KR102038560B1 - A preparation method of an porous hemostatic agent using wood based-oxidized cellulose and silk fibroin - Google Patents

Abstract

The present invention relates to a method for preparing a porous support for wound healing or hemostasis comprising an aqueous solution of silk fibroin and oxidized cellulose nanofiber solution.
The porous hemostatic agent is excellent in biocompatibility, cell proliferation effect, swelling ability, and excellent wound healing effect to absorb blood and wound exudates when implanted in vivo and promote tissue regeneration, wound healing agent, It can be used as a hemostatic or wound dressing.

Description

A preparation method of an porous hemostatic agent using wood based-oxidized cellulose and silk fibroin}

The present invention relates to a method for producing a porous support for wound treatment or hemostasis comprising an aqueous solution of silk fibroin and oxidized cellulose nanofiber solution.

Delayed wound healing is one of the most common postoperative complications. In general, wound treatment consists of an inflammatory stage, an epithelial stage, a proliferation stage, and a maturation stage. If the inflammatory stage lasts for a long time, the next stage is delayed. Short-term inflammatory reactions reduce the chance of bacterial penetration and cause the production of several types of cytokines and growth factors associated with wound healing, while long-term inflammatory reactions exacerbate wound sites and increase chronic infection.

The goal of wound healing is to absorb wound exudates, induce wound healing as soon as possible and accelerate the healing process. In order to produce wound dressings that speed up the healing and healing of aesthetics, there have been a number of studies to develop novel bioactive compounds and polymers. In addition, many wound healing agents have been developed that can absorb wound exudate, prevent infection, and easily circulate oxygen in the wound site. However, it was still not satisfactory.

Commercially available wound dressings, on the other hand, can be classified as passive, interactive or bioactive products. Passive products include gauze dressings that are non-obstructive and are used to absorb wound exudates and cover the wound to restore skin function. Interactive dressings can be used in the form of films, foams, hydrogels and hydrocolloids and are semi-closed or closed. Such dressings act as a barrier against bacteria from penetrating the wound.

Wound healing agents must be compatible with the tissue, are non-toxic, do not penetrate external microorganisms, have water vapor permeability similar to normal skin, adhere to the wound surface quickly and consistently, and can withstand linear and shear stresses, It has to be acceptable, long life and low cost. In addition, improved wound healing agents should be used to improve wound healing properties.

Thus, in the present invention, a novel porosity that quickly absorbs wound exudates, retains moisture at the wound site, promotes epidermal growth, and can be readily removed after application, and maintains tissue temperature and blood flow at the wound site To develop a porous support for wound healing or hemostasis of the structure.

Republic of Korea Patent Registration 10-0875136

It is an object of the present invention to provide a method for preparing a wound support or hemostatic porous support, comprising the step of preparing a wound support or hemostatic porous support.

Another object of the present invention is to provide a porous support for wound treatment or hemostasis prepared by the above method.

The inventors confirmed that the porous support including the aqueous solution of silk fibroin and the oxidized cellulose nanofiber solution has excellent swelling property, no cytotoxicity, excellent cell proliferation effect, and excellent wound healing effect in vivo, It was confirmed that the hemostatic agent or wound dressing can be used to complete the present invention.

As one aspect for achieving the above object,

Dissolving silk fibroin in water to prepare an aqueous silk fibroin solution;

Mixing the silk fibroin aqueous solution and the cellulose oxide nanofiber solution to form a slurry;

Filling the slurry into a mold;

Lyophilizing the packed slurry to prepare a porous support; And

Crosslinking the lyophilized porous support with a crosslinking agent to prepare a porous support for wound treatment or hemostasis,

Provided is a method of preparing a porous support for wound healing or hemostasis.

Method for producing a porous support for wound treatment or hemostasis of the present invention comprises the step of dissolving silk fibroin in water to prepare a silk fibroin aqueous solution.

In the present invention, the silk fibroin was dissolved in water to prepare a silk fibroin aqueous solution, the aqueous solution of the silk fibroin prepared may have a concentration of 1 to 12% (w / v), specifically 2% (w / v) It may have a concentration of.

In one embodiment of the present invention specifically prepared 2%, 5% and 10% (w / v) silk fibroin aqueous solution.

Method for producing a porous support for wound healing or hemostasis of the present invention comprises the step of mixing the silk fibroin aqueous solution and oxidized cellulose nanofiber solution to form a slurry.

In the present invention, the silk fibroin aqueous solution and the oxidized cellulose nanofiber solution may be mixed in a volume ratio of 1 to 3: 1, specifically, may be mixed in a volume ratio of 2: 1. The slurry may be formed by stirring for 20 to 28 hours at 45 ~ 55 ℃.

