Acellular-based wound dressing

Abstract

A method of forming an acellular sample is disclosed. The method includes forming a submucosal-free tissue and decellularization of cells existed among an interconnected framework of the submucosal-free tissue by applying a cold atmospheric plasma to the submucosal-free tissue. Forming the submucosal-free tissue includes decellularization of a submucosal layer of a tissue sample by soaking the tissue sample in an enzyme solution. The acellular sample is used for producing a wound dressing patch and a wound dressing gel.

Description

ACELLULAR-BASED WOUND DRESSINGTECHNICAL FIELD

[0001] The present disclosure generally relates to a method of decellularization of a tissue sample using cold plasma, and more particularly, relates to a method of forming a wound dressing based on acellular structures.BACKGROUND ART

[0002] Wounds are injuries that damage skin or other body tissues. Wounds are divided into different categories of intensity and healing time. Wounds with high intensity of damage can harm body tissues which need more time and a sterile environment for healing. Some factors may cause chronic wounds which may last several years. These factors may include lack of oxygen supply, infection, age, sex hormones, stress, diabetes, obesity, drugs, alcoholism, smoking, and nutrition. White blood cells need oxygen for fighting against infectious organisms. Lacking oxygen may cause white blood cells malfunction and subsequent infections.

[0003] Wound and tissue healing is a programmed, overlapping, dynamic, and natural four- step biological process in body that includes hemostasis, inflammation, proliferation, and maturation. The last stage of wound healing is maturation stage, which requires a precise balance between apoptosis (programmed death) of existing cells and production of new cells. Many factors cause this type of cell death, including hormones, toxins, cytokines, radiation, heat, viral infection etc. gradual destruction of extracellular matrix and immature type III collagen and formation of mature type I collagen is very important in this stage, which lasts for several months and years. Any deviation in this step may lead to excessive healing of wounds or chronic wounds.

[0004] Acellular structure is a framework of a tissue without cells. Acellular structures are used as a framework for cell proliferation with no body immune responses due to being produced using a tissue of a body. Acellular structures can be formed by decellularization using physical and chemical approaches. In physical approach, cell membrane may be under action of force, pressure, or temperature. In chemical approach, cell membrane is in contact with chemical solvents. Physical and chemical approaches both suffer from different drawbacks including destruction of acellular framework, remaining toxic solvents within acellular framework, etc.Drawbacks