Scaffold formulation enabling skin regeneration

Abstract

To provide skin tissue regenerators and scaffold materials for skin tissue regeneration.SOLUTION: The present invention provides skin tissue regenerators containing gelatin sponge, and scaffold materials for skin tissue regeneration containing gelatin sponge.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a skin tissue regeneration agent and a scaffold for skin tissue regeneration. [Background technology]

[0002] Bone cortex exposed wounds accompanied by skin and soft tissue defects often occur after trauma or extensive resection of malignant tumors, and are difficult to treat. Because the bone cortex is exposed, it can easily progress to sequestrum, which increases the possibility of infection, and there is also a risk of sepsis, making it an appropriate surgical treatment using a free skin flap. However, this is a long operation that requires microvascular anastomosis under a microscope, and involves the risk of donor site sacrifice and flap necrosis, making it a stressful operation for both the surgeon and the patient. Moreover, even if the flap survives, the scars around the flap and the scars at the donor site do not regenerate skin appendages, and many patients suffer from keloids and ulcers because they have no sensation or temperature regulation.

[0003] Treatment that leaves no scars (tissue regeneration) is also known as scarless wound healing, but in humans, this is only possible during the fetal stage; after birth, all wounds with full-thickness or greater skin defects are treated with scarless wound healing. Therefore, complications such as itching, sensory paralysis, and recurrent ulcers caused by scarring are inevitable, and scarless wound healing after birth has been an urgent issue for many years.

[0004] The use of scaffolds in wound healing is an alternative treatment. However, although conventional scaffolds claim to promote wound healing, the goal of treatment is scar healing.

[0005] Gelatin sponges are used as hemostatic agents in clinical settings. Other examples of the use of such gelatin sponges are reported in Patent Documents 1 and 2, Non-Patent Document 1, etc.

[0006] Patent Document 1 reports a composition for treating facial nerve paralysis that is inserted through the external auditory canal and placed at an open site of the facial nerve canal located at a position that reaches the facial nerve from within the tympanic cavity, and that contains a substance having a nerve regeneration effect supported on a carrier made of a bioabsorbable polymer. Gelatin sponge is cited as an example of the carrier made of a bioabsorbable polymer.

[0007] Patent Document 2 reports an agent for regenerating tympanic membrane or ear canal, which is a combination of basic fibroblast growth factor, gelatin sponge, and a covering material, and during use, the gelatin sponge is placed at the regeneration site while carrying the basic fibroblast growth factor, and the covering material isolates the regeneration site from the outside.

[0008] Non-Patent Document 1 describes a method for treating tympanic membrane perforation using a gelatin sponge scaffold. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2020-176072 A [Patent Document 2] Patent No. 5398712 [Non-patent literature]

[0010] [Non-Patent Document 1] Jpn Otolaryngology 125: 933-939,2022 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a skin tissue regeneration agent and a scaffold material for skin tissue regeneration, typically for deep tissue defect wounds in which the cortical bone is exposed. [Means for solving the problem]

[0012] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they found that when gelatin sponge (hereinafter sometimes referred to as "GS" in this specification) was used as a scaffold in a rat head model with extensive bone exposure, tissue regeneration of the layered structure of the periosteum, soft tissue, and skin was possible. Furthermore, not only the layered structure of the skin but also the regeneration of skin appendages such as hair follicles and peripheral nerves was possible.

[0013] The present invention has been completed based on these findings and through further investigation, and provides the following skin tissue regeneration agent and scaffold material for skin tissue regeneration.

[0014] Item 1. A skin tissue regeneration agent containing a gelatin sponge. Item 2. The regenerative agent according to Item 1, for treating skin defects involving soft tissue. Item 3. The regenerative agent according to Item 1, which is for treating wounds in which bone cortex is exposed. Item 4. The regenerative agent according to Item 3, wherein the wound with exposed bone cortex is a wound with a loss of periosteum. Item 5. The regenerative agent according to Item 1, which is used for regenerating the layered structures of periosteum, soft tissue and skin. Item 6. A scaffold material for skin tissue regeneration comprising a gelatin sponge. Item 7. The scaffold according to Item 6, for treating skin defects involving soft tissue. Item 8. The scaffold according to Item 6, which is for treating wounds where bone cortex is exposed. Item 9. The scaffold according to Item 8, wherein the bone cortex exposure wound is a wound in which the periosteum is missing. Item 10. The scaffold according to Item 6, which is for regenerating the layered structure of periosteum, soft tissue and skin. Effect of the Invention

[0015] According to the present invention, it is possible to regenerate tissues, not scars, even in the most severe cases where the skin, soft tissues, and even the periosteum are lost. Furthermore, it is possible to regenerate skin appendages such as hair follicles and sebaceous glands. Furthermore, the skin tissue regenerator and the scaffold for skin tissue regeneration of the present invention do not require a donor or complicated surgery, and are therefore procedurally easy.