In an embodiment of the present invention, the silk fibroin aqueous solution and TOCN (TEMPO-oxidized cellulose nanofibers, TEMPO-oxidized cellulose nanofibril, TOCN) solution is mixed at a volume ratio of 2: 1 by a magnetic stirrer at 50 ° C. for 24 hours. Mixing to form a slurry.

Biocompatibility, biodegradability and recyclability are properties that make biological macromolecules beneficial for wound healing. The silk fibroin is a protein derived from silkworm (silk moth; Bombyx mori), which has remarkable mechanical properties, controllable biodegradability and proper biocompatibility. In addition, silk fibroin derived from a porous material may be an excellent candidate for skin tissue repair and wound healing.

In the present invention, the oxidized cellulose nanofibers may be derived from wood. Nanocellulose is a biomaterial candidate. Nanocellulose can be used in regenerative medicine and wound healing applications such as tissue engineered meniscus, scaffolds such as blood vessels, ligaments and tendon substitutes, which have significant physical properties, special surface chemistry, biodegradability and bioavailability. This is due to good biological properties such as suitability and low toxicity.

To make cellulose nanofibers, it is necessary to separate the cellulose fibers of wood. Cellulose fibers were difficult to separate with high efficiency because the fibers and the fibers are strongly bonded, but the functional catalyst 2,2,6,6-tetramethyl-piperidine-1-oxyl (2,2,6,6 When oxidized with -tetramethyl-piperidin-1-oxyl (TEMPO), the cellulose fibers can be made into uniform cellulose nanofibers. The oxidized cellulose nanofibril (OCN) is biodegradable from wood-based biomass. In addition, since it has various excellent properties such as high crystallinity, excellent heat resistance, and high transparency, it can be used for polymer composite materials, medical engineering materials, and membranes.

In the present invention, a porous support for wound treatment or hemostasis was prepared using oxidized cellulose nanofibers obtained by TEMPO mediated oxidation of nanocellulose. The oxidized cellulose is suitable for treating bleeding conditions and healing wounds due to its good water absorption.

Method for producing a porous support for wound treatment or hemostasis of the present invention comprises the step of filling the slurry into a mold (Mold).

The mold may have a diameter of 4.3 to 5.3 cm and a thickness of 0.4 to 0.6 cm.

In one embodiment of the present invention, the slurry was filled in a polyethylene mold having a diameter of 4.8 cm and a thickness of 0.5 cm.

Method for producing a porous support for wound healing or hemostasis of the present invention comprises the step of lyophilizing the filled slurry to prepare a porous support.

In one embodiment of the present invention, the slurry filled in the mold is frozen at -20 ° C for 12 hours and then placed in a lyophilizer for 48 hours to lyophilize to prepare a porous support.

Lyophilization is very useful for preparing porous structures. By the lyophilization process of the packed slurry, pores are generated to form a porous structure. In other words, when the colloidal suspension is lyophilized, a porous material is formed and the porous structure is actually a replica of the frozen crystals of ice. The porous structure is effective to maintain biological activity, and when the porous structure is derived from water rather than an organic solvent, it is confirmed that the spreading of fibroblasts is improved, so that the silk fibroin is dissolved in water using water as a solvent. Was used.

Method for producing a porous support for wound treatment or hemostasis of the present invention comprises the step of crosslinking the lyophilized porous support with a crosslinking agent to prepare a porous support for wound treatment or hemostasis.

In one embodiment of the present invention by cross-linking the lyophilized porous support with a crosslinking agent to prepare a porous support for wound treatment or hemostasis. Specifically, porous scaffolds were cross-linked in 80% ethanol containing EDC and NHS in a molar ratio of 5: 2 overnight at 4 ° C. for 24 hours. Then, the support was washed with distilled water to remove the remaining EDC, frozen and freeze-dried. By the freeze-drying process as described above, the structure of the porous support for wound treatment or hemostasis of the present invention is not broken, and the structure can be maintained.

In one embodiment of the present invention, TOCN-silk fibroin 2% support, TOCN-silk fibroin 5% support and TOCN-silk fibroin 10% support, including 2%, 5% and 10% (w / v) aqueous solution of silk fibroin It was confirmed that the swelling property was increased in the TOCN-silk fibroin 2% support and the TOCN-silk fibroin 5% support, and the high PBS absorption ability was confirmed, that it can actually absorb blood and wound exudates well in vivo. Could know. Thus, the TOCN-silk fibroin support can be used as a wound treatment, hemostatic agent or wound dressing.

In addition, in one embodiment of the present invention it was confirmed that the cell viability, biocompatibility and L929 supportability (suportiveness) in the support containing silk fibroin.

