WO2022138206A1

WO2022138206A1 - Wound dressing material

Info

Publication number: WO2022138206A1
Application number: PCT/JP2021/045361
Authority: WO
Inventors: 田畑泰彦; 夏原久美子; 島田直樹
Original assignee: 日本毛織株式会社; 田畑泰彦
Priority date: 2020-12-22
Filing date: 2021-12-09
Publication date: 2022-06-30
Also published as: JPWO2022138206A1

Abstract

The present invention relates to a wound dressing material including a hydrogel nonwoven fabric having gelatin as a main component, wherein the hydrogel nonwoven fabric has a thickness retention rate of 86% or higher after six hours of collagenase treatment. The hydrogel nonwoven fabric preferably has a compressive strength retention rate of 70% or higher after six hours of collagenase treatment. Additionally, the hydrogel nonwoven fabric preferably does not include a cell growth factor. Provided thereby is a wound dressing material having improved early cell invasion and vascular invasion into the wound dressing material during use.

Description

Wound dressing

The present invention relates to a wound dressing used for repairing a wound such as a defect in a tissue such as skin or a wound.

In a wound dressing used for repairing a wound such as a defect in a tissue such as skin or a wound, a hydrogel-forming component capable of providing a moist environment is used on the side in contact with the wound site. As such a wound dressing, a sponge using a hydrogel-forming component such as collagen is widely used. Further, Patent Document 1 proposes a wound dressing containing a hydrogel fiber layer containing an organic fiber capable of forming a hydrogel.

Japanese Unexamined Patent Publication No. 2020-103502

However, wound healing requires early cell invasion and blood vessel invasion into the wound covering material after treating the wound site with the wound covering material, such as collagen sponge and the wound covering material described in Patent Document 1. Conventional wound dressings are required to improve early invasion of cells and blood vessels into the interior.

The present invention provides a wound dressing in which early cell invasion and blood vessel invasion into the inside of the wound dressing are improved during use in order to solve the above-mentioned conventional problems.

The present invention is a wound dressing containing a hydrogel nonwoven fabric containing gelatin as a main component, wherein the hydrogel nonwoven fabric has a thickness retention rate of 86% or more after 6 hours of treatment with collagenase. Regarding the covering material.

By using the wound dressing of the present invention, early cell invasion and blood vessel invasion into the inside of the wound dressing can be improved.

It is a photograph of the scanning electron microscope (100 times) of the hydrogel non-woven fabric in the dry state of Example 2. It is a photograph of the scanning electron microscope (100 times) of the collagen sponge in the dry state of Comparative Example 2. It is a photograph (40 times) showing the result of hematoxylin-eosin staining (HE staining) of the cross section (depth 200-400 μm) of the transplant material on the 10th day of transplantation using the hydrogel non-woven fabric of Example 1. The scale bar is 100 μm. same as below. It is a photograph (40 times) showing the result of immunostaining of CD31 (platelet endothelial cell adhesion molecule-1) of the cross section (depth 200-400 μm) of the transplant material on the 10th day of transplantation using the hydrogel non-woven fabric of Example 1. .. It is a photograph (40 times) showing the result of hematoxylin / eosin staining of the cross section (depth 200-400 μm) of the transplant material on the 10th day of transplantation using the collagen sponge of Comparative Example 2. It is a photograph (40 times) showing the result of immunostaining of CD31 (platelet endothelial cell adhesion molecule-1) of the cross section (depth 200-400 μm) of the transplant material on the 10th day of transplantation using the collagen sponge of Comparative Example 2. It is a photograph (40 times) showing the result of hematoxylin / eosin staining of the cross section (depth 400-600 μm) of the transplant material on the 7th day of transplantation using the hydrogel non-woven fabric of Example 2. It is a photograph (40 times) showing the result of immunostaining of CD31 (platelet endothelial cell adhesion molecule-1) of the cross section (depth 400-600 μm) of the transplant material on the 7th day of transplantation using the hydrogel non-woven fabric of Example 2. .. It is a photograph (40 times) showing the result of hematoxylin / eosin staining of the cross section (depth 400-600 μm) of the transplant material on the 7th day of transplantation using the collagen sponge of Comparative Example 2. It is a photograph (40 times) showing the result of immunostaining of CD31 (platelet endothelial cell adhesion molecule-1) of the cross section (depth 400-600 μm) of the transplant material on the 7th day of transplantation using the collagen sponge of Comparative Example 2. It is a schematic explanatory drawing of the nonwoven fabric manufacturing apparatus.

The inventors of the present invention have repeated studies in order to solve the above-mentioned problems. As a result, by using a hydrogel non-woven fabric containing gelatin as a main component and having a thickness retention rate of 86% or more after treatment with collagenase for 6 hours, early cell invasion and blood vessel invasion into the wound dressing during use ( It was found that angiogenesis) was improved.
In particular, by using a hydrogel non-woven fabric containing gelatin as a main component, blood vessels can invade the inside of the wound dressing, that is, angiogenesis can be promoted without using a cell growth factor, and the wound site can be affected. It induces angiogenesis and can be applied to diabetic patients and malignant tumor patients. In addition, the thickness retention rate after 6 hours of treatment with collagenase is 86% or more, so that when the wound dressing is used, for example, 1 to 2 weeks after transplanting the wound dressing, the wound dressing is covered. The communication hole structure that allows the invasion of cells and blood vessels into the material is maintained, and early angiogenesis can be induced.

