JP7691682B2 - wound dressing - Google Patents

Description

The present invention relates to a wound dressing used to repair wounds such as defects and cuts in tissues such as skin.

In wound dressings used to repair tissue defects and wounds such as those in the skin, a hydrogel-forming component capable of providing a moist environment is used on the side that comes into contact with the wound site. Sponges using hydrogel-forming components such as collagen are widely used as such wound dressings. In addition, Patent Document 1 proposes a wound dressing that includes a hydrogel fiber layer that includes organic fibers capable of forming a hydrogel.

JP 2020-103502 A

However, for wound healing, early cell and blood vessel penetration into the wound dressing after treating the wound site with the wound dressing is necessary, and with conventional wound dressings such as collagen sponge and the wound dressing described in Patent Document 1, there is a need to improve early cell and blood vessel penetration into the interior.

In order to solve the above-mentioned problems of the conventional art, the present invention provides a wound dressing material that improves early cell and blood vessel penetration into the interior of the wound dressing material during use.

The present invention relates to a wound dressing material comprising a hydrogel nonwoven fabric mainly composed of gelatin, the hydrogel nonwoven fabric being characterized in that it has a thickness retention rate of 86% or more after treatment with collagenase for 6 hours.

By using the wound dressing material of the present invention, it is possible to improve early cell penetration and blood vessel penetration into the interior of the wound dressing material.

1 is a scanning electron microscope (100x magnification) photograph of the hydrogel nonwoven fabric in a dry state in Example 2. 1 is a scanning electron microscope photograph (100x) of the dried collagen sponge of Comparative Example 2. 1 is a photograph (40x) showing the results of hematoxylin-eosin staining (HE staining) of a cross section (depth 200-400 μm) of a transplant material on the 10th day after transplantation using the hydrogel nonwoven fabric of Example 1. The scale bar is 100 μm. The same applies below. 1 is a photograph (40x magnification) showing the results of CD31 (platelet endothelial cell adhesion molecule-1) immunostaining of a cross section (depth 200-400 μm) of a graft material using the hydrogel nonwoven fabric of Example 1 on the 10th day after grafting. 1 is a photograph (40x) showing the results of hematoxylin-eosin staining of a cross section (depth 200-400 μm) of a transplant material using the collagen sponge of Comparative Example 2 on the 10th day after transplantation. 1 is a photograph (40x) showing the results of CD31 (platelet endothelial cell adhesion molecule-1) immunostaining of a cross section (depth 200-400 μm) of a transplant material using the collagen sponge of Comparative Example 2 on the 10th day after transplantation. 1 is a photograph (40x magnification) showing the results of hematoxylin-eosin staining of a cross section (depth 400-600 μm) of a graft material using the hydrogel nonwoven fabric of Example 2 on the 7th day after grafting. 1 is a photograph (40x magnification) showing the results of CD31 (platelet endothelial cell adhesion molecule-1) immunostaining of a cross section (depth 400-600 μm) of a graft material using the hydrogel nonwoven fabric of Example 2 on the 7th day after grafting. 1 is a photograph (40x) showing the results of hematoxylin-eosin staining of a cross section (depth 400-600 μm) of a transplant material using the collagen sponge of Comparative Example 2 on the 7th day after transplantation. 1 is a photograph (40x) showing the results of CD31 (platelet endothelial cell adhesion molecule-1) immunostaining of a cross section (depth 400-600 μm) of a transplant material using the collagen sponge of Comparative Example 2 on the 7th day after transplantation. FIG. 2 is a schematic explanatory diagram of a nonwoven fabric manufacturing apparatus.

The inventors of the present invention have conducted extensive research to solve the above-mentioned problems, and as a result, have found that the use of a hydrogel nonwoven fabric containing gelatin as a main component and having a thickness retention rate of 86% or more after 6 hours of treatment with collagenase improves early cell infiltration and blood vessel infiltration (angiogenesis) into the wound dressing during use.
In particular, the use of a hydrogel nonwoven fabric mainly composed of gelatin allows blood vessels to penetrate into the wound dressing without the use of cell growth factors, i.e., promotes angiogenesis, and induces angiogenesis at the wound site, making it applicable to diabetes patients and malignant tumor patients. Furthermore, since the thickness retention rate after 6 hours of treatment with collagenase is 86% or more, when the wound dressing is used, for example, as early as 1 to 2 weeks after the wound dressing is implanted, the interconnected pore structure that allows cells and blood vessels to penetrate into the wound dressing is maintained, making it possible to induce early angiogenesis.

