ideal hemostatic agents should be highly effective, easy to use, sterile, stable, non-irritating, biodegradable and cost effective. Collagen and collagen fibers, cellulose oxide, fiber proteins and regenerated cellulose, alginates, medical hemostats, gelatin and chitosan (CS) are commonly used as absorbent hemostats. In addition, QuikClot, QuikClot ACS ⁺ (Z-Medica), HemCon, self-expanding hemostatic polymer (SEHP), WoundStat (Marum Polymer Technologies Inc.), CombatGauze (Z- Medica), FastAct / SeraSeal Inc. and TraumaDex have been approved by the US Food and Drug Administration (FDA) and Conformity European (CE). However, these artificial hemostatic agents have poor exothermic or biodegradable properties, have a risk of transmission of infectious factor, formation of terminal thrombosis, and an allergic reaction.

Collagen-based products have recently been developed and approved in the hemostatic agent market. As active microfibrillar collagen hemostat, avitene derived from bovine collagen was first introduced in the United States in the early 1970s. Avitene was first introduced in powder form and is still commonly used in powder form, but recently it has expanded to nonwoven fabric and sponge form. The most recently added Avitene Ultra Foam Collagen was approved by the US FDA in May 2000 in the form of a sponge. Avitene has been shown to promote clot formation through platelet aggregation and release of fibrinolytic proteins. However, even when Avitene is used as a successful hemostatic agent, there are some limitations, such as abscess formation, hematoma, wound healing, allergic reactions, and reinforcement of infections, including foreign bodies.

On the other hand, commercially available collagen-based materials are generally derived from cattle and pigs. Bovine collagen is involved in the transmission of BSE and infectious spongiform encephalopathy (TSE). Pork collagen can cause ethical concerns in some religious communities.

Therefore, in order to overcome physiological problems and ethical problems, it is required to develop a hemostatic agent having the same or superior performance as a conventional collagen hemostatic agent while obtaining much safer collagen.

Korean Patent Publication No. 10-2016-0087033

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems, and it is an object of the present invention to provide a porous sponge for hemostasis, which can be produced by mixing collagen derived from duck foot and silk, .

These and other objects and advantages of the present invention will become apparent from the following description of a preferred embodiment.

The above object can be achieved by a porous sponge for hemostasis comprising a collagen-silk hybrid structure.

At this time, the collagen may be collagen derived from an animal or collagen derived from a duck foot.

The above object can also be achieved by a method for producing a collagen solution, comprising: a first step of dissolving an animal collagen in an acid solution to prepare a collagen solution; A second step of mixing a silk solution with a collagen solution to prepare a collagen-silk mixed solution; And a third step of lyophilizing the collagen-silk mixed solution to prepare a collagen-silk hybrid porous sponge.

In the first step, the animal collagen may be collagen derived from duck foot, and the collagen solution is preferably 2-5% w / v (g / ml).

In the second step, the silk solution and the collagen solution can be mixed at a volume ratio of 1: 1. In the third step, the collagen-silk mixed solution can be lyophilized at minus 70 to 90 ° C for 12 to 48 hours.

INDUSTRIAL APPLICABILITY According to the present invention, by using collagen and silk in combination, there is an effect that porosity is high, hemostatic properties and solidification characteristics are excellent.

In addition, by using collagen derived from duck foot, it has an effect of overcoming physiological problems or ethical problems such as infection or infection caused by using collagen derived from cattle or pigs.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

1 is a diagram showing an optical digital photograph and an SEM image of Examples and Comparative Examples. A, C, E and G are external forms of Avitene (Comparative Example 1), DFC (Comparative Example 2), Silk (Comparative Example 3) and DFC / Silk (Scale Bar: 1 mm) of Avitene (Comparative Example 1), DFC (Comparative Example 2), Silk (Comparative Example 3) and DFC / Silk (Example).
2 is a view showing the FTIR spectra of Examples and Comparative Examples.
3 is a graph showing the porosity (A) and the water absorption degree (B) of Examples and Comparative Examples.
FIG. 4 is a graph showing cytotoxicity evaluation for 1, 3, and 5 days of Examples and Comparative Examples. FIG.
5 is a graph showing the results of whole blood coagulation tests of Examples and Comparative Examples.
6 is a photograph showing hemostatic effects of femoral artery wounds in Examples and Comparative Examples. A and E are before and after Avitene (Comparative Example 1), B and F are before and after DFC (Comparative Example 2), C and G are before and after Silk (Comparative Example 3), D and H are DFC / Silk (Example).