In one or more embodiments of the present invention, the wound dressing comprises a hydrogel non-woven fabric containing gelatin as a main component. In one or more embodiments of the present invention, "having gelatin as a main component" means that gelatin is contained in an amount of 90% by mass or more. The hydrogel nonwoven fabric may contain 95% by mass or more of gelatin, or may be substantially composed of 100% by mass of gelatin. In addition to gelatin, the hydrogel nonwoven fabric may contain 10% by mass or less of other components or 5% by mass or less, if necessary. Other components may be other biocompatible polymers, cross-linking agents, agents, plasticizers, other additives and the like.

The type and site of the animal from which collagen, which is the raw material of the gelatin, is derived are not particularly limited. Collagen may be, for example, derived from spinal animals or fish. In addition, collagen derived from various organs and tissues such as dermis, ligaments, tendons, bones and cartilage can be appropriately used. Further, the method for preparing gelatin from collagen is not particularly limited, and examples thereof include acid treatment, alkali treatment, and enzyme treatment. The molecular weight of the gelatin is not particularly limited, and gelatin having various molecular weights can be appropriately selected and used. In addition, one type of gelatin may be used, or two or more types may be used in combination.

The gelatin is not particularly limited, but has appropriate flexibility and hardness, and from the viewpoint of enhancing the handleability of the hydrogel nonwoven fabric, the jelly strength is preferably 100 g or more and 400 g or less, and more preferably 150 g or more and 360 g. It is as follows. In one or more embodiments of the present invention, the jelly strength is measured according to JIS K 6503: 2001. The gelatin may be a commercially available product.

The other biocompatible polymer is not particularly limited, but for example, a natural polymer or a synthetic polymer can be used. Examples of natural polymers include proteins and polysaccharides. Examples of the protein include collagen, fibronectin, fibrinogen, laminin, fibrin and the like. Examples of polysaccharides include natural high polymers such as chitosan, calcium alginate, heparan sulfate, chondroitin sulfate, hyaluronic acid, heparin, starch, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, and dieutan gum. A molecule may be used, or a derivative of a natural polymer such as carboxymethyl cellulose may be used. Examples of the synthetic polymer include polyethylene glycol, polypropylene glycol, polyethylene terephthalate, polyvinyl alcohol, thermoplastic elastomer, polypropylene, polyethylene, polystyrene, polymethyl methacrylate, polycarbonate, polydimethylsiloxane, cycloolefin polymer, and amorphous fluororesin. Examples thereof include non-absorbable synthetic polymers and bioabsorbable polymers such as polylactic acid, polyglucuric acid, polycaprolactone and polydioxanone. As the other biocompatible polymers described above, one type may be used, or two or more types may be used.

The hydrogel non-woven fabric has a thickness retention rate (hereinafter, also referred to as an initial thickness retention rate) of 86% or more after being treated with collagenase for 6 hours. The initial thickness retention rate of the hydrogel non-woven fabric is preferably 88% or more, more preferably 90% or more. This maintains a communication hole structure that allows cells and blood vessels to enter the wound dressing 1 to 2 weeks after treatment with the wound dressing, and can induce early angiogenesis. ..

The initial thickness retention rate of the hydrogel nonwoven fabric is calculated as follows based on the thickness (Ha) before the treatment with collagenase and the thickness (Hb) after the 6-hour treatment with collagenase in the swollen hydrogel nonwoven fabric. .. Treatment with collagenase can be performed using 2 mL of a PBS (+) solution of collagenase D at 1.25 μg / mL per 1 mg of hydrogel non-woven fabric.
Thickness retention rate (%) = Hb / Ha × 100

In one or more embodiments of the present invention, "swelling" means swelling to saturation with one or more liquids selected from the group consisting of water or buffers (phosphate buffers, etc.). For example, the hydrogel nonwoven fabric can be swollen by immersing it in one or more liquids selected from the group consisting of water or a buffer solution for about 10 minutes or more.

The hydrogel non-woven fabric preferably has a compressive strength retention rate (hereinafter, also referred to as an initial compressive strength retention rate) after 6 hours of treatment with collagenase of 70% or more, more preferably 80% or more, and 90%. % Or more is more preferable. This allows for the invasion of cells and blood vessels into the wound dressing early 1-2 weeks after transplantation, even when compressed by surrounding tissue at the wound site, i.e. the transplant site. Is easy to maintain and it is easy to induce early angiogenesis. In one or more embodiments of the invention, "compressive strength" means the stress at 70% strain in a swollen hydrogel nonwoven fabric.

The initial compressive strength retention rate of the hydrogel non-woven fabric is the stress (Fa) at 70% strain before treatment with collagenase and the stress at 70% strain (Fb) after 6-hour treatment with collagenase in the swollen hydrogel non-woven fabric. ), Calculate as follows. Treatment with collagenase can be performed using 2 mL of a PBS (+) solution of collagenase D at 1.25 μg / mL per 1 mg of hydrogel non-woven fabric.
Compressive strength retention rate (%) = Fb / Fa × 100

The compressive strength of the hydrogel nonwoven fabric is not particularly limited, but is preferably 5000 Pa or more, more preferably 10,000 Pa or more, from the viewpoint of enhancing the invasion of cells and blood vessels into the hydrogel nonwoven fabric at an early stage. It is more preferably 15,000 Pa or more. Further, from the viewpoint of ease of handling at the time of transplantation and the decomposability of the hydrogel nonwoven fabric in the later stage, the compressive strength of the hydrogel nonwoven fabric is preferably 38000 Pa or less, more preferably 36000 Pa or less, and 31000 Pa or less. Is more preferable.