In one or more embodiments of the present invention, the wound dressing material includes a hydrogel nonwoven fabric mainly composed of gelatin. In one or more embodiments of the present invention, "mainly composed of gelatin" means containing 90% or more by mass of gelatin. The hydrogel nonwoven fabric may contain 95% or more by mass of gelatin, or may be substantially composed of 100% by mass of gelatin. In addition to gelatin, the hydrogel nonwoven fabric may contain 10% or less by mass of other components, or 5% or less by mass of other components, as necessary. The other components may be other biocompatible polymers, crosslinkers, drugs, plasticizers, other additives, etc.

The type or part of the animal from which the collagen, which is the raw material for the gelatin, is derived is not particularly limited. The collagen may be derived from, for example, a spinal animal or a fish. Collagen derived from various organs and tissues, such as the dermis, ligament, tendon, bone, and cartilage, may be used as appropriate. The method for preparing gelatin from collagen is also not particularly limited, and examples of such methods include acid treatment, alkali treatment, and enzyme treatment. The molecular weight of the gelatin is also not particularly limited, and gelatin of various molecular weights may be appropriately selected and used. 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 improving the handleability of the hydrogel nonwoven fabric, the jelly strength is preferably 100 g or more and 400 g or less, more preferably 150 g or more and 360 g or less. In one or more embodiments of the present invention, the jelly strength is measured in accordance with JIS K 6503:2001. The gelatin may be a commercially available product.

The other biocompatible polymers are not particularly limited, and may be, for example, natural polymers or synthetic polymers. Examples of natural polymers include proteins and polysaccharides. Examples of proteins include collagen, fibronectin, fibrinogen, laminin, and fibrin. Examples of polysaccharides include natural 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 diutan gum, and derivatives of natural polymers such as carboxymethylcellulose may also be used. Examples of synthetic polymers include non-absorbable synthetic polymers such as polyethylene glycol, polypropylene glycol, polyethylene terephthalate, polyvinyl alcohol, thermoplastic elastomers, polypropylene, polyethylene, polystyrene, polymethyl methacrylate, polycarbonate, polydimethylsiloxane, cycloolefin polymers, and amorphous fluororesins, and bioabsorbable polymers such as polylactic acid, polyglycolic acid, polycaprolactone, and polydioxanone. The other biocompatible polymers described above may be used alone or in combination of two or more.

The hydrogel nonwoven fabric has a thickness retention rate (hereinafter also referred to as initial thickness retention rate) of 86% or more after 6 hours of treatment with collagenase. The initial thickness retention rate of the hydrogel nonwoven fabric is preferably 88% or more, and more preferably 90% or more. This maintains a communicating pore structure that allows cells and blood vessels to enter the wound dressing as early as 1 to 2 weeks after treatment with the wound dressing, making it possible to induce early angiogenesis.

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

In one or more embodiments of the present invention, "swelling" means swelling to a saturated state with one or more liquids selected from the group consisting of water or buffer solutions (e.g., phosphate buffer solution). 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 buffer solutions for about 10 minutes or more.

The hydrogel nonwoven fabric preferably has a compressive strength retention rate (hereinafter also referred to as initial compressive strength retention rate) of 70% or more after 6 hours of treatment with collagenase, more preferably 80% or more, and even more preferably 90% or more. This makes it easier to maintain a communicating pore structure that allows cells and blood vessels to enter the wound dressing material early after transplantation, 1 to 2 weeks after transplantation, even when the wound is compressed by surrounding tissues at the wound site, i.e., the transplantation site, and makes it easier to induce early angiogenesis. In one or more embodiments of the present invention, "compressive strength" means the stress at 70% strain in a swollen hydrogel nonwoven fabric.

The initial compressive strength retention rate of the hydrogel nonwoven fabric is calculated as follows based on the stress (Fa) at 70% strain in the swollen hydrogel nonwoven fabric before collagenase treatment and the stress (Fb) at 70% strain after 6 hours of collagenase treatment. The collagenase treatment can be performed using 2 mL of 1.25 μg/mL collagenase D in PBS(+) per 1 mg of hydrogel nonwoven fabric.
Compressive strength retention rate (%) = Fb / Fa x 100

The compressive strength of the hydrogel nonwoven fabric is not particularly limited, but from the viewpoint of enhancing early cell and blood vessel penetration into the hydrogel nonwoven fabric, it is preferably 5,000 Pa or more, more preferably 10,000 Pa or more, and even more preferably 15,000 Pa or more. Furthermore, from the viewpoint of ease of handling during transplantation and the later degradability of the hydrogel nonwoven fabric, the compressive strength of the hydrogel nonwoven fabric is preferably 38,000 Pa or less, more preferably 36,000 Pa or less, and even more preferably 31,000 Pa or less.