In addition, in one embodiment of the present invention confirmed the wound healing effect of the TOCN-silk fibroin support in vivo, after 7 days, the wound closure rate of the control group was 15 ± 0.5%, but the treatment group TOCN, The wound closure rate of TOCN-silk fibroin 2% and TOCN-silk fibroin 5% increased to 18 ± 0.33%, 36.66 ± 0.6% and 33.3 ± 0.3%, respectively. After 14 days, the wound closure rate of the control group was 28.33 ± 0.3 %, But the wound closure rates of the treatment groups TOCN, TOCN-silk fibroin 2%, and TOCN-silk fibroin 5% increased to 38.33 ± 0.3%, 78.66 ± 0.3%, and 71.66 ± 0.6%, respectively, compared to TOCN- The wound healing rate of the silk fibroin support was fast, and specifically, the wound healing effect of 2% TOCN-silk fibroin was confirmed to be the best.

In addition, as a result of confirming the wound healing effect through the tissue staining picture, the wound of the control group was not completely closed (FIG. 6A), and the formation of new neo-epidermis was not uniform throughout the wound site. However, in the TOCN-silk fibroin 2% (FIG. 6B) group and the TOCN-silk fibroin 5% (FIG. 6C) group, a thick new epidermis appeared 14 days after the support was treated, and silk fibroin addition improved the wound healing performance of TOCN. It could be seen to promote.

 Therefore, it was confirmed in vitro and in vivo that the wound support or hemostatic porous support of the present invention was excellent in biocompatibility, cell proliferation effect, and swellability, and excellent in wound healing effect, and absorbed blood and wound exudates when implanted in vivo. And it has the advantage of promoting tissue regeneration, it can be used as a wound healing agent, hemostatic agent or wound dressing.

As another aspect for achieving the above object, there is provided a porous support for wound treatment or hemostasis produced by the manufacturing method.

In the present invention, the description of the porous support for wound treatment or hemostasis is as described above.

The present invention relates to a method for producing a porous support for wound treatment or hemostasis comprising an aqueous solution of silk fibroin and oxidized cellulose nanofiber solution.

The wound support or hemostatic porous support is excellent in biocompatibility, cell proliferation effect, and excellent in wound healing effect to absorb blood and wound exudates when implanted in vivo and promote tissue regeneration. It can be used as a wound healing agent, hemostatic agent or wound dressing.

1: scanning electron micrograph of TOCN (A), TOCN-silk fibroin 2% (B), TOCN-silk fibroin 5% (C), TOCN-silk fibroin 10%; (D) EDS profile (inset); Pore Diameter Curve (E) for Incremental Pore Volume
2: FT-IR spectrum (A) and PBS absorption behavior (B) of various TOCN and silk fibroin compositions
Figure 3: L929 MTT assay (A) and cell proliferation (B) for various TOCN-silk fibroin compositions after different incubation times.
4: Promoting wound treatment of TOCN-silk fibroin in a rat incision wound model (A); Effect of wound healing on the aspect of wound closure over time in the rat incisional wound model (*: p <0.001) (B)
5: Light micrographs (A, B, C) showing wound treated tissues of various treatment groups after 7 days. H & E stained pictures are A, B panels and Mason Trichrome stained pictures are C panels. In the figure, black circles represent unrecovered wounds. GT = granular tissue, NE = neocortex
6: Light micrographs (A, B, C) showing wound treatment of various treatment groups after 14 days. In the figure, black circles represent unrecovered wounds. GT = granular tissue, NE = neocortex
7: Representative blots of protein concentrations (changes in protein expression fold relative to control) of fibronectin, collagen I, PECAM1, GAPDH obtained in Western blot analysis after 7 days (A) and 14 (B). The results were obtained from three separate experiments. SF = silk fibroin and **** P <0.001 means statistically significant.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

Example  One: TOCN - silk  Preparation of Fibroin Porous Support

In the present invention, a porous support having a different content of silk fibroin was prepared as follows.