The fibers constituting the hydrogel nonwoven fabric are not particularly limited, but the average fiber diameter in the swollen state is preferably 10 μm or more and 200 μm or less, more preferably 20 μm or more and 150 μm or less, and further preferably 30 μm or more and 100 μm or less. It is particularly preferably 40 μm or more and 80 μm or less. When the average fiber diameter of the fibers is within the above range, cells and blood vessels tend to invade the inside of the hydrogel non-woven fabric at an early stage of transplantation when transplanted to the wound site. In one or more embodiments of the present invention, "average fiber diameter" means the average value of the diameters of 50 fibers arbitrarily selected from the swollen hydrogel nonwoven fabric.

It is preferable that the fibers constituting the hydrogel non-woven fabric are partially welded at the fiber intersections. This partial welding is not particularly limited, but can be developed, for example, by depositing fibers in a completely unsolidified state blown off by a pressure fluid during the production of the hydrogel nonwoven fabric, as described later. By this partial welding, the hydrogel non-woven fabric has a bridge structure, which is easy to mold into a desired shape and has high molding stability. Further, since the fiber intersections are partially welded, the hydrogel non-woven fabric does not become flat even after swelling. Further, in the hydrogel nonwoven fabric, some of the fiber intersections may be welded, or all of the fiber intersections may be welded.

The thickness of the hydrogel non-woven fabric is not particularly limited and can be appropriately determined depending on the site to which the hydrogel non-woven fabric is applied, etc. The thickness in the swollen state is preferably 0.1 mm or more, more preferably 0.2 mm or more, further preferably 0.3 mm or more, and particularly preferably 0.4 mm or more. Further, from the viewpoint of ease of handling at the time of transplantation and the decomposability of the hydrogel nonwoven fabric in the later stage, the thickness of the hydrogel nonwoven fabric in the swollen state is preferably 5 mm or less, more preferably 3 mm or less, and more preferably 2 mm. It is more preferably 1 mm or less, and even more preferably 1 mm or less. The thickness of the hydrogel nonwoven fabric is not particularly limited, but more specifically, the thickness in the swollen state is preferably 0.1 mm or more and 5 mm or less, more preferably 0.2 mm or more and 3 mm or less, and 0. It is more preferably 3 mm or more and 2 mm or less, further preferably 0.4 mm or more and 2 mm or less, and even more preferably 0.4 mm or more and 1 mm or less.

The basis weight of the hydrogel non-woven fabric is not particularly limited and can be appropriately determined depending on the site to which the hydrogel non-woven fabric is applied. From the viewpoint, the basis weight is preferably 40 g / m ² or more, more preferably 50 g / m ² or more, and even more preferably 60 g / m ² or more. Further, from the viewpoint of handleability and decomposability of the hydrogel nonwoven fabric in the later stage, the texture of the hydrogel nonwoven fabric is preferably 500 g / m ² or less, more preferably 400 g / m ² or less, and 350 g. It is even more preferable that it is / m ² or less, and it is more preferable that it is 300 g / m ² or less. The basis weight of the hydrogel nonwoven fabric is not particularly limited, but more specifically, it is preferably 40 g / m ² or more and 500 g / m ² or less, and more preferably 50 g / m ² or more and 400 g / m ² or less. It is preferably 60 g / m ² or more and 350 g / m ² or less. In one or more embodiments of the present invention, the basis weight of the hydrogel non-woven fabric is measured according to JIS L 1913: 2010.

The pore size of the hydrogel nonwoven fabric is not particularly limited and can be appropriately determined depending on the site to which the hydrogel nonwoven fabric is applied, etc. The pore diameter is preferably 30 μm or more, more preferably 60 μm or more, and further preferably 100 μm or more. Further, from the viewpoint that the number of fiber intersections is within an appropriate range and the strength of the hydrogel can be easily maintained, the pore diameter in the swollen state is preferably 700 μm or less, more preferably 600 μm or less, and more preferably 500 μm or less. Is even more preferable. The pore size of the hydrogel nonwoven fabric is not particularly limited, but more specifically, the pore size in the swollen state is preferably 30 μm or more and 700 μm or less, more preferably 60 μm or more and 600 μm or less, and 100 μm or more and 500 μm or less. It is more preferable to have. In one or more embodiments of the present invention, the pore size of the gelatin nonwoven fabric can be calculated by the following formula (1) based on the assumption of Wrotnowski.

As described above, the hydrogel nonwoven fabric has at least an initial thickness retention rate within a predetermined range, preferably an initial thickness retention rate, and an initial compressive strength retention rate within a predetermined range, more preferably an initial thickness. By setting the fiber diameter, pore diameter, and texture in the predetermined ranges in addition to the retention rate and the initial thickness retention rate, angiogenesis can be induced even if the cell growth factor is not contained. Since the use of cell growth factors is contraindicated in diabetic patients and malignant tumor patients, the hydrogel non-woven fabric and wound covering material are also referred to as cell growth factors (also referred to as cell growth factors) from the viewpoint of application to diabetic patients and malignant tumor patients. It is preferable that it does not contain substances such as). Examples of the cell growth factor include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and the like.