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, even more preferably 30 μm or more and 100 μm or less, and particularly preferably 40 μm or more and 80 μm or less. If the average fiber diameter of the fibers is within the above range, cells and blood vessels are likely to penetrate into the hydrogel nonwoven fabric early after transplantation when transplanted into a wound site. In one or more embodiments of the present invention, the "average fiber diameter" means the average value of the diameters of 50 fibers arbitrarily selected from the hydrogel nonwoven fabric in a swollen state.

It is preferable that the fibers constituting the hydrogel nonwoven fabric are partially welded at their fiber intersections. This partial welding is not particularly limited, but can be achieved, for example, by depositing fibers in a state where they are not completely solidified, which are blown away by a pressure fluid during the production of the hydrogel nonwoven fabric, as described below. This partial welding gives the hydrogel nonwoven fabric a bridge structure, which makes it easy to mold into a desired shape and also provides high molding stability. In addition, since the fiber intersections are partially welded, the hydrogel nonwoven fabric does not sag even after swelling. In addition, 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 nonwoven fabric is not particularly limited and can be appropriately determined depending on the site of application, etc., but from the viewpoint of improving the handling property and the early penetration of cells and blood vessels into the hydrogel nonwoven fabric, the thickness in the swollen state is preferably 0.1 mm or more, more preferably 0.2 mm or more, even more preferably 0.3 mm or more, and particularly preferably 0.4 mm or more. In addition, from the viewpoint of ease of handling during 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, even more preferably 2 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, even more preferably 0.3 mm or more and 2 mm or less, even more 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 nonwoven fabric is not particularly limited and can be appropriately determined depending on the site to be applied, etc., but for example, from the viewpoint of improving handleability and early cell and blood vessel penetration into the hydrogel nonwoven fabric, the basis weight is preferably 40 g/m ² or more, more preferably 50 g/m ² or more, and more preferably 60 g/m ² or more. Also, from the viewpoint of handleability and later decomposition of the hydrogel nonwoven fabric, the basis weight of the hydrogel nonwoven fabric is preferably 500 g/m ² or less, more preferably 400 g/m ² or less, even more preferably 350 g/m ² or less, and more preferably 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/m2 or ^more and 500 g/ ^m2 or less, more preferably 50 g/ ^m2 or more and 400 g/ ^m2 or less, and even more preferably 60 g/ ^m2 or more and 350 g/ ^m2 or less. In one or more embodiments of the present invention, the basis weight of the hydrogel nonwoven fabric is measured in accordance with 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 of application, etc., but from the viewpoint of further increasing the early penetration of cells and blood vessels into the hydrogel nonwoven fabric, the pore size in the swollen state is preferably 30 μm or more, more preferably 60 μm or more, and even more preferably 100 μm or more. In addition, from the viewpoint of keeping the number of fiber intersections within an appropriate range and easily maintaining the strength of the hydrogel, the pore size in the swollen state is preferably 700 μm or less, more preferably 600 μm or less, and even more preferably 500 μm or less. 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 even more preferably 100 μm or more and 500 μm or less. In one or more embodiments of the present invention, the pore size of the gelatin nonwoven fabric can be calculated based on Wrotnowski's assumptions using the following calculation formula (1).

As described above, the hydrogel nonwoven fabric can induce angiogenesis even without containing cell growth factors by setting at least the initial thickness retention rate within a predetermined range, preferably the initial compressive strength retention rate in addition to the initial thickness retention rate, and more preferably the fiber diameter, pore size, and basis weight in addition to the initial thickness retention rate within a predetermined range. Since the use of cell growth factors is contraindicated for diabetes patients and malignant tumor patients, it is preferable that the hydrogel nonwoven fabric and wound dressing material do not contain substances such as cell growth factors (also called cell growth factors) from the viewpoint of application to diabetes patients and malignant tumor patients. Examples of cell growth factors include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), etc.

The hydrogel nonwoven fabric may be coated with cell adhesion factors, cell induction factors, cell growth factors, substances that provide nutrients or energy to cells, substances that suppress or enhance cell function, etc., as necessary, and may be immersed in a solution containing such substances to allow the substances to penetrate into the interior of the fibers. Examples of cell adhesion factors include, but are not limited to, fibronectin, etc. Examples of substances that provide nutrients or energy to cells include, but are not limited to, ATP, pyruvic acid, glutamine, etc.

The hydrogel nonwoven fabric is preferably produced by, but not limited to, extruding a spinning solution containing gelatin into the air from a nozzle outlet from the viewpoint of suppressing the generation of impurities and preventing product contamination, injecting a pressurized fluid forward from a fluid injection port located behind the nozzle outlet and not in contact with the nozzle outlet, forming fibers by causing the extruded spinning solution to accompany the pressurized fluid, and accumulating the obtained fibers to form a nonwoven fabric. When the fibers are accumulated (piled) after spinning, the fibers are stacked in a moist state, so that the fibers are fused together or entangled with each other to be integrated. The density of the nonwoven fabric can be easily changed by changing the collection distance when the fibers are piled up. 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 even more preferably 30 cm or more and 150 cm or less.