[0016] The gelatin sponge used in the present invention has been used clinically as a hemostatic agent for many years, and is therefore highly safe. [Brief description of the drawings]

[0017] [Figure 1] Figure 1 shows that gelatin sponge enables skin regeneration in deep wounds with exposed bone cortex. (af) Photographs showing the appearance of skin / subcutaneous defects with / without periosteal defect when gelatin sponge was administered or not (after 0 and 4 weeks), (gl) Photographs showing H&E staining of deep wounds with periosteal defect after 4 weeks, (mp) Immunofluorescent staining images with POSTIN, CD31 and SMA after 4 weeks of wounding. Scale bars: (af) 5 mm, (g,j) 1 mm, (h,i,k,l) ​​100 μm, (mp) 10 μm, d: days, w: weeks, pw: post-wounding, B: bone, Wounds with brackets in g and j: initially excised area, dotted line: apical border, dashed line: surface of bone cortex, POSTIN: periostin, SMA: smooth muscle actin [Diagram 2] Figure showing tissue regeneration by gelatin sponge in deep wounds with periosteal defects in a BMT rat model. (a) Outline of preparation of BMT rats, (b) Appearance of deep wounds at each time point with and without gelatin sponge administration, (c) Graph showing wound area with and without gelatin sponge administration, (dt) Photograph showing H&E stained image of wounds with and without gelatin sponge administration, Scale bar: (b) 5 mm, (d, g, j, m, p, r) 1 mm, (e, f, h, i, k, l, n, o, q, s, t) 100 μm, B: cortical bone, *P<0.05, **P<0.01, ***P<0.005 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Hereinafter, an embodiment of the present invention will be described in detail.

[0019] The skin tissue regenerating agent and scaffold for skin tissue regeneration of the present invention are characterized by containing a gelatin sponge (hereinafter, also referred to as "the regenerating agent and scaffold of the present invention").

[0020] Gelatin sponge is a porous structure made of gelatin. The type of gelatin used as the raw material for gelatin sponge is not particularly limited, and may be any gelatin that is usually available. For example, crude collagen obtained by treating bones, ligaments, tendons, and skin of cows, pigs, chickens, salmon, etc. with acid or alkali, and then heating and extracting it with water, etc. Gelatin may be used alone, or may be used by appropriately mixing gelatin of different raw materials and physical properties such as solubility, molecular weight, isoelectric point, etc.

[0021] The gelatin sponge may be crosslinked to improve water resistance. The crosslinking method is not particularly limited, and examples thereof include a method using a crosslinking agent, a gamma ray irradiation method, an ultraviolet ray irradiation method, an electron beam irradiation method, an X-ray irradiation method, a vacuum heat dehydration method, and a dry heat method.

[0022] Gelatin sponge has many fine pores and has a higher degree of freedom than collagen. When used as a scaffold material, the presence of many fine pores allows surrounding cells to easily enter the sponge, enabling good skin tissue regeneration.

[0023] Considering that cells can easily enter the interior of the sponge and good cell adhesiveness can be obtained, the average pore size of the gelatin sponge is preferably about 10 to about 500 μm, and more preferably about 100 to about 400 μm.

[0024] The gelatin sponge can be produced by a known method (for example, the method described in International Publication No. 2009 / 157558). Specifically, it can be produced by the steps of (I) dissolving gelatin in heated water and filtering through a filter with a pore size of 0.2 μm while maintaining the temperature at 45° C. or higher, (II) vigorously stirring the obtained gelatin aqueous solution using a homogenizer or the like to foam it, (III) immediately freeze-drying the foamed gelatin aqueous solution by a known method, and (IV) cutting the obtained freeze-dried product into a sheet of a desired thickness. In addition to the above steps, a step of (V) heating the obtained sheet to further thermally crosslink the gelatin may be added. This step can increase the strength of the obtained gelatin sponge. The gelatin concentration in the gelatin aqueous solution can be appropriately adjusted so as to obtain the desired physical properties. Specifically, the water absorption amount in a water absorption test (the mass after immersion in water divided by the mass before immersion) may be adjusted to about 40 to 50 times, for example, 5.5 to 6.5% by mass.