The hydrogel non-woven fabric may be coated with a cell adhesion factor, a cell inducing factor, a cell proliferation factor, a substance that gives nutrition or energy to cells, a substance that suppresses or enhances the function of cells, or the like, if necessary. It may be immersed in a solution containing the substance and penetrated into the fiber. The cell adhesion factor is not particularly limited, and examples thereof include fibronectin and the like. The substance that gives nutrition and energy to the cells is not particularly limited, and examples thereof include ATP, pyruvic acid, and glutamine.

The hydrogel non-woven fabric is not particularly limited, but from the viewpoint of suppressing the generation of impurities and preventing product contamination, the spinning fluid containing gelatin is extruded into the air from the nozzle discharge port and is located behind the nozzle discharge port. , The pressure fluid is injected forward from the fluid injection port in a non-contact state with the nozzle discharge port, and the extruded spinning liquid is associated with the pressure fluid to form fibers, and the obtained fibers are accumulated. It is preferable to make it into a non-woven fabric. When the fibers are accumulated (deposited) after spinning, the fibers are laminated in a state of containing water, so that the fibers are welded together or entangled with each other to be integrated. By changing the collection distance when depositing fibers, the density of the non-woven fabric can be easily changed. The collection distance is, for example, preferably 10 cm or more and 200 cm or less, more preferably 20 cm or more and 180 cm or less, and further preferably 30 cm or more and 150 cm or less.

FIG. 11 is a schematic explanatory view of a hydrogel nonwoven fabric manufacturing apparatus. In the nonwoven fabric manufacturing apparatus 10, the spinning liquid 2 containing gelatin contained in the heating tank 1 is extruded into the air from the nozzle discharge port 3. A predetermined pressure is applied to the heating tank 1 by the compressor 4. Reference numeral 12 is a heat insulating container.
Further, the pressure fluid 7 is injected forward from the fluid injection port 5 which is located behind the nozzle discharge port 3 and is in a non-contact state with the nozzle discharge port 3. A pressure fluid (for example, compressed air) is supplied from the compressor 6 to the fluid injection port 5. The distance between the fluid injection port 5 and the nozzle discharge port 3 is preferably 5 mm or more and 30 mm or less, and more preferably 5 mm or more and 15 mm or less.
The extruded spinning liquid is accompanied by the pressure fluid 7 to become gelatin fibers 8, and is deposited as gelatin nonwoven fabric 9 on the take-up roll 11. At this time, since the deposited fibers contain water and are not completely solidified, the fibers in contact with each other at at least a part of the fiber intersections are welded to each other. In addition, other collecting means such as a net may be used instead of the take-up roll.

First, gelatin alone or, if necessary, gelatin and other biocompatible polymers that can be used as the above-mentioned other components are dissolved in a solvent, preferably water, to prepare a spinning solution. The dissolution temperature (temperature of a solvent such as water) is preferably 20 ° C. or higher and 90 ° C. or lower, and more preferably 40 ° C. or higher and 90 ° C. or lower. If necessary, gelatin may be dissolved in a solvent such as water and then filtered to remove foreign substances and dust. Further, if necessary, the dissolved air may be subsequently removed by depressurization or vacuum defoaming. From the viewpoint of efficiently removing gas (air bubbles), the degree of vacuum at the time of defoaming under reduced pressure is preferably 5 kPa or more and 30 kPa or less. Since gelatin is water-soluble, it can be spun in an aqueous solution as a spinning solution, and its safety to the living body is improved. As the water, for example, pure water, distilled water, ultrapure water and the like can be appropriately used. When another biocompatible water-soluble polymer is used as another component, a spinning solution can be prepared by dissolving it in water at the same time as gelatin.

The temperature of the spinning liquid is preferably 20 ° C. or higher and 90 ° C. or lower, and more preferably 40 ° C. or higher and 90 ° C. or lower. Within the above range, gelatin can maintain a stable sol state. Further, the gelatin concentration of the gelatin aqueous solution is preferably 30% by mass or more and 55% by mass or less when the gelatin aqueous solution is 100% by mass. A more preferable concentration is 35% by mass or more and 50% by mass or less. At the above concentration, a stable sol state can be maintained. The viscosity of the gelatin aqueous solution (spinning solution) is preferably 500 mPa · s or more and 3000 mPa · s or less. If the viscosity of the gelatin aqueous solution is within the above range, stable spinning can be achieved.

The spinning liquid is discharged from the nozzle of the spinning machine, a pressure fluid is supplied from around the nozzle, the discharged gelatin aqueous solution is associated with the pressure fluid to form fibers, and the obtained gelatin fibers are accumulated to accumulate the obtained gelatin non-woven fabric. (Hydrogel non-woven fabric). The discharge pressure of the nozzle is not particularly limited, but may be, for example, 0.1 MPa or more and 1 MPa or less.

The temperature of the pressure fluid is preferably 20 ° C. or higher and 120 ° C. or lower, and more preferably 80 ° C. or higher and 120 ° C. or lower. Although it depends on the flow velocity of the pressure fluid and the temperature of the ambient atmosphere, stable spinning can be achieved within the above temperature range. It is preferable to use air as the pressure fluid, and the pressure is preferably 0.1 MPa or more and 1 MPa or less. Within the above range, the spinning liquid extruded into the air from the nozzle discharge port can be blown off to form fibers.