11 is a schematic explanatory diagram of a hydrogel nonwoven fabric manufacturing apparatus. In the nonwoven fabric manufacturing apparatus 10, a spinning solution 2 containing gelatin placed in a heating tank 1 is extruded into the air from a nozzle outlet 3. A predetermined pressure is applied to the heating tank 1 by a compressor 4. 12 is a heat-retaining container.
In addition, a pressurized fluid 7 is sprayed forward from a fluid ejection port 5 located behind the nozzle ejection port 3 and not in contact with the nozzle ejection port 3. A pressurized fluid (e.g., compressed air) is supplied to the fluid ejection port 5 from a compressor 6. The distance between the fluid ejection port 5 and the nozzle ejection 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 solution is entrained by the pressurized fluid 7 to become gelatin fibers 8, which are then piled up on a take-up roll 11 as a gelatin nonwoven fabric 9. At this time, the piled up fibers contain moisture and are not completely solidified, so that fibers that are in contact with each other at least at some of the fiber intersections are fused together. Note that other collecting means such as a net may be used instead of the take-up roll.

First, gelatin alone, or gelatin and other biocompatible polymers that can be used as the other components described above, are dissolved in a solvent, preferably water, to prepare a spinning solution. The dissolution temperature (temperature of the solvent, such as water) is preferably 20°C or higher and 90°C or lower, 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 matter and dust. If necessary, the solution may then be degassed under reduced pressure or vacuum to remove dissolved air. From the viewpoint of efficiently removing gas (bubbles), the degree of vacuum during degassing under reduced pressure is preferably 5 kPa or higher and 30 kPa or lower. Since gelatin is water-soluble, it can be spun in the form of an aqueous solution as a spinning solution, and safety for living organisms is increased. As the water, for example, pure water, distilled water, ultrapure water, etc. can be appropriately used. In addition, when other biocompatible water-soluble polymers are used as other components, the spinning solution can be prepared by dissolving the gelatin in water at the same time.

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

The spinning solution is discharged from the nozzle of a spinning machine, a pressurized fluid is supplied from around the nozzle, the discharged gelatin aqueous solution is caused to accompany the pressurized fluid to form fibers, and the resulting gelatin fibers are accumulated to form a gelatin nonwoven fabric (hydrogel nonwoven fabric). The nozzle discharge pressure is not particularly limited, but may be, for example, 0.1 MPa or more and 1 MPa or less.

The temperature of the pressurized fluid is preferably 20°C or higher and 120°C or lower, and more preferably 80°C or higher and 120°C or lower. Stable spinning can be achieved within the above temperature range, although this depends on the flow rate of the pressurized fluid and the temperature of the surrounding atmosphere. It is preferable to use air as the pressurized fluid, and the pressure is preferably 0.1 MPa or higher and 1 MPa or lower. Within the above range, the spinning solution extruded into the air from the nozzle outlet can be blown away to produce fibers.

The hydrogel nonwoven fabric is preferably crosslinked. This can improve the dimensional stability and water resistance. The crosslinking may be chemical crosslinking using a compound such as a crosslinking agent, but from the viewpoint of biological safety, crosslinking using a crosslinking agent having biological safety and/or crosslinking without a crosslinking agent is preferable. Examples of crosslinking without a crosslinking agent include thermal crosslinking, radiation crosslinking such as electron beams and gamma rays, and ultraviolet crosslinking. In the case of radiation irradiation such as electron beams and gamma rays, sterilization and crosslinking can be performed simultaneously. From the viewpoint of easily obtaining the desired crosslinking effect, thermal crosslinking is preferable, and thermal dehydration crosslinking is more preferable. Thermal crosslinking 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 crosslinking time may be, for example, 24 hours or longer and 96 hours or shorter. Thermal dehydration crosslinking may be performed, for example, at 100°C or higher and 180°C or lower for 24 hours or longer and 96 hours or shorter, or at 100°C or higher and 160°C or lower for 24 hours or longer and 96 hours or shorter. The thermal dehydration crosslinking may be carried out under a vacuum of, for example, 1 kPa or less. Before the crosslinking, the material may be dried. The drying method is not particularly limited, and may be, for example, air drying at room temperature or freeze drying.