[0025] As the gelatin sponge, commercially available hemostatic agents such as Spongel (registered trademark) (Astellas Pharma Inc., LTL Pharma Co., Ltd.) and Gelfoam (registered trademark) (Pfizer) can be used.

[0026] The shape and size of the gelatin sponge are not particularly limited as long as they are large enough to cover the skin defect site of the patient.

[0027] The regenerative agent and scaffold of the present invention may contain other components besides the gelatin sponge, as long as they do not adversely affect the function of the regenerative agent and scaffold. Examples of such components include synthetic peptides and bacteriophages for the purpose of preventing infection.

[0028] The content of gelatin sponge in the regenerating agent and scaffold of the present invention may be, for example, 0.001 to 100% by mass of the total amount of the regenerating agent and scaffold of the present invention. The upper or lower limit of the range is, for example, 0.01% by mass, 0.1% by mass, 1% by mass, 5% by mass, 10% by mass, 20% by mass, 30% by mass, 40% by mass, 50% by mass, 60% by mass, 70% by mass, 80% by mass, 90% by mass, 95% by mass, 99% by mass, or 99.9% by mass.

[0029] When the regenerator and scaffold material of the present invention are sterilized for use, the sterilization method is not particularly limited, and any applicable known method can be used as appropriate, such as high-pressure steam sterilization, filtration sterilization, dry heat sterilization, electron beam (EB) sterilization, gamma ray sterilization, ethylene oxide gas (EOG) sterilization, and hydrogen peroxide gas sterilization.

[0030] The regenerating agent and scaffold of the present invention can be used by being placed directly on the damaged site of the skin. After the regenerating agent and scaffold of the present invention are placed on the damaged site of the skin, the gelatin sponge can be fixed by performing a procedure such as suturing to the surrounding tissue or applying pressure from above with tape.

[0031] The regenerating agent and scaffold of the present invention can be used for the treatment of skin defects due to trauma, after extensive resection of malignant tumors, etc., and can be preferably used for the treatment of skin defects accompanied by soft tissue, and can be particularly preferably used for the treatment of wounds with exposed bone cortex. Here, the wounds with exposed bone cortex are particularly used to mean wounds with a defect in the periosteum. Conventionally, there has been no method for regenerating skin from wounds with a defect in the periosteum. However, by using the regenerating agent and scaffold of the present invention, even in wounds where the periosteum is defective and the bone cortex is exposed, it is possible to regenerate while maintaining the layered structure of the skin. Therefore, the regenerating agent and scaffold of the present invention can be used for the regeneration of the layered structure of the periosteum, soft tissue, and skin.

[0032] The regenerator and scaffold of the present invention are intended for use in patients with skin defects due to trauma, extensive resection of malignant tumors, etc., and are particularly suitable for use in patients with skin defects involving soft tissue, and in particular for patients with wounds exposing bone cortex (especially wounds in which the periosteum is missing).

[0033] The regenerative agent and scaffold of the present invention are administered to mammals including humans, monkeys, rats, mice, guinea pigs, rabbits, goats, sheep, horses, cows and pigs, and are particularly administered to humans.

[0034] The dosage of the regenerative agent and scaffold of the present invention is appropriately determined taking into consideration the size of the site of skin defect in the patient, and can be, for example, a rectangle with each side measuring approximately 1 to 5 cm.

[0035] The regenerating agent and scaffold of the present invention can regenerate tissues without scarring in the most severe cases of exposed bone wounds in which the skin, soft tissues, and even the periosteum are missing. In other words, the present invention allows for scarless wound healing, a treatment that does not leave a wound. Furthermore, it is possible to regenerate skin appendages such as hair follicles and sebaceous glands. In the absence of skin appendages, there is no sweating, no hair follicles, and no sebaceous glands, resulting in dry skin and itching. In addition, the regenerating agent and scaffold of the present invention do not require a donor, do not require complicated surgery, and are easy to use. Thus, the present invention is less invasive and does not require long-term surgery or long-term hospitalization.