The hydrogel non-woven fabric is preferably crosslinked. This makes it possible to improve morphological stability and water resistance. The cross-linking may be a chemical cross-linking using a compound such as a cross-linking agent, but from the viewpoint of biosafety, the cross-linking shall be a cross-linking using a cross-linking agent having bio-safety and / or a cross-linking without using a cross-linking agent. Is preferable. Examples of the cross-linking without using a cross-linking agent include thermal cross-linking, radiation cross-linking such as electron beam and γ-ray, and ultraviolet cross-linking. In the case of irradiation with electron beam or γ-ray, sterilization and cross-linking can be performed at the same time. From the viewpoint of easily obtaining the desired crosslinking effect, thermal crosslinking is preferable, and thermal dehydration crosslinking is more preferable. The thermal cross-linking may be performed, for example, at 100 ° C. or higher and 180 ° C. or lower, or at 100 ° C. or higher and 160 ° C. or lower. The cross-linking time may be, for example, 24 hours or more and 96 hours or less. The thermal dehydration crosslinking may be performed, for example, at 100 ° C. or higher and 180 ° C. or lower for 24 hours or more and 96 hours or less, or at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or shorter. Further, the thermal dehydration crosslinking may be performed under a vacuum of 1 kPa or less, for example. It may be dried before cross-linking. The drying is not particularly limited, but can be performed by, for example, air-drying at room temperature or freeze-drying.

Within the above range, the discharge amount of the spinning liquid, the nozzle diameter (inner diameter), the nozzle discharge pressure, the pressure of the pressure fluid, the temperature of the pressure fluid, the distance between the fluid injection port and the nozzle discharge port, the collection distance, the bridging conditions, etc. By adjusting the above, a hydrogel non-woven fabric having a desired initial thickness retention rate, initial compression strength retention rate, thickness, grain size, fiber diameter, pore diameter and the like can be obtained. As an example, by increasing the nozzle discharge pressure and increasing the fiber diameter, the initial thickness retention rate and the initial compressive strength retention rate can be increased. As an example, the initial thickness retention rate and the initial compressive strength retention rate can be increased by shortening the distance between the fluid injection port and the nozzle discharge port and increasing the fiber intersection points.

The hydrogel non-woven fabric may be cut into a predetermined shape and size, if necessary. The hydrogel non-woven fabric may be sterilized by ethylene oxide gas sterilization, steam (autoclave), electron beam irradiation, irradiation with γ-rays or the like, or may be sterilized by ethanol treatment or the like. In the case of irradiation with electron beam or γ-ray, cross-linking can be performed at the same time as sterilization.

In one or more embodiments of the present invention, the wound dressing is used so that one surface of the hydrogel nonwoven fabric is in contact with the wound site. That is, in the wound dressing, one surface of the hydrogel non-woven fabric is on the wound surface side. The wound dressing may contain a protective film in addition to the hydrogel non-woven fabric. The protective film is preferably made of a waterproof material. This makes it possible to prevent moisture from the outside other than the wound site from invading the hydrogel non-woven fabric. Since the protective film has self-adhesiveness, it may be adhered to the hydrogel non-woven fabric, or may be adhered to the hydrogel non-woven fabric via the pressure-sensitive adhesive layer. The protective film can be placed on one or both surfaces of the hydrogel non-woven fabric. When using the wound dressing, one protective film may be peeled off and transplanted so that one surface of the hydrogel non-woven fabric is in contact with the wound site of the tissue.

It is preferable that the manufacturing process of the hydrogel non-woven fabric and the wound dressing is performed aseptically in, for example, a clean bench or a clean room. It is possible to prevent the hydrogel non-woven fabric and the wound dressing from being contaminated by the propagation of various germs during the work. As the manufacturing equipment to be used, for example, it is preferable to use an autoclave, one that has been sterilized by irradiation with an electron beam, γ-ray, or the like.

The wound dressing may be applied to a wound site on the surface of the skin, or may be applied to a wound site of subcutaneous tissue.

Hereinafter, one or more embodiments of the present invention will be described in more detail with reference to examples. The present invention is not limited to the following examples. Unless otherwise specified in the present specification, the operation is performed at room temperature (20 ± 5 ° C.).

The measurement / evaluation method is as follows.
<Thickness and thickness retention rate>
In the swollen hydrogel nonwoven fabric, the thickness before treatment with collagenase (Ha) and the thickness after treatment with collagenase (Hb) were measured with a creep meter manufactured by Sanyo Electric Railway Co., Ltd. In addition, the thickness retention rate was calculated as follows.
Thickness retention rate (%) = Hb / Ha × 100
<Compressive strength>
In the swollen hydrogel non-woven fabric, the stress at 70% strain (Fa) before treatment with collagenase and the stress (Fb) at 70% strain after treatment with collagenase are measured with a creep meter manufactured by Yamaden Co., Ltd. and compressed. The strength was set. In addition, the compressive strength retention rate was calculated as follows.
Compressive strength retention rate (%) = Fb / Fa × 100
<Treatment with collagenase>
(1) Collagenase D (Sigma Aldrich) was added to D-PBS (+) (Dalvecolinic acid buffered saline (D-PBS (-) (1x) from Nakaraitesk), and calcium chloride from Fujifilm Wako Junyaku Co., Ltd. It was dissolved in D-PBS (+) supplemented with (100 μg / mL) and magnesium chloride (46.8 μg / mL) to obtain a collagenase D / PBS (+) solution having a concentration of 1.25 μg / mL.
(2) A swollen hydrogel non-woven fabric (dry mass of about 1 mg) was immersed in 2 mL of a solution of collagenase D and incubated at 37 ° C. for 6 hours.
<Average fiber diameter>
The hydrogel non-woven fabric in the swollen state was observed with a microscope (Keyence, BZ-X700), the fiber diameters of 50 arbitrarily selected fibers were measured, and the average fiber diameters in the swollen state were obtained. Calculated.
<Metsuke (mass per unit area)>
The basis weight of the gelatin non-woven fabric was measured according to JIS L 1913: 2010.
<Hole diameter>
The pore size of the gelatin nonwoven fabric was calculated by the following formula 1 based on the assumption of Wrotnowski.