Within the above ranges, by adjusting the amount of spinning solution discharged, the nozzle diameter (inner diameter), the nozzle discharge pressure, the pressure of the pressurized fluid, the temperature of the pressurized fluid, the distance between the fluid injection port and the nozzle discharge port, the collection distance, and the crosslinking conditions, etc., it is possible to obtain a hydrogel nonwoven fabric having the desired initial thickness retention, initial compressive strength retention, thickness, basis weight, fiber diameter, pore size, etc. As an example, the initial thickness retention and initial compressive strength retention can be increased by increasing the nozzle discharge pressure and thickening the fiber diameter. As an example, the initial thickness retention and initial compressive strength retention can be increased by shortening the distance between the fluid injection port and the nozzle discharge port and increasing the number of fiber intersections.

The hydrogel nonwoven fabric may be cut to a predetermined shape and size as necessary. The hydrogel nonwoven fabric may be sterilized by ethylene oxide gas sterilization, steam (autoclave), electron beam irradiation, gamma ray irradiation, or other radiation exposure, or may be sterilized by ethanol treatment, etc. In the case of irradiation with electron beams or gamma rays, crosslinking can be performed simultaneously with sterilization.

In one or more embodiments of the present invention, the wound dressing is used such 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 nonwoven fabric is the wound surface side. The wound dressing may include a protective film in addition to the hydrogel nonwoven fabric. The protective film is preferably made of a waterproof material. This can prevent moisture from outside the wound site from penetrating into the hydrogel nonwoven fabric. The protective film may be self-adhesive and adhere to the hydrogel nonwoven fabric, or may be adhered to the hydrogel nonwoven fabric via an adhesive layer. The protective film may be disposed on one or both surfaces of the hydrogel nonwoven fabric. When using the wound dressing, one protective film may be peeled off, and the hydrogel nonwoven fabric may be transplanted and used such that one surface of the hydrogel nonwoven fabric is in contact with the wound site of the tissue.

The manufacturing process of the hydrogel nonwoven fabric and wound dressing material is preferably carried out aseptically, for example, on a clean bench or in a clean room. This can prevent the hydrogel nonwoven fabric and wound dressing material from being contaminated by the proliferation of bacteria during the process. It is preferable to use manufacturing tools that have been sterilized, for example, by autoclave or exposure to radiation such as electron beams or gamma rays.

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

One or more embodiments of the present invention will be described in more detail below using examples. Note that the present invention is not limited to the following examples. In this specification, unless otherwise specified, operations are performed at room temperature (20±5°C).

The measurement and evaluation methods are as follows.
<Thickness and thickness retention rate>
The thickness (Ha) of the swollen hydrogel nonwoven fabric before and after collagenase treatment (Hb) was measured using a creep meter manufactured by Yamaden Co., Ltd. The thickness retention rate was calculated as follows:
Thickness retention rate (%) = Hb/Ha x 100
<Compressive strength>
For the swollen hydrogel nonwoven fabric, the stress at 70% strain before collagenase treatment (Fa) and the stress at 70% strain after collagenase treatment (Fb) were measured using a creep meter manufactured by Yamaden Co., Ltd., and these were taken as compressive strengths. The compressive strength retention rate was calculated as follows:
Compressive strength retention rate (%) = Fb / Fa x 100
Treatment with Collagenase
(1) Collagenase D (Sigma-Aldrich) was dissolved in D-PBS(+) (Dulbecco's phosphate buffered saline (D-PBS(-) (1x) from Nacalai Tesque, Inc., to which calcium chloride (100 μg/mL) and magnesium chloride (46.8 μg/mL) from Fujifilm Wako Pure Chemical Industries, Ltd. were added D-PBS(+)) to obtain a collagenase D/PBS(+) solution with a concentration of 1.25 μg/mL.
(2) The swollen hydrogel nonwoven fabric (dry mass: approximately 1 mg) was immersed in 2 mL of collagenase D solution and incubated at 37° C. for 6 hours.
<Average fiber diameter>
The hydrogel nonwoven fabric in the swollen state was observed under a microscope (Keyence Corporation, BZ-X700), and the fiber diameters of 50 arbitrarily selected fibers were measured. These were averaged to calculate the average fiber diameter in the swollen state.
<Weight per unit area>
The basis weight of the gelatin nonwoven fabric was measured in accordance with JIS L 1913:2010.
<Pore diameter>
The pore size of the gelatin nonwoven fabric was calculated using the following calculation formula 1 based on the Wrotnowski assumption.