Hereinafter, the present invention will be described in detail with reference to embodiments and drawings of the present invention. It will be apparent to those skilled in the art that these embodiments are provided by way of illustration only for the purpose of more particularly illustrating the present invention and that the scope of the present invention is not limited by these embodiments .

Also, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains and, where contradictory, Will be given priority.

In order to clearly illustrate the claimed invention, parts not related to the description are omitted, and like reference numerals are used for like parts throughout the specification. And, when a section is referred to as "including " an element, it does not exclude other elements unless specifically stated to the contrary. In addition, "part" described in the specification means one unit or block performing a specific function.

In each step, the identification code (first, second, etc.) is used for convenience of explanation, the identification code does not describe the order of each step, and each step does not explicitly list a specific order in the context May be performed differently from the above-described sequence. That is, each of the steps may be performed in the same order as described, or may be performed substantially concurrently or in the reverse order.

The porous sponge for hemostasis according to an embodiment of the present invention includes a collagen-silk hybrid structure. The collagen-silk hybrid structure is a composite structure formed by mixing a collagen solution and a silk solution and then lyophilizing the collagen-silk hybrid structure. The present invention has an excellent hemostatic and coagulation characteristics by including a collagen-silk hybrid structure having a large porosity .

In one embodiment, the collagen can be animal-derived animal collagen, but it is preferred to use collagen derived from duck foot to overcome physiological problems or ethical problems such as infection or transmission. The collagen derived from the foot of ducks is removed from the bones of the duck foot, then divided into small pieces with the husks, and then quenched at a temperature of minus 20 ° C. Removing bleaching and excess fat with sodium hydroxide; Preparing a soluble collagen extract using an acid and sodium chloride; A desalting step of removing the salt used for the extraction; And drying it by concentration and drying to obtain a collagen-containing dried product.

In one embodiment, the silk is a natural fibrous polymer extracted from Bombyx mori silk-worm and consists of two proteins, fibroin and sericin. Silk can be mixed with collagen to increase porosity, improve hemostatic and coagulant properties, as well as improve mechanical properties. In particular, fibroin, which is a main component of silk protein, has been widely utilized in the biomedical field due to its excellent tensile strength, low antigenicity, noninflammatory property and controllable biodegradability. Therefore, silk has been used only for fibroin (called silk fibroin) It is more preferable to use it.

Next, a method of manufacturing a porous sponge for hemostasis will be described. In the description, overlapping portions will not be described.

A method of manufacturing a porous sponge for hemostasis according to an embodiment of the present invention includes: a first step of dissolving an animal collagen in an acid solution to prepare a collagen solution; A second step of mixing a silk solution with a collagen solution to prepare a collagen-silk mixed solution; And a third step of lyophilizing the collagen-silk mixed solution to prepare a collagen-silk hybrid porous sponge. According to the present invention, a collagen-silk hybrid structure having a high porosity is formed by mixing an animal collagen and silk, followed by lyophilization, thereby producing a porous sponge for hemostasis, which is excellent in hemostatic properties and solidification characteristics.

In one embodiment, the first step is a step of dissolving animal collagen in an acid solution to prepare a collagen solution, and the acid solution may be a solution of various acids such as hydrochloric acid. Animal collagen is preferably collagen derived from duck foot to overcome physiological problems or ethical problems such as infection or transmission, and the collagen solution is prepared by dissolving collagen in an acid solution to a concentration of 2-5% w / v (g / ml). < / RTI >

In one embodiment, the second step is a step of mixing the silk solution with the collagen solution prepared in the first step to prepare a collagen-silk mixed solution. The silk solution and the collagen solution are mixed at a volume ratio of 1: 1 desirable. The silk solution means that the silk protein is dissolved in a mixed solvent of calcium chloride, water and ethanol. Fibroin, a major component of silk proteins, has been used extensively in biomedical applications due to its excellent tensile strength, low antigenicity, noninflammatory properties and controllable biodegradability. Due to this advantage of fibroin, silk solution is superior to sericin protein It is preferable to use a silk fibroin solution obtained by dissolving impurity-removed silk fibroin protein in the mixed solvent.

In one embodiment, the third step is a step of lyophilizing the collagen-silk mixed solution prepared in the second step to prepare a collagen-silk hybrid porous sponge. The collagen- It is preferable to remove moisture by freeze-drying for 48 hours.