(Example 1)
Nitta Gelatin Co., Ltd. (jelly strength 262 g, raw material: alkaline-treated beef bone) was used as gelatin, and the mass ratio was gelatin: water = 3: 5 (gelatin concentration 37.5 mass%) and dissolved at a temperature of 60 ° C. .. The viscosity of the aqueous gelatin solution at 60 ° C. was 960 to 970 mPa · s. This gelatin aqueous solution was used as a spinning solution, and a gelatin nonwoven fabric was produced using the manufacturing apparatus shown in FIG. The temperature of the spinning liquid is 60 ° C, the nozzle diameter (inner diameter) is 250 μm, the discharge pressure is 0.2 MPa, the nozzle height is 5 mm, the air pressure is 0.375 MPa, the air temperature is 100 ° C, and the distance between the fluid injection port and the nozzle discharge port is 5 mm. The collection distance was 100 cm. The gelatin non-woven fabric was air dried overnight at room temperature and then dehydrated and crosslinked by heating. The cross-linking conditions were a temperature of 140 ° C. and 48 hours. The basis weight of the obtained gelatin non-woven fabric was about 65 g / m ² .
Next, the dried gelatin non-woven fabric was punched into a disk shape having a diameter of about 5 mm (dry mass: about 1 mg), and placed in Dulbeccoline phosphate buffered saline (D-PBS (-) (1x), Nakaraitesku Co., Ltd.) at room temperature. It was allowed to stand for 10 minutes to swell. The diameter of the swollen hydrogel nonwoven fabric was about 7 mm.

(Example 2)
A gelatin nonwoven fabric was produced in the same manner as in Example 1 except that the collection distance was set to 50 cm. The gelatin non-woven fabric was air dried overnight at room temperature and then dehydrated and crosslinked by heating. The cross-linking conditions were a temperature of 140 ° C. and 48 hours. The basis weight of the obtained gelatin non-woven fabric was about 100 g / m ² .
Next, the dried gelatin non-woven fabric was punched into a disk shape having a diameter of about 4 mm (dry mass: about 1 mg), and placed in Dulbeccoline phosphate buffered saline (D-PBS (-) (1x), Nakaraitesk Co., Ltd.) at room temperature. It was allowed to stand for 10 minutes to swell. The diameter of the swollen hydrogel nonwoven fabric was about 6 mm.

(Example 3)
<Making hydrogel non-woven fabric>
A gelatin nonwoven fabric was produced in the same manner as in Example 2 except that the discharge amount of the spinning liquid was increased. The gelatin non-woven fabric was air dried overnight at room temperature and then dehydrated and crosslinked by heating. The cross-linking conditions were a temperature of 140 ° C. and 48 hours. The basis weight of the obtained gelatin non-woven fabric was about 150 g / m ² .
Next, the dried gelatin non-woven fabric was punched into a disk shape having a diameter of about 3 mm, and 1.25 sheets (dry mass of about 1 mg), Dulbeccoline acid buffered saline (D-PBS (-) (1x), Nakaraitesk) were used. The company) was allowed to stand at room temperature for 10 minutes to swell. The diameter of the swollen hydrogel nonwoven fabric was about 5 mm.

(Example 4)
A gelatin nonwoven fabric was produced in the same manner as in Example 2 except that the discharge pressure was 0.1 MPa and the air pressure was 0.275 MPa. The gelatin non-woven fabric was air dried overnight at room temperature and then dehydrated and crosslinked by heating. The cross-linking conditions were a temperature of 140 ° C. and 48 hours. The basis weight of the obtained gelatin non-woven fabric was about 300 g / m ² .
Next, the dried gelatin non-woven fabric was punched into a disk shape having a diameter of about 2 mm (dry mass: about 1 mg), and placed in Dulbeccoline phosphate buffered saline (D-PBS (-) (1x), Nakaraitesku Co., Ltd.) at room temperature. It was allowed to stand for 10 minutes to swell. The diameter of the swollen hydrogel nonwoven fabric was about 3 mm.

(Comparative Example 1)
A gelatin nonwoven fabric was produced in the same manner as in Example 2 except that the discharge amount of the spinning liquid was reduced. The gelatin non-woven fabric was air dried overnight at room temperature and then dehydrated and crosslinked by heating. The cross-linking conditions were a temperature of 140 ° C. and 48 hours. The basis weight of the obtained gelatin non-woven fabric was about 50 g / m ² .
Next, the dried gelatin non-woven fabric was punched into a disk shape having a diameter of about 6 mm (dry mass: about 1 mg), and placed in Dulbeccoline phosphate buffered saline (D-PBS (-) (1x), Nakaraitesku Co., Ltd.) at room temperature. It was allowed to stand for 10 minutes to swell. The diameter of the swollen hydrogel nonwoven fabric was about 9 mm.