Example 1
Gelatin manufactured by Nitta Gelatin Co., Ltd. (jelly strength 262 g, raw material: alkali-treated cow bone) was used, and gelatin:water = 3:5 mass ratio (gelatin concentration 37.5 mass%) was used and dissolved at a temperature of 60 ° C. The viscosity of the gelatin aqueous 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 device shown in Figure 11. The temperature of the spinning solution was 60 ° C., the nozzle diameter (inner diameter) was 250 μm, the discharge pressure was 0.2 MPa, the nozzle height was 5 mm, the air pressure was 0.375 MPa, the air temperature was 100 ° C., the distance between the fluid injection port and the nozzle discharge port was 5 mm, and the collection distance was 100 cm. The gelatin nonwoven fabric was air-dried at room temperature overnight, and then heated to dehydrate and crosslink. The crosslinking conditions were a temperature of 140 ° C. and 48 hours. The resulting gelatin nonwoven fabric had a basis weight of about 65 g/m ² .
Next, the dry gelatin nonwoven fabric was punched out into a disk shape with a diameter of about 5 mm (dry mass about 1 mg), and allowed to stand at room temperature for 10 minutes in Dulbecco's phosphate buffered saline (D-PBS(-)(1x), Nacalai Tesque) to swell. The diameter of the swollen hydrogel nonwoven fabric was about 7 mm.

Example 2
A gelatin nonwoven fabric was prepared in the same manner as in Example 1, except that the collection distance was set to 50 cm. The gelatin nonwoven fabric was air-dried at room temperature overnight, and then heated to dehydrate and crosslink. The crosslinking conditions were a temperature of 140°C and 48 hours. The basis weight of the obtained gelatin nonwoven fabric was about 100 g/ ^m2 .
Next, the dry gelatin nonwoven fabric was punched out into a disk shape with a diameter of about 4 mm (dry mass about 1 mg), and allowed to stand at room temperature for 10 minutes to swell in Dulbecco's phosphate buffered saline (D-PBS(-)(1x), Nacalai Tesque, Inc.). The diameter of the swollen hydrogel nonwoven fabric was about 6 mm.

Example 3
<Preparation of hydrogel nonwoven fabric>
A gelatin nonwoven fabric was produced in the same manner as in Example 2, except that the amount of the spinning solution discharged was increased. The gelatin nonwoven fabric was air-dried at room temperature overnight, and then heated to dehydrate and crosslink. The crosslinking conditions were a temperature of 140°C and 48 hours. The basis weight of the obtained gelatin nonwoven fabric was about 150 g/ ^m2 .
Next, the gelatin nonwoven fabric in a dry state was punched out into a disk shape with a diameter of about 3 mm, and 1.25 pieces (dry mass about 1 mg) were left to stand at room temperature for 10 minutes in Dulbecco's phosphate buffered saline (D-PBS(-)(1x), Nacalai Tesque) 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 nonwoven fabric was air-dried at room temperature overnight, and then heated to dehydrate and crosslink. The crosslinking conditions were a temperature of 140°C and 48 hours. The basis weight of the obtained gelatin nonwoven fabric was about 300 g/ ^m2 .
Next, the gelatin nonwoven fabric in a dry state was punched out into a disk shape with a diameter of about 2 mm (dry mass about 1 mg), and allowed to stand at room temperature for 10 minutes to swell in Dulbecco's phosphate buffered saline (D-PBS(-)(1x), Nacalai Tesque, Inc.). 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 amount of the spinning solution discharged was reduced. The gelatin nonwoven fabric was air-dried at room temperature overnight, and then heated to dehydrate and crosslink. The crosslinking conditions were a temperature of 140°C and 48 hours. The basis weight of the obtained gelatin nonwoven fabric was about 50 g/ ^m2 .
Next, the dry gelatin nonwoven fabric was punched out into a disk shape with a diameter of about 6 mm (dry mass about 1 mg), and allowed to stand at room temperature for 10 minutes to swell in Dulbecco's phosphate buffered saline (D-PBS(-)(1x), Nacalai Tesque, Inc.). The diameter of the swollen hydrogel nonwoven fabric was about 9 mm.

(Comparative Example 2)
The collagen sponge was punched out into a disk shape with a diameter of about 8 mm (dry mass about 1 mg) and allowed to swell in Dulbecco's phosphate buffered saline (D-PBS(-)(1x), Nacalai Tesque) at room temperature for 10 minutes. The diameter of the swollen collagen sponge was about 8 mm.

In Examples 1 to 4 and Comparative Examples 1 and 2, the thickness and stress at 70% strain (compressive strength) of the swollen hydrogel nonwoven fabric or collagen sponge before and after treatment with collagenase were measured as described above. For Comparative Example 2, it was dissolved before 6 hours had elapsed, so the compressive strength after 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. In addition, the average fiber diameter of the swollen hydrogel nonwoven fabric was measured as described above. These results are shown in Table 1 below.

Figure 1 is a scanning electron microscope (100x magnification) photograph of the dried hydrogel nonwoven fabric of Example 2. Figure 2 is a scanning electron microscope (100x magnification) photograph of the dried collagen sponge of Comparative Example 2. As can be seen from Figure 1, in the hydrogel nonwoven fabric of Example 2, the intersections between the fibers are fused together, resulting in an interconnected pore structure.