Silk fibroin was dissolved in water at three different concentrations (2%, 5% and 10% (w / v)) to prepare an aqueous silk fibroin solution. The aqueous solution of silk fibroin and TOCN (TEMPO-oxidized cellulose nanofiber, TEMPO-oxidized cellulose nanofibril, TOCN) solution was mixed at a volume ratio of 2: 1, and stirred at a magnetic stirrer at 50 ° C for 24 hours to form a slurry. . Thereafter, the slurry was filled into a polyethylene mold having a diameter of 4.8 cm and a thickness of 0.5 cm. The packed slurry was frozen at −20 ° C. for 12 hours and then placed in a lyophilizer for 48 hours to be lyophilized to prepare a porous support. The lyophilized porous support was crosslinked with N-Ethyl-N '-(3-dimethylaminopropy) carbodiimide hydrochloride (EDC) and NHS (Hydroxy-2,5-dioxopyrolidine-3-sulfonic acid sodium) in a molar ratio of 5: 2. Cross-linked for 24 hours at 4 ° C. in 80% ethanol containing overnight. This process was repeated three times (every 8 hours) to crosslink the support to prepare a porous support for wound healing or hemostasis. The support was washed 5 times with distilled water for 10 minutes to remove the remaining EDC, and frozen at -20 ° C for 8 hours and then freeze-dried.

Example  2: PBS Uptake Behavior

The water intake capacity of the four support groups was determined by conventional mass spectrometry. A 1 × 1 cm size and 0.4 mm thick support was prepared. The mass (W 0) of the triplicated samples was each measured and placed in a suitable well plate and equilibrated at room temperature. 2 ml of PBS was added to the support and wetted. The well plates were then placed in a 37 ° C. CO 2 incubator. At some point (1 hr, 2 hr, 5 hr, 10 hr, 24 hr, 36 hr, 48 hr), the support was removed from the well plate and excess water was wiped off with filter paper. Thereafter, the mass of the support was measured. Thereafter, the mass change amount Wt was recorded. PBS absorption was calculated according to the following equation.

[Equation 1]

Where W ₀ is the initial mass and W _t is the mass of the sample at various time points. PBS absorption rate (P _α ) is measured by the above equation (1).

Example  3: cell culture

L929 fibroblasts were passaged in primary medium consisting of 500 ml RPMI, 10% FBS, and 1% PS. The experiment was performed after four passages of cells and the medium was changed three times a week.

Example  4: TOCN - silk  Fibroin Support Sterilization Process

Various types of support were placed in a 24-well plate. Thereafter, the support was sterilized in a simple and convenient manner. In summary, the support was sterilized by soaking in 70% ethanol for 30 minutes under UV irradiation. Thereafter, the support was washed three times for 10 minutes with PBS to remove ethanol from the support. 1 mL of basic RPMI medium was added to the sample to increase the hydrophilicity of the support. After equilibrating the support with the medium for 24 hours, the medium was removed from the support and cells were seeded on the support.

Example  5: MTT  analysis

MTT assays were performed using standard test protocols (ISO10993-5: 2009 (E)) to determine cell viability in various TOCN-silk fibroin compositions. Samples were inoculated with 1 mL of L929 fibroblast suspension (about 2.5 × 10 ⁴ ). Cells inoculated on the sterile support were allowed to stand for 1 day, 3 days and 7 days under 37 ° C., 5% CO 2 conditions. After incubation for the above time, MTT solution (5 mg / ml, 100 μL) was added to the wells and the plates were incubated at 37 ° C. for 4 hours. Finally, the solution was removed from the wells, 1000 μL of DMSO was added to the wells and incubated for 1 hour to dissolve formazan crystals. The absorbance of the solution at 595 nm wavelength was determined using an ELISA reader. The support was tested three times.

Example  6: cell proliferation

The sterilized support was inoculated with 1 mL of L929 fibroblast suspension (about 2.4 × 10 ⁴ ). These supports were then maintained at 37 ° C. under 5% CO 2 conditions for 1 day, 3 days and 7 days. After incubation for the above time, the support was washed twice with PBS (37 ° C.) and then fixed with warm (37 ° C.) 4% formaldehyde (Sigma Aldrich) at room temperature for 10 minutes. After washing three times with PBS, 0.5% Triton X-100 (Sigma Aldrich) was passed through the cells for 10 minutes. After this procedure, 2.5% bovine serum albumin was added as a blocking reagent for 60 minutes. Cells were immunostained in dark at 4 ° C. overnight using fluorescein isothiocyanate-conjugated Phalloidin (FITC, 25 μg / mL, Sigma Aldrich) combined with fluorescein isothiocyanate. The nucleus is Hoechst 33342 (2 ′-[4-ethoxyphenyl] -5- [4-methyl-1-piperazinyl] -2,5'-bi-1H-benzimidazole trihydrochloridetrihydrate; invitrogen ) Stain for 10 minutes at 1 μg / mL. The support was washed three times and then naturally dried. The support was then visualized with confocal fluorescence microscopy (Olympus, FV10i-W, Tokyo, Japan) and analyzed with the FV10i-ASW 2.0 viewer software.