(Comparative Example 2)
The collagen sponge was punched into a disk shape with a diameter of about 8 mm (dry mass: about 1 mg), and allowed to stand in Dulbeccoline phosphate buffered saline (D-PBS (-) (1x), Nakaraitesku) at room temperature for 10 minutes. And swollen. The diameter of the swollen collagen sponge was about 8 mm.

In Examples 1 to 4 and Comparative Examples 1 to 2, the thickness of the hydrogel non-woven fabric or collagen sponge in a swollen state before and after treatment with collagenase and the stress (compressive strength) at 70% strain were measured as described above. As for Comparative Example 2, since it was dissolved before the lapse of 6 hours, the compressive strength after the treatment could not be measured. In Examples 1 to 4 and Comparative Example 1, the basis weight (mass per unit area) and pore size of the gelatin nonwoven fabric were measured as described above. Further, in the hydrogel non-woven fabric in the swollen state, the average fiber diameter was measured as described above. These results are shown in Table 1 below.

FIG. 1 is a photograph of a scanning electron microscope (100 times) of a hydrogel non-woven fabric in a dry state of Example 2. FIG. 2 is a photograph of a collagen sponge of Comparative Example 2 in a dry state with a scanning electron microscope (100 times). As can be seen from FIG. 1, in the hydrogel nonwoven fabric of Example 2, the intersections between the fibers are fused and have a communication hole structure.

(Experimental Example 1)
A disk-shaped hydrogel non-woven fabric of Example 1 having a swollen diameter of about 6 mm and a hydrogel of Comparative Example 1 under the skin of the back of a normal mouse (C57BL / 6J, 8 weeks old, male, obtained from Shimizu Laboratory Materials Co., Ltd.) The non-woven fabric and the collagen sponge of Comparative Example 2 were transplanted. The disk-shaped hydrogel nonwoven fabric of Example 1, the hydrogel nonwoven fabric of Comparative Example 1 and the collagen sponge of Comparative Example 2 having a swollen diameter of about 6 mm were sterilized with ethylene oxide gas in a dry state before transplantation. Sterilized.
On the 10th day after transplantation, the transplanted material was collected, and a frozen section having a thickness of 10 μm was prepared on the maximum split plane in the thickness direction of the disc-shaped transplanted material.
HE staining and CD31 immunostaining were performed using the frozen sections, and the total number of cells (HE staining) and the number of CD31-positive cells (CD31 immunostaining) were measured within a range of 250 μm to the left and right from the center of the section. In addition, the ratio of cells by depth and the ratio of CD31-positive cells in all cells were calculated. n was set to 3. The results are shown in Table 2 below. In Table 2 below, depth means the distance from the surface in contact with the subcutaneous tissue.
CD31 immunostaining was performed by a peroxidase coloring method using DAB using a CD31 antibody (“Rabbit Monoclonal Antibody CST 77699” manufactured by Cell Signaling Technology Co., Ltd.).

FIG. 3 is a photograph (40 times) showing the result of HE staining of the cross section (depth 200-400 μm) of the transplant material on the 10th day of transplantation using the hydrogel non-woven fabric of Example 1 as the transplant material.
FIG. 4 is a photograph (40 times) showing the result of CD31 immunostaining of a cross section (depth 200-400 μm) of the transplant material on the 10th day of transplantation using the hydrogel non-woven fabric of Example 1 as the transplant material.
FIG. 5 is a photograph (40 times) showing the result of HE staining of the cross section (depth 200-400 μm) of the transplanted material on the 10th day of transplantation using the collagen sponge of Comparative Example 2 as the transplanted material.
FIG. 6 is a photograph (40 times) showing the results of CD31 immunostaining of a cross section (depth 200-400 μm) of the transplant material on the 10th day of transplantation using the collagen sponge of Comparative Example 2 as the transplant material.

As can be seen from Table 2 and FIGS. 3 to 6, when the hydrogel non-woven fabric of Example 1 having a predetermined initial thickness retention rate is used, when the collagen sponge of Comparative Example 2 is used, and the initial thickness retention rate is low. Compared with the case of using the hydrogel non-woven fabric of Comparative Example 1, the proportion of cells inside (depth 200 μm ~) is larger than that on the surface (depth 0 to 200 μm) of the transplant material, and the proportion of CD31 positive cells in all cells is large. The ratio was also high. It was confirmed that Example 1 has a tendency to have better cell invasion and vascular invasion (angiogenesis) into the transplant material at an early stage of transplantation than Comparative Example 1 and Comparative Example 2, and can be suitably used for wound healing. Guessed.

(Experimental Example 2)
Under the skin of the back of a normal mouse (C57BL / 6J, 8 weeks old, male, obtained from Shimizu Laboratory Materials Co., Ltd.), a disk-shaped hydrogel non-woven fabric having a swollen diameter of about 6 mm and collagen of Comparative Example 2 I transplanted a sponge.
The transplanted material was collected on the 7th and 14th days after the transplantation, and frozen sections having a thickness of 10 μm were prepared on the maximum split plane in the thickness direction of the disc-shaped transplanted material.
HE staining and CD31 immunostaining were performed using the frozen sections, and the total number of cells (HE staining) and the number of CD31-positive cells (CD31 immunostaining) were measured within a range of 250 μm to the left and right from the center of the section. In addition, the ratio of cells by depth and the ratio of CD31-positive cells in all cells were calculated. n was set to 3. The results on the 7th day after transplantation are shown in Table 3 below, and the results on the 14th day after transplantation are shown in Table 4 below. In Tables 3 and 4 below, depth means the distance from the surface in contact with the subcutaneous tissue.
CD31 immunostaining was performed by a peroxidase coloring method using DAB using a CD31 antibody (“Rabbit Monoclonal Antibody CST 77699” manufactured by Cell Signaling Technology Co., Ltd.).