[0036] The gelatin sponge used in the present invention has been used clinically as a hemostatic agent for many years, and is therefore highly safe. Therefore, the process of clinical application of the gelatin sponge, which is a repositioning method, is relatively easy.

[0037] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present invention includes all arbitrary combinations of the constituent elements described in this specification.

[0038] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present invention above may be combined in any way to specify the subject matter included in the present invention. In other words, the present invention includes all subject matter consisting of all combinations of the combinable characteristics described in this specification. EXAMPLES

[0039] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0040] method <Animal> Wild-type Sprague-Dawley (SD) rats and SD-Tg(CAG-EGFP)CZ-004Osb(GFP) rats (Japan SLC Co., Ltd.) were used in this study. GFP rats were crossed with SD rats, and their offspring were used as donors for bone marrow transplantation (BMT). Seven-week-old male wild-type SD rats (Japan SLC Co., Ltd.) were used as recipients. During the experimental period, rats had free access to water and CLEA Rodent Diet CE-2 (Japan CLEA Co., Ltd.). This study was approved by the Animal Experiment Ethics Committee of Shiga University of Medical Science and was carried out in accordance with the Shiga University of Medical Science Animal Experiment Guidelines and the Act on the Welfare and Management of Animals.

[0041] <Bone marrow transplant (BMT)> Recipient rats were irradiated with 9 Gy total body radiation from an X-ray source (FUJIFILM Healthcare Co., Ltd.). Bone marrow was harvested from the femur, tibia, and humerus of transgenic donor rats by flushing with phosphate buffered saline (PBS). Six hours after X-ray irradiation, bone marrow-derived cells (BMDCs) were harvested at 1.0 × 10 8 was injected into the tail vein of the recipient rats. Four weeks after bone marrow transplantation, peripheral leukocytes from the recipient rats were analyzed for GFP expression by flow cytometry (FACScant II, Becton Dickinson), and it was confirmed that more than 88% of blood cells had been replaced by those derived from the donor.

[0042] <Animal model of deep wound with periosteal defect> Under general anesthesia with 2% isoflurane combined with intraperitoneal injection of medetomidine hydrochloride (0.15 mg / kg), midazolam (2 mg / kg), and butorphanol tartrate (2.5 mg / kg), a 10 mm × 10 mm (100 mm) thick piece of full-thickness skin with or without periosteum was placed in the graft. 2 ) square defects were created in the calvaria of wild-type or BMT rats. Gelatin sponge (LTL Pharma Co., Ltd.) was administered to the wound in one group, while the wound in the other group was left open. Animals without wounds were used as controls. The wounds were photographed at 0, 2, 4, and 6 weeks, and the wound areas were measured using ImageJ software (National Institutes of Health). Three rats were prepared in each group, and the experiment was repeated twice.

[0043] <Tissue structure and immunohistochemistry> After exsanguination and transcardial perfusion with 4% PFA in 0.1 M PBS, calvaria were decalcified with 10% EDTA for 24 h at 4°C. Calvaria sections were prepared at 7 μm thickness using a cryostat for staining with hematoxylin and eosin. For immunohistochemistry, sections were treated with 5% normal goat serum for 1 h at 25°C, incubated with primary antibodies overnight at 4°C, and then incubated with secondary antibodies for 2 h at 25°C. The primary antibodies used were: anti-smooth actin (1:200, ab5694, Abcam), anti-CD31 (1:50, ab281583, Abcam), and anti-POSTN (1:200, ab215199, Abcam). The secondary antibody used was Goat anti-rabbit Alexa 555 (1:400, A27039, Thermo Fisher Scientific).

[0044] Sections for immunofluorescence were mounted with Vector Shield using 4'-6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and photographed using a confocal laser microscope (Leica TCS SP8, Leica Microsystems).

[0045] <Statistical analysis> Data were evaluated using t-test to analyze differences between two groups. Data are expressed as mean ± SE. P<0.05 was considered significant.

[0046] result The wound healing process was compared in a rat model of skin and subcutaneous tissue defects with or without a periosteal defect. First, a deep wound without a periosteal defect was created in the rat calvaria (Fig. 1a). The wound was left open in one group, while a gelatin sponge was administered to the wound in the other group (Fig. 1b, c). As a result, the wound healed with few hair follicles after 4 weeks, regardless of the administration of gelatin sponge.