Example  7: animal experiment

In the present invention, 24 healthy adult male rattus norvegicus rats (average weight ¼ 230 g) were selected. Animals were placed in standard cages, housed in a room with controlled humidity and temperature (12 ° C) with a 12-hour contrast cycle, and free to eat the same amount of standard food and water according to the Soonchunhyang National University Animal Ethics Committee It was. Mice were adapted to this environment 7 days before the experiment. In the present invention, mice were randomly divided into four groups: control group, TOCN, TOCN-silk fibroin 2%, and TOCN-silk fibroin 5%. Each group included three rats. The control group was not treated.

Upon wound formation, mice were anesthetized with isoflurane (pyramal, USP). The incision was washed with povidone iodine solution. Scissors and forceps were used to form a full thickness 1 × 1 cm incision wound at the back of the rat. In order to fix the support to the wound, each type of support was sutured to the edge of the wound using a surgical suture.

Example  8: wound healing active

Wound shrinkage was observed using a digital camera to capture a photo of the full thickness of the wound. Photos were taken 7 and 14 days after wound formation (Fig. 4A). The wound area was photographed and the wound diameter was measured to quantify the wound closure rate (%) according to Equation 2.

[Equation 2]

Skin biopsies were performed on days 7 and 14. To this end, rats were euthanized using an excess of diethyl ether (Pop., Korea). Skin samples of full thickness were taken from the wound. Tissue samples were immediately fixed at room temperature for 48 hours using 10% formaldehyde (Duksan Pharmaceutical Co., Ltd.) solution. Tissue samples were taken from the animals and stored at −80 ° C. until used for Western blot protein analysis.

Example  9: H & E dyeing and Mason Trichrome  dyeing ( Masson ’s Trichrome  staining)

Tissue samples were taken out of formaldehyde solution and washed with deionized water for 1 hour. It was then dehydrated with 50%, 70%, 80%, 90%, 95% and 100% ethanol solutions. Xylene was used to remove the alcohol before embedding in paraffin wax. Samples were cut into 5 ± 2 micrometer thick sections using a microtome (Thermo Scientific, USA), deparaffinized, hematoxylin and eosin (H & E) staining and Mason Trichrome staining ( Sarker, Amirian, Min, and Lee, 2015) method. Tissue sections were observed with a BX53 light microscope (Olympus) and photographed with an Olympus DP72 camera. Images were analyzed using the accompanying Cellsens Software.

Example  10: Weston Blot  analysis

For western blot analysis, use a PRO-PREP buffer (20 mg / 600 μl, Intron, Korea) to dissolve frozen skin sections on ice for 30 minutes and centrifuge the lysates for 20 minutes at 12000 rpm and 4 ° C. Separated and separated. Bradford analyzer (Biorad, UAS) was used to determine the protein concentration of the lysate. Equal amounts of total protein for skin lysate were subjected to 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by polyvinylidene fluoride (PVDF) membrane (0.22 μm, Millipore) Was electrically transferred to. Blots were blocked with blocking buffer (3% BSA with 0.05% Tween-20 in TBS) at room temperature for 1 hour and then incubated with primary antibody overnight at 4 ° C. After incubation for 1 hour with HRP-bound secondary antibody (1: 10000), the bands were visualized and visualized using an improved chemiluminescent sample (Ez-Western Lumi Pico, Donggen, Korea) and X-ray film. . Primary antibodies used in Western blot analysis were goat anti-PECAM1 (1: 500; Santa Cruz Biotechnology, USA), rabbit anti-Fibronectin (1: 1000; Abcam, UK), rat anti-Collagen1 (1: 1000; Santa Cruz Biotechnology, USA) and rabbit anti-GAPDH (1: 5000; Santa Cruz Biotechnology, USA) were used diluted.

Experimental Example  One: TOCN - silk  Morphology and Pore Size Analysis of Fibroin

1 shows scanning electron micrographs of TOCN (A), TOCN-silk fibroin 2% (B), TOCN-silk fibroin 5% (C), TOCN-silk fibroin 10%, (D) EDS profile (inset) Indicates.

The support prepared after freeze drying is porous and looks like a sponge. (Figures 1A, 1B, 1C and 1D). Changing the ratio of TOCN and silk fibroin also changes the sample porosity. As the silk fibroin ratio increased, the support formed pores of larger size. Morphology as well as morphology and interspace interactions are equally important for cell inoculation, migration, good cell adhesion, tissue growth, gene expression and nutrient transport inside the support.

Porosity is a variable that measures the voids (pores) inside a material and is expressed as the ratio of pore volume to total volume. Most human cells have a size in the range of 2-120 μm. The small pore size of surgical wound dressings is important in preventing the migration of fibroblasts and keratin to the pores during wound treatment. It is also possible to prevent secondary damage when removing the dressing. The average pore diameter of the TOCN-silk fibroin support that can be used as a wound treatment is 40 to 80 μm.