FIG. 7 is a photograph (40 times) showing the result of HE staining of the cross section (depth 400-600 μm) of the transplant material on the 7th day of transplantation using the hydrogel non-woven fabric of Example 2 as the transplant material.
FIG. 8 is a photograph (40 times) showing the result of CD31 immunostaining of a cross section (depth 400-600 μm) of the transplant material on the 7th day of transplantation using the hydrogel non-woven fabric of Example 2 as the transplant material.
FIG. 9 is a photograph (40 times) showing the result of HE staining of the cross section (depth 400-600 μm) of the transplanted material on the 7th day of transplantation using the collagen sponge of Comparative Example 2 as the transplanted material.
FIG. 10 is a photograph (40 times) showing the results of CD31 immunostaining of a cross section (depth 400-600 μm) of the transplant material on the 7th day of transplantation using the collagen sponge of Comparative Example 2 as the transplant material.

As can be seen from Tables 3 and 7 to 10 above, when the hydrogel non-woven fabric of Example 2 having a predetermined thickness retention rate was used, compared with the case of using the collagen sponge of Comparative Example 2, on the 7th day of transplantation. In addition, the proportion of CD31-positive cells in all cells was high. It is presumed that Example 2 has a tendency to be more excellent in blood vessel invasion (angiogenesis) into the inside of the transplant material at an early stage of transplantation than Comparative Example 2, and can be suitably used for wound healing.

As can be seen from Table 4 above, when the hydrogel non-woven fabric of Example 2 having a predetermined thickness retention rate was used, as compared with the case of using the collagen sponge of Comparative Example 2, the transplant material was transferred on the 14th day of transplantation. The proportion of cells inside (depth from 200 μm) was larger than that on the surface (depth of 0 to 200 μm), and the proportion of CD31-positive cells in all cells was also high. It is confirmed that Example 2 is more excellent in cell invasion into the inside of the transplant material and blood vessel invasion (angiogenesis) in the early stage of transplantation than in Comparative Example 2, and it is presumed that it can be suitably used for wound healing.

The present invention is not particularly limited, but preferably includes the following aspects.
[1] A wound dressing containing a hydrogel non-woven fabric containing gelatin as a main component.
The hydrogel non-woven fabric is a wound dressing having a thickness retention rate of 86% or more after being treated with collagenase for 6 hours.
[2] The wound dressing according to [1], wherein the hydrogel nonwoven fabric has a compressive strength retention rate of 70% or more after being treated with collagenase for 6 hours.
[3] The wound dressing according to [1] or [2], wherein the hydrogel nonwoven fabric has a thickness of 0.1 mm or more and 5 mm or less in a swollen state and a basis weight of 40 g / m ² or more and 500 g / m ² or less. Material.
[4] The wound dressing according to any one of [1] to [3], wherein the hydrogel nonwoven fabric has a swollen pore diameter of 30 μm or more and 700 μm or less.
[5] The wound dressing according to any one of [1] to [4], wherein the fibers constituting the hydrogel nonwoven fabric have an average fiber diameter of 10 μm or more and 200 μm or less in a swollen state.
[6] The wound dressing according to any one of [1] to [5], wherein the hydrogel nonwoven fabric is thermally dehydrated and crosslinked.
[7] The wound dressing according to any one of [1] to [6], wherein the hydrogel nonwoven fabric does not contain a cell growth factor.
[8] The wound dressing according to any one of [1] to [7], wherein a protective film is arranged on one or both surfaces of the hydrogel nonwoven fabric.

1 Heating tank 2 Spinning liquid 3

Nozzle discharge port

4, 6 Compressor 5 Fluid injection port 7 Pressure fluid 8 Gelatin fiber 9 Hydrogel non-woven fabric 10 Non-woven fabric manufacturing equipment 11 Winding roll 12 Heat insulation container

Claims

A wound dressing containing a hydrogel non-woven fabric containing gelatin as a main component.
The hydrogel non-woven fabric is a wound dressing having a thickness retention rate of 86% or more after being treated with collagenase for 6 hours.
The wound dressing according to claim 1, wherein the hydrogel non-woven fabric has a compressive strength retention rate of 70% or more after being treated with collagenase for 6 hours.
The wound dressing according to claim 1 or 2, wherein the hydrogel nonwoven fabric has a thickness of 0.1 mm or more and 5 mm or less in a swollen state and a basis weight of 40 g / m 2 or more and 500 g / m 2 or less.
The wound dressing according to any one of claims 1 to 3, wherein the hydrogel non-woven fabric has a swollen pore diameter of 30 μm or more and 700 μm or less.
The wound dressing according to any one of claims 1 to 4, wherein the fibers constituting the hydrogel non-woven fabric have an average fiber diameter of 10 μm or more and 200 μm or less in a swollen state.
The wound dressing according to any one of claims 1 to 5, wherein the hydrogel non-woven fabric is thermally dehydrated and crosslinked.
The wound dressing according to any one of claims 1 to 6, wherein the hydrogel non-woven fabric does not contain a cell growth factor.
The wound dressing according to any one of claims 1 to 7, wherein a protective film is arranged on one or both surfaces of the hydrogel non-woven fabric.