Also, after the third step, the collagen-silk hybrid porous sponge is immersed in a glutaraldehyde solution for 12 to 36 hours; Removing the collagen-silk hybrid porous sponge and washing with deionized water or distilled water; Immersing the washed collagen-silk hybrid porous sponge in an aqueous glycine solution; And a collagen-silk hybrid porous sponge, followed by washing with deionized water or distilled water, followed by freeze-drying at a temperature of 80 to 100 ° C for 12 to 24 hours. Mechanical properties and the like can be improved.

Hereinafter, the structure and effects of the present invention will be described in more detail with reference to specific examples and comparative examples. However, this embodiment is intended to explain the present invention more specifically, and the scope of the present invention is not limited to these embodiments.

[Manufacturing Example]

Material preparation

Duck 's feet derived collagen (DFC) was purchased from Sewon Cellontech, Seoul, and NIH3T3 fibroblasts were purchased from ATCC (Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), antibiotics / antibiotics (A / A) and trypsin were purchased from Welgene, Fresh MediaTM (Dalseogu, Daegu, Korea). Cell Count Kit-8 (CCK-8) was purchased from EnzoLifeSciences (Switzerland).

Preparation of silk fibroin solution

Heating the cocoons of the Bombyx mori to 100 ℃ for 30 minutes in 0.02M Na ₂ CO ₃ aqueous solution, and by washing with deionized water three times to remove the sericin proteins and impurities to give pure silk fibroin. The refined silk fibroin was dissolved in a mixed solvent of CaCl ₂ : H ₂ O: EtOH = 1: 8: 2 mol ratio at 98 ° C for 1 hour and then dialyzed in distilled water for 72 hours to prepare pure silk fibroin solution (SF) Respectively. The final concentration of the silk fibroin produced was 6 wt%.

[Example]

DFC was dissolved in 0.1 M hydrochloric acid (HCl) and neutralized with 1 M sodium hydroxide (NaOH) to prepare a 3% (w / v) collagen solution. 20 ml of silk fibroin solution (SF) and 20 ml of collagen solution were mixed, and 40 ml was poured into a Petri dish. After freezing at minus 80 ° C for 12 hours, it was lyophilized for 24 hours to prepare a porous sponge for hemostasis. The porous sponge was immersed in an aqueous solution of 0.25% v / v glutaraldehyde diluted in 95% ethanol (EtOH) for 24 hours, and then taken out and washed with distilled water two or three times, followed by the addition of 0.1 M glycine aqueous solution Lt; / RTI > for 24 hours. Thereafter, the porous sponge was taken out, sufficiently washed with distilled water, frozen at -80 ° C for 12 hours, and lyophilized to prepare a porous sponge for hemostasis (example: DFC / Silk) containing a collagen-silk hybrid structure Respectively.

[Comparative Example 1]

Avetene sold on the market was purchased, and this was designated as Comparative Example 1 (Avetene).

[Comparative Example 2]

DFC was dissolved in 0.1 M hydrochloric acid (HCl) and neutralized with 1 M sodium hydroxide (NaOH) to prepare a 3% (w / v) collagen solution, and 40 ml was added to the Petri dish. After freezing at minus 80 ° C for 12 hours, it was lyophilized for 24 hours to prepare a porous sponge for hemostasis. The porous sponge was immersed in an aqueous solution of 0.25% v / v glutaraldehyde diluted in 95% ethanol (EtOH) for 24 hours, and then taken out and washed with distilled water two or three times, followed by the addition of 0.1 M glycine aqueous solution Lt; / RTI > for 24 hours. Thereafter, the porous sponge was taken out, sufficiently washed with distilled water, frozen at -80 ° C for 12 hours, and lyophilized to prepare a porous sponge for hemostasis (Comparative Example 2, DFC) containing a collagen construct.

[Comparative Example 3]

The silk fibroin solution of the above preparation example was poured into a Petri dish at 40 ml, and then freeze-dried at -80 캜 for 12 hours. Subsequently, an ethanol solution of 100% to 50% (100, 90, 80, 70, 50%) (mixing volume ratio of ethanol and water, in the case of 80%, ethanol: water at a volume ratio of 80:20 ) Was washed with deionized water and lyophilized to prepare a porous sponge (Comparative Example 3, Silk).

[Experimental Example]

Appearance observation

In order to observe the structures of Examples and Comparative Examples, each sponge was made into a square having a size of 1 cm x 1 cm, coated with Au-Pd, and observed with a scanning electron microscope (VP-FESEM) ) Is expressed by 1 mm). Scanning electron microscopy was performed at the Basic Science Research Institute (Chuncheon).