(Experimental Example 1)
The hydrogel nonwoven fabric of Example 1, the hydrogel nonwoven fabric of Comparative Example 1, and the collagen sponge of Comparative Example 2, each having a disk-like shape with a diameter of about 6 mm in a swollen state, were subcutaneously transplanted into the back of a normal mouse (C57BL/6J, 8-week-old, male, obtained from Shimizu Experimental Materials Co., Ltd.). The hydrogel nonwoven fabric of Example 1, the hydrogel nonwoven fabric of Comparative Example 1, and the collagen sponge of Comparative Example 2, each having a disk-like shape with a diameter of about 6 mm in a swollen state, were sterilized with ethylene oxide gas in a dry state before being transplanted.
The graft material was collected 10 days after transplantation, and a frozen section having a thickness of 10 μm was prepared at the maximum cut surface in the thickness direction of the disk-shaped graft material.
The frozen sections were subjected to HE staining and CD31 immunostaining, and the total cell count (HE staining) and the CD31 positive cell count (CD31 immunostaining) were counted within a range of 250 μm from the center of the section to the left and right. The ratio of cells by depth and the ratio of CD31 positive cells to 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 using CD31 antibody (Cell Signaling Technology, "Rabbit Monoclonal Antibody CST 77699") by the peroxidase color development method using DAB.

FIG. 3 is a photograph (40x) showing the results of HE staining of a cross section (depth 200-400 μm) of a transplant material on the 10th day after transplantation, in which the hydrogel nonwoven fabric of Example 1 was used as the transplant material.
FIG. 4 is a photograph (40x) showing the results of CD31 immunostaining of a cross section (depth 200-400 μm) of a transplant material on the 10th day after transplantation, in which the hydrogel nonwoven fabric of Example 1 was used as the transplant material.
FIG. 5 is a photograph (40x) showing the results of HE staining of a cross section (depth 200-400 μm) of a transplant material on the 10th day after transplantation, in which the collagen sponge of Comparative Example 2 was used as the transplant material.
FIG. 6 is a photograph (40x) showing the results of CD31 immunostaining of a cross section (depth 200-400 μm) of a transplant material on the 10th day after transplantation, in which the collagen sponge of Comparative Example 2 was used as the transplant material.

As can be seen from Table 2 and Figures 3 to 6 above, when the hydrogel nonwoven fabric of Example 1 having a predetermined initial thickness retention rate was used, the proportion of cells inside (depth 200 μm or more) of the graft material was greater than the surface (depth 0 to 200 μm) and the proportion of CD31 positive cells among all cells was also greater, compared to when the collagen sponge of Comparative Example 2 was used and when the hydrogel nonwoven fabric of Comparative Example 1 having a low initial thickness retention rate was used. A tendency was confirmed in Example 1 that cell infiltration and blood vessel infiltration (angiogenesis) into the interior of the graft material early after grafting was superior to Comparative Examples 1 and 2, and it is presumed that Example 1 can be used favorably for wound healing.

(Experimental Example 2)
The hydrogel nonwoven fabric of Example 2 and the collagen sponge of Comparative Example 2 in a disk shape with a diameter of about 6 mm in a swollen state were implanted subcutaneously on the back of a normal mouse (C57BL/6J, 8 weeks old, male, obtained from Shimizu Experimental Materials Co., Ltd.).
The grafts were collected on the 7th and 14th days after transplantation, and frozen sections having a thickness of 10 μm were prepared from the maximum cut surface in the thickness direction of the disk-shaped grafts.
The frozen sections were subjected to HE staining and CD31 immunostaining, and the total cell number (HE staining) and the CD31 positive cell number (CD31 immunostaining) were counted within a range of 250 μm from the center of the section to the left and right. The ratio of cells by depth and the ratio of CD31 positive cells in the total 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 using CD31 antibody (Cell Signaling Technology, "Rabbit Monoclonal Antibody CST 77699") by the peroxidase color development method using DAB.

FIG. 7 is a photograph (40x) showing the results of HE staining of a cross section (depth 400-600 μm) of a transplant material on the 7th day after transplantation, in which the hydrogel nonwoven fabric of Example 2 was used as the transplant material.
FIG. 8 is a photograph (40x magnification) showing the results of CD31 immunostaining of a cross section (depth 400-600 μm) of a transplant material on the 7th day after transplantation, in which the hydrogel nonwoven fabric of Example 2 was used as the transplant material.
FIG. 9 is a photograph (40x) showing the results of HE staining of a cross section (depth 400-600 μm) of a transplant material on the 7th day after transplantation, in which the collagen sponge of Comparative Example 2 was used as the transplant material.
FIG. 10 is a photograph (40x) showing the results of CD31 immunostaining of a cross section (depth 400-600 μm) of a transplant material on the 7th day after transplantation, in which the collagen sponge of Comparative Example 2 was used as the transplant material.