In the present invention, the pore sizes of the TOCN-silk fibroin 2% support, the TOCN-silk fibroin 5% support, and the TOCN-silk fibroin 10% support are 55.95 μm, 41.75 μm and 81.22 μm, respectively, and the TOCN-silk fibroin 5% support is The pore size was the smallest. In the case of TOCN-silk fibroin 5% support, the smallest pore size was expected to be good capillary action. Smaller pore sizes promote maintenance of wound exudates. In addition, the excellent liquid absorption / retention properties of the support are one of the most important physical properties of the dressing material. The good liquid absorption / retention capacity of the support helps to effectively absorb wound exudates and to provide a consistent and humid wound environment in the dressing.

Experimental Example  2: TOCN - silk  FT-IR Analysis of Fibroin

The FTIR spectra of TOCN and TOCN-silk fibroin are shown in FIG. 2 (A). The most pronounced spectral bands at 3326 cm ⁻¹ and 1040 cm ⁻¹ correspond to the stretching vibrations of OH and CO ethers. The peak at 1154-1159 cm ⁻¹ represents the CC ring stretching band and was found in all four samples. The FTIR spectra provide information to identify proteins and their related structures. Silk fibroin is a protein and its absorbance by various samples varies with protein chain morphology. The spectrum of TOCN is obtained at 1606 cm ⁻¹ by the C═O functional group. Characteristic absorption bands are shown. In addition, the spectra of 2% TOCN-silk fibroin, 5% of TOCN-silk fibroin and 10% of TOCN-silk fibroin show characteristic absorption bands representing amides I, II. Amide I is associated with C═O (carbonyl) stretching and occurs at 1580-1590 cm ⁻¹ . Amide II ranges from 1540-1520 cm ⁻¹ and is associated with NH bending and CH stretching vibrations. TOCN-silk fibroin supports have characteristic vibration bands for amide I in the range 1622-1629 cm ^-1 (CO stretch) and characteristic vibration bands for amide II (secondary NH bending) at 1540-1520 cm ^-1 ). This confirmed that the β-sheet form and the secondary structure of silk fibroin were maintained even when TOCN was added (FIG. 2A).

Experimental Example  3: TOCN - silk  Fibroin Swelling force  analysis

2B shows the PBS absorption behavior of various TOCN and silk fibroin compositions. Swelling of the support was observed every hour until mass gain equilibrium was achieved. After 48 hours, the support showed no significant mass increase or swelling, demonstrating equilibrium of swelling conditions. The high porosity allowed the support to expand to 3000% of its initial mass (FIG. 2B). This range is advantageous for maintaining the moisture content of the wound site and preventing drying.

Referring to FIG. 2B, TOCN itself is swellable and increases in swelling when silk fibroin is added. Swelling of the support was increased in 2% support of TOCN-silk fibroin and 5% support of TOCN-silk fibroin. However, when a large amount of silk fibroin (greater than 5%) is added, the support is easily broken by PBS addition and degrades. Successful wound dressings can prevent the accumulation of exudate at the wound site, so it is important to quickly absorb blood and wound exudate. As a result, the TOCN-silk fibroin support is suitable for use in hemostatic agents and wound dressings due to its high swelling power. Every hour TOCN-silk fibroin 2% support and TOCN-silk fibroin 5% support showed the maximum PBS absorption. In fact, it was found that it can absorb blood and wound exudate well in vivo.

Experimental Example  4: TOCN - silk  Cytotoxicity and Cell Proliferation Analysis of Fibroin

L929 cells were used to assess the cytotoxicity of different forms of TOCN-silk fibroin scaffolds using colorimetric MTT assay. The results are expressed in optical density over days. Cell culture plate (TCP) was used as a control. 3A shows MTT analysis of L929 for various TOCN-silk fibroin compositions after different incubation times. As a result, L929 cultured on two kinds of supports showed a growth pattern of cell number increasing with time during the culture period (FIG. 3A). In addition, the cell viability of the support comprising 5% and 10% silk fibroin was higher compared to the cell viability of L929 on the support comprising 2% silk fibroin. Cell survival data of the support including silk fibroin showed that silk fibroin has improved biocompatibility and L929 support. Sericin sericin is believed to play an important role in the biocompatibility of silk fibroin-derived biomaterials.

All materials that can be used to treat wounds must be biocompatible and capable of tissue regeneration. Three types of scaffolds were used for cell proliferation studies with L929. 3B shows cell proliferation of L929 for various TOCN-silk fibroin compositions after different incubation times. In the case of a support with a high content of silk fibroin, better cell proliferation was observed, confirming that the results were consistent with the MTT analysis.