From the overall image, it was confirmed that the porous structure was formed in all the sponges. Both Avitene and DFC sponges showed amorphous interconnection pores, and silk sponges showed a strip-like structure. The DFC / Silk sponge showed larger pore size than the other sponge, and it was found that the addition of silk to the DFC formed a hybrid sponge with a larger porous structure. However, DFC-based sponges showed uniformly interconnected pore distributions over other sponges.

FTIR spectrum observation

For structural analysis of the examples and comparative examples, crystallization analysis was performed using an ATR-FTIR measuring device (GX PerkinElmer Inc., USA), and the results are shown in FIG. The spectral range was from 2000 to 1000 cm ^-1 .

FTIR spectra of the amide Avitene Ⅰ and Ⅱ ^band showed a strong peak in the (1653 and 1547 cm ^1). DFC is much wider amide Ⅰ and Ⅱ ^band are shown (1650 and 1553 cm ^1). The difference between Avitene and DFC appears to be due to the structural differences between bovine collagen and duck-foot collagen. Or the extraction method of collagen. Silk exhibited strong absorption peaks at 1628 and 1520 cm ^-1 . DFC / Silk showed absorption bands at 1629 cm ^-1 (amide I) and 1523 cm ^-1 (amide II).

Check porosity (porosity) and water absorption

The weight of the dried sponge was measured (W _d ) to ascertain the water absorption of the examples and comparative examples. The sponge was then immersed in distilled water for 3 hours, and the distilled water from the surface of the fully immersed sponge was removed and weighed (W _s ). The water absorption was calculated by the following equation (1), and the results are shown in FIG. 3 (A).

[Equation 1]

In addition, a liquid displacement process was used to measure the porosity of the examples and comparative examples. A sponge was immersed in a constant volume of distilled water (V ₁ ) for 10 minutes. The volume of distilled water in which the sponge was immersed was then checked (V ₂ ). After the immersed sponge was removed, the volume of the remaining distilled water was measured (V ₃ ). The porosity (P) was calculated by the following equation (2), and the results are shown in FIG. 3 (B).

&Quot; (2) "

The values in Figure 3 are expressed as mean ± standard deviation (n = 3) and are * p <0.05 and ** p <0.01 compared to Avitene and DFC.

Porosity plays an important role in biological sponges. The DFC / Silk sponge showed higher porosity (73.79 ± 5.380) than the other sponges, indicating that the addition of silk has a significant effect on the porosity of the DFC sponge, These results were consistent with the SEM image of the cross section of the sponge.

Water uptake was also an important factor in the evaluation of hemostatic and wound dressing materials. The water uptake of all sponges investigated was about 90%, indicating that silk had no particular effect on the water uptake of the DFC sponge.

Evaluation of cell viability by CCK-8 assay

The sterilized sponges of Examples and Comparative Examples were standardized to a circle having a diameter of 8 mm, and NIH 3T3 fibroblasts were inoculated in one well at a density of 2 x 10 ⁴ . Each sponge was placed in the bottom of the Transwell culture system, and the transwell culture system was transferred to the cell culture medium for 1, 3, and 5 days. For each period, the culture solution: CCK-8 solution was mixed at a ratio of 9: 1 by volume and incubated for 2 hours. Absorbance was measured at 450 nm using an ELISA microplate reader (Molecular Devices, SpectraMax ^® Plus 384, Sunnyvale, Calif., USA) and is shown in FIG. The values in Fig. 4 were expressed as mean + standard deviation (n = 3).

NIH3T3 fibroblasts gradually increased cell growth, which means that each sponge provided an appropriate environment for the cell cycle. All sponges showed similar growth patterns during incubation time. From these results, it can be seen that each sponge has no toxic effect.

The whole blood clotting experiment

The examples and the comparative examples were prepared to have a size of 0.5 cm x 0.5 cm, and 0.2 ml of rat blood was dropped and placed in a tube. Then, 20 μl of CaCl ₂ solution (0.2 M) was dropped and solidified. After reacting for 10 minutes at 37 rpm at 30 rpm, 25 ml of distilled water was added to the tube to dissolve erythrocytes without thrombus formation. The negative control was prepared by adding 25 ml of distilled water to 0.2 ml of blood. The absorbance (D _s ) of the hemoglobin solution at 540 nm was measured and the absorbance (D _o ) of the hemolyzed whole blood at 0.2 ml was measured at 540 nm. The blood clotting index (BCI) was calculated by the following equation (3), and the results are shown in FIG. The results in Fig. 5 are expressed as mean ± standard deviation (n = 3) and are * p <0.05 and ** p <0.01 compared to Avitene and DFC.