As can be seen from Table 3 and Figures 7 to 10 above, when the hydrogel nonwoven fabric of Example 2 having a specified thickness retention rate was used, the proportion of CD31 positive cells among all cells was higher on the 7th day after transplantation compared to when the collagen sponge of Comparative Example 2 was used. It was confirmed that Example 2 tended to have better blood vessel invasion (angiogenesis) into the graft material early after transplantation than Comparative Example 2, and it is presumed that it can be used preferably for wound healing.

As can be seen from Table 4 above, when the hydrogel nonwoven fabric of Example 2 having a predetermined thickness retention rate was used, the proportion of cells inside (depth 200 μm or more) of the graft material was greater than the surface (depth 0-200 μm) of the graft material on the 14th day after transplantation, and the proportion of CD31 positive cells among all cells was also higher, compared to when the collagen sponge of Comparative Example 2 was used. A tendency was confirmed in Example 2 that cell infiltration and blood vessel infiltration (angiogenesis) into the graft material early after transplantation was superior to that of Comparative Example 2, and it is presumed that it can be used favorably for wound healing.

The present invention is not particularly limited, but preferably includes the following aspects.
[1] A wound dressing comprising a hydrogel nonwoven fabric mainly composed of gelatin,
The hydrogel nonwoven fabric is a wound dressing material characterized in that it has a thickness retention rate of 86% or more after treatment with collagenase for 6 hours.
[2] The wound dressing material according to [1], wherein the hydrogel nonwoven fabric has a compressive strength retention rate of 70% or more after treatment with collagenase for 6 hours.
[3] The wound dressing material according to [1] or [2], wherein the hydrogel nonwoven fabric has a thickness in a swollen state of 0.1 mm or more and 5 mm or less, and a basis weight of 40 g/ ^m2 or more and 500 g/m2 or ^less .
[4] The wound dressing material according to any one of [1] to [3], wherein the hydrogel nonwoven fabric has a pore size in a swollen state of 30 μm or more and 700 μm or less.
[5] The wound dressing material according to any one of [1] to [4], wherein the fibers constituting the hydrogel nonwoven fabric have an average fiber diameter in a swollen state of 10 μm or more and 200 μm or less.
[6] The wound dressing material according to any one of [1] to [5], wherein the hydrogel nonwoven fabric is thermally crosslinked by dehydration.
[7] The wound dressing material according to any one of [1] to [6], wherein the hydrogel nonwoven fabric does not contain a cell growth factor.
[8] The wound dressing material according to any one of [1] to [7], wherein a protective film is disposed on one or both surfaces of the hydrogel nonwoven fabric.

Reference Signs List 1 Heating tank 2 Spinning solution 3 Nozzle outlet 4, 6 Compressor 5 Fluid injection port 7 Pressurized fluid 8 Gelatin fiber 9 Hydrogel nonwoven fabric 10 Nonwoven fabric manufacturing device 11 Winding roll 12 Thermal insulation container

Claims (7)

A wound dressing material comprising a hydrogel nonwoven fabric mainly composed of gelatin,
The hydrogel nonwoven fabric has a thickness retention rate of 86% or more after treatment with collagenase for 6 hours;
The hydrogel nonwoven fabric is a wound dressing material characterized in that the pore size of the hydrogel nonwoven fabric in a swollen state obtained by immersing the hydrogel nonwoven fabric in one or more liquids selected from the group consisting of water and buffer solutions for 10 minutes or more is 30 μm or more and 700 μm or less . The wound dressing material according to claim 1, wherein the hydrogel nonwoven fabric has a compressive strength retention rate of 70% or more after treatment with collagenase for 6 hours. 3. The wound dressing according to claim 1, wherein the hydrogel nonwoven fabric has a thickness in the swollen state of 0.1 mm or more and 5 mm or less, and a basis weight of 40 g/ ^m2 or more and 500 g/m2 or ^less . The wound dressing material according to any one of claims 1 to 3 , wherein the fibers constituting the hydrogel nonwoven fabric have an average fiber diameter in the swollen state of 10 µm or more and 200 µm or less. The wound dressing according to any one of claims 1 to 4 , wherein the hydrogel nonwoven fabric is thermally crosslinked by dehydration. The wound dressing material according to any one of claims 1 to 5 , wherein the hydrogel nonwoven fabric does not contain a cell growth factor. The wound dressing according to any one of claims 1 to 6 , wherein a protective film is disposed on one or both surfaces of the hydrogel nonwoven fabric.

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