[0047] Next, the same wound, including the excision of the periosteum, was created in the rat calvaria (Fig. 1d). The wound was left open in one group (GS(-)) and the other group was administered gelatin sponge into the wound (GS(+)). After 4 weeks, the wound was covered with a scab in GS(-) rats, whereas the administration of gelatin sponge caused the wound to heal similarly to the wound with periosteum (Fig. 1b, c, e, f). H&E staining showed that bone defects were observed in the center due to osteolysis caused by osteomyelitis (Fig. 1g, brackets), and the bone cortex was thinner in GS(-) rats than in GS(+) rats (Fig. 1g, j, dashed lines). Higher magnification revealed the presence of immune cells and cellular debris adjacent to the bone in GS(-) rats, and that multinucleated cells, presumably osteoclasts, had infiltrated the bone cortex (Fig. 1h, i, arrowheads). In contrast, in GS(+) rats, the thickness of the bone in the center of the wound was comparable to that of the surrounding bone (Fig. 1j, brackets), and surprisingly, the exposed bone was covered with membranous tissue (Fig. 1k, arrowheads). In addition, hair follicles were observed in the wound (Fig. 1l, arrowheads). We found that the membranous tissue covering the bone cortex in GS(+) rats after 4 weeks was positive for periostin, which is selectively expressed in the periosteum, but was absent in the wounded bone of GS(-) rats (Fig. 1m, n). We next investigated whether angiogenesis occurs in the skin formed in deep wounds with periosteal defects. As a result, CD31- and smooth muscle actin (SMA)-positive vascular structures were observed in the new dermis of GS(+) rats after 4 weeks, but not in GS(-) rats (Fig. 1o, p). In particular, the CD31-positive cells were surrounded by SMA-positive cells, and considering that CD31 is an endothelial cell marker and SMA is expressed in vascular smooth muscle cells, the observed blood vessels are considered to be functional in GS(+) rats.

[0048] After 9 Gy irradiation, allogeneic bone marrow cells were transplanted from GFP rats, followed by the creation of deep wounds including the excision of the periosteum, and they were divided into two groups, namely GS(-) and GS(+) rats (Fig. 2a). A similar course was observed in GS(+)BMT rats as in Fig. 1, and scabs remained in GS(-) rats after 6 weeks (Fig. 2b). Analysis of the wound area over time showed that the wound area was significantly reduced in GS(+)BMT rats compared with GS(-)BMT rats (Fig. 2c). H&E staining showed no significant difference between GS(-)BMT rats and GS(+)BMT rats in low magnification images after 2 weeks, except that the degradation of the gelatin sponge had already started in GS(+)BMT rats (Fig. 2d, g). However, high magnification images revealed that osteolysis had already developed in the center of the exposed bone cortex in GS(-)BMT rats after 2 weeks, and infiltration of osteoclast-like cells into the bone cortex was observed at the wound edge (Fig. 2e, f). Conversely, in GS(+)BMT rats after 2 weeks, not only was cell infiltration into the bone cortex not observed at the wound edge, but many cavities surrounded by cells were observed (Fig. 2h, i). After 4 weeks, similar images were obtained as shown in Fig. 1g-l, indicating that BMT rats recapitulated normal wound healing (Fig. 2j-o). After 6 weeks, fibrous tissue was observed on the bone cortex in GS(-)BMT rats, and surprisingly, a layered skin with new hair follicles / sebaceous glands was reconstituted at the center of the wound in GS(+)BMT rats (Fig. 2p-s). Moreover, the accumulation of cells on the bone cortex suggested the regeneration of the periosteum (Fig. 2t).

Claims

1. A skin tissue regenerator comprising a gelatin sponge.

2. The regenerative agent according to claim 1, which is for treating skin defects involving soft tissue.

3. The regenerative agent according to claim 1 , which is used for treating wounds where bone cortex is exposed.

4. The regenerative agent according to claim 3 , wherein the wound in which bone cortex is exposed is a wound in which the periosteum is missing.

5. The regenerating agent according to claim 1, which is for regenerating the layered structures of periosteum, soft tissue and skin.

6. A scaffold material for skin tissue regeneration comprising a gelatin sponge.

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