&Quot; (3) &quot;

BCI = D _s / D _o

Here, D _s means hemoglobin solution absorbance, and D _o means absorbance of hemolyzed whole blood. The BCI value reflects free red blood cells that are not trapped by clotting factors, while the lower BCI value represents better coagulation capacity.

The BCI values of Avitene, DFC and Silk were measured as 0.045 ± 0.0024, 0.0519 ± 0.0069 and 0.051 ± 0.0024, respectively. On the other hand, the DFC / Silk sponge was measured at the lowest BCI value of 0.0329 ± 0.0034. This means that the DFC / Silk sponge has the best blood coagulation capacity than any other sponge. From this result, it can be seen that the addition of the silk fibers to the DFC increases the blood coagulation capacity of the DFC sponge. Collagen fibers are known to promote platelet adhesion, coagulation factor binding and induce rapid coagulation. It has been found that the addition of silk fibers can increase blood coagulation capacity in vitro.

The femoral artery hemorrhage rat model

In the hemostasis experiments of the Examples and Comparative Examples, male Sprague-Dawley rats weighing 250-300 g were used as experimental animals. The animal room was adjusted to a temperature of 25 ± 1 ° C, and the contrast was automatically adjusted for 12 hours day and night. In addition, feed and water were freely taken and all experimental animals were adapted to the animal room environment for 1 week and used in the experiment. 3% isoflurane was used to induce respiratory anesthesia in rats. The unilateral inguinal part of each rat was dissected to expose the femoral artery and the femoral vein, and the bleeding was induced by cutting the blood vessel using a surgical mass. 1cm x 1cm covering the size sponge of the bleeding site by using a tensile compression testing machine (Digital Push Pull Gauge ^®, SUNDOO, China) to exert a constant pressure of 5N press 30 seconds was observed for bleeding effect, in Figure 6 the results Respectively.

The blood absorption rates were in the order of Silk, Avitene, DFC / Silk and DFC respectively. In Figure 6E, the bleeding was completely stopped after treatment with Avitene and the bleeding site was observed to be clean. When the wound was covered with a DFC sponge, no hemostasis was achieved during the experiment (B, F). Similarly, bleeding did not completely stop when the wound was treated with a silk sponge (C, G). On the other hand, in the case of DFC / Silk sponge, it was observed that the bleeding was completely stopped by the transparent wound area (D, H). From this, it was found that the incorporation of silk into the DFC increases the hemostatic properties of the DFC sponge in vivo.

All statistical data from the above example were analyzed using Graphpad Prism 7.0 and all statistics were evaluated using ANOVA and post hoc Bonferroni tests. All data were expressed as mean ± SD, and p <0.05 was considered statistically significant.

Through these experiments, it was found that the DFC / Silk sponge (example) had structural characteristics of larger interconnected pores than all other sponges, and the incorporation of SF significantly increased the porosity of the DFC sponge, Of the total weight of the sample. From the results of the whole blood coagulation experiment, it was found that the DFC / Silk sponge showed better coagulation ability than Avitene in vitro. From the rat femoral bleeding test, DFC-based sponge showed similar hemostatic ability to avetene I could.

It is to be understood that the present invention is not limited to the above embodiments and various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims (8)

A porous sponge for hemostasis, comprising a collagen-silk hybrid structure. The method according to claim 1,
A porous sponge for hemostasis, characterized in that it is collagen derived from an animal. 3. The method according to claim 2,
A porous sponge for hemostasis, characterized in that it is a collagen derived from a duck foot. A first step of dissolving an animal collagen in an acid solution to prepare a collagen solution;
A second step of mixing a silk solution with a collagen solution to prepare a collagen-silk mixed solution; And
And a third step of lyophilizing the collagen-silk mixed solution to prepare a collagen-silk hybrid porous sponge. 5. The method of claim 4,
Wherein the animal collagen in the first step is a collagen derived from a duck foot. 5. The method of claim 4,
Wherein the collagen solution in the first step is 2-5% w / v (g / ml). 5. The method of claim 4,
Wherein the silk solution and the collagen solution are mixed at a volume ratio of 1: 1 in the second step. 5. The method of claim 4,
Wherein the collagen-silk mixed solution is freeze-dried at 70 to 90 ° C for 12 to 48 hours in the third step.

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