In conclusion, silk fibroin nanofibers have been shown to have remarkable results in cell adhesion and proliferation of fibroblasts in vitro.

Experimental Example  5: raw vivo ) TOCN - silk  Fibroin's Wound Healing Effects

The present inventors investigated the effect of treating the wound on rats of the TOCN-silk fibroin support. In the present invention, a wound of a circular full thickness of 10 mm in diameter was formed on the back of the rat. 4 promotes wound healing of TOCN-silk fibroin in a rat incision wound model (A); Wound healing effects in terms of wound closure over time in the rat incisional wound model. Representative wound treatment pictures at various time points are shown in FIG. 4 (A). Wound contractions usually occur between one and two weeks after injury. Most of the wound tissue in the control and three other groups contracted within 14 days. New epidermis (neoepidermise) appeared 14 days after wound formation. Skin scabs were removed after 7 days in all groups (FIG. 4A). Epithelial cells grew gradually under tissue scab. The wound shrinkage effect of various types of support is expressed as percentage wound closure (%) over time (FIG. 4B). After 7 days, the wound closure rate of the control group was 15 ± 0.5%, while the wound closure rates of the treated groups TOCN, TOCN-silk fibroin 2% and TOCN-silk fibroin 5% were 18 ± 0.33%, 36.66 ± 0.6% and 33.3, respectively. Increased to ± 0.3% (FIG. 4B). After 14 days, the wound closure rate of the control group was 28.33 ± 0.3%, but the wound closure rates of the treated groups TOCN, TOCN-silk fibroin 2% and TOCN-silk fibroin 5% were 38.33 ± 0.3%, 78.66 ± 0.3% and 71.66, respectively. Increased to ± 0.6% (FIG. 4B). The healing rate of the TOCN treated wound was lower than the TOCN-silk fibroin support. In particular, wounds treated with 2% TOCN-silk fibroin were the fastest healed.

In the present invention, the TOCN-silk fibroin 5% scaffold has the smallest pore size and excellent cell proliferation effect in vitro, so that the wound healing effect is expected to be the best, but in animal experiments, TOCN-silk fibroin 2 The wound healing effect of% scaffold was the best.

Experimental Example  6: TOCN - silk  Fibroin Wound Treatment Activity Measurement (Tissue Staining)

As mentioned earlier, there are four basic steps in wound healing. This is a complex process that involves a series of overlapping steps, aimed at complete remodeling. In the present invention, wound tissue was treated by ablation for further histological examination of the wound healing process. 5 is a light micrograph showing wound treatment tissues of various treatment groups after 7 days. H & E stained pictures are A, B panels and Mason Trichrome stained pictures are C panels. In the figure, black circles represent unrecovered wounds. GT = granular tissue, NE = neocortex. 5 and 6 show representative tissue sections of four groups of rat skin, showing evidence of wound treatment. Of the four groups, wound shrinkage of 2% TOCN-silk fibroin and 5% of TOCN-silk fibroin was better than the other groups.

When a wound occurs, a series of events occur to repair the wound. Skin cell migration occurs from the edge of the wound toward the center of the wound for re-epithelialization, an important component of wound healing. After 2 to 3 days, fibroblasts migrate to the wound for granulation tissue formation. 6 is a light micrograph (A, B, C) showing wound treatment of various treatment groups after 14 days. In the figure, black circles represent unrecovered wounds. GT = granular tissue, NE = neocortex. In the present invention, the development of new epidermis was significant after 14 days, but the wound of the control group was not completely closed (FIG. 6A), and the formation of new neo-epidermis was not uniform throughout the wound site. In addition, high magnification photographs of the 2% TOCN-silk fibroin (FIG. 6B) group and the 5% TOCN-silk fibroin (FIG. 6C) group showed a thick new epidermis 14 days after treatment of the support. This demonstrates that the addition of silk fibroin to TOCN promotes wound healing performance of TOCN.

Collagen is the most abundant protein in the extracellular matrix (ECM). Tissue sections were subjected to Mason Trichrome staining to confirm collagen deposition on the skin defects for 7 days (FIG. 5C), at which time the collagen fibers were stained blue. After treatment with three kinds of support, collagen deposition was seen in 2% of TOCN-silk fibroin. High deposition of collagen fibers is evidence of wound healing. After the fibroblasts migrate, granule tissue is formed, and new epidermis is formed, the collagen is synthesized at the site of injury continuously for several weeks.

Experimental Example  7: TOCN - silk  Fibroin Weston Blot  analysis

Western blot analysis was performed in the wound treatment model to check the state of fibronectin and collagen on days 7 and 14 post-surgery (FIG. 7). FIG. 7 shows representative blots of protein concentrations (changes in protein expression fold relative to control) of fibronectin, collagen I, PECAM1, GAPDH obtained in Western blot analysis after 7 days (A) and 14 (B).

The results are expressed as changes in protein expression fold over the control. Western blot analysis of collagen and fibronectin showed that both of them were markedly synthesized at the wound site within 7 days (FIG. 7A). However, when the inflammatory phase of wound treatment was completed within 7 days, fibronectin levels decreased in the TOCN-silk fibroin 2% treatment group and the TOCN-silk fibroin 5% treatment group compared to the control group (FIG. 7A). Thus, the inflammatory phase was effectively completed in these TOCN-silk fibroin treated groups within 7 days. This demonstrates that silk fibroin has a significant effect on the expression of these proteins. However, fibronectin plays an important role in the movement and growth of cells during granulation tissue development and organization. Fibronectin expression was higher (1.62 fold) compared to the control group on day 14 after treatment with 2% TOCN-silk fibroin, because myofibroblasts attract fibronectin and reactivate the fibronectin during wound contraction. In addition, granulation tissue is rich in fibronectin, which helps mediate wound contraction through various interactions with collagen and other matrix proteins.

As can be seen in FIG. 7, collagen expression was increased at wound sites (1.01417 fold) treated with 2% TOCN-silk fibroin compared to the control, consistent with Mason Trichrome staining. These findings also demonstrate that silk fibroin is effective in treating wounds by the collagen synthesis pathway because silk fibroin promotes collagen synthesis and accelerates tissue remodeling of fibroblasts.

It has long been known that blood supply is an important factor in wound healing. Thus, the formation of new blood vessels is a basic step that allows mediators and control agents to reach the center of the treatment process. On days 7 and 14, PECAM1 (CD31) was markedly expressed at 1.03 fold (FIG. 7A) on day 7 and 1.7 fold (FIG. 7B) on day 14 in the 2% group of TOCN-silk fibroin compared to control. This is because upregulation of angiogenic growth factor and vascular endothelial growth factor coexists with ECM formation. In addition, silk fibroin can stimulate the release of epidermal growth factor and subsequent wound healing (HaeYong et al., 2008). Western blot analysis showed that CD31 (PECAM1) protein was significantly expressed in rat skin treated with TOCN-silk fibroin 2% support, which showed better angiogenesis with less amount of silk fibroin in wound healing material. Means to provide.

The expression of these proteins (fibronectin, collagen, PECAM1) changes at various stages of wound healing, and it is expected that the fibrous network will be restored during the long-term remodeling phase, resulting in a structure close to normal skin.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to the scope of rights.

Claims (9)

Dissolving silk fibroin in water to prepare an aqueous silk fibroin solution;
Mixing the silk fibroin aqueous solution and the cellulose oxide nanofiber solution to form a slurry;
Filling the slurry into a mold;
Lyophilizing the packed slurry to prepare a porous support;
Cross-linking the lyophilized porous support with a crosslinking agent to prepare a porous support for wound treatment or hemostasis,
The crosslinking agent is N-Ethyl-N '-(3-dimethylaminopropy) carbodiimide hydrochloride (EDC) and hydroxy-2,5-dioxopyrolidine-3-sulfonic acid sodium (NHS),
Method of producing a porous support for wound treatment or hemostasis with increased swelling. The method of claim 1, wherein the aqueous solution of silk fibroin has a concentration of 1 to 12% (w / v). The method of claim 1, wherein the aqueous solution of silk fibroin has a concentration of 2% (w / v). The method of claim 1, wherein the silk fibroin aqueous solution and the cellulose oxide nanofiber solution are mixed at a volume ratio of 1 to 3: 1, wherein the porous support for wound treatment or hemostasis. The method of claim 1, wherein the slurry is formed by stirring for 20 to 28 hours at 45 ~ 55 ℃, wound or hemostatic porous support for producing a method. The method of claim 1, wherein the oxidized cellulose nanofibers are derived from wood. The method of claim 1, wherein the mold has a diameter of 4.3 to 5.3 cm and a thickness of 0.4 to 0.6 cm. The method of claim 1, wherein the crosslinking agent is a molar ratio of N-Ethyl-N '-(3-dimethylaminopropy) carbodiimide hydrochloride (EDC) and NHS (Hydroxy-2,5-dioxopyrolidine-3-sulfonic acid sodium) in a molar ratio of 5: 2. A method for producing a porous support for wound treatment or hemostasis containing. A porous support for wound treatment or hemostasis with increased swelling property prepared by the method of any one of claims 1 to 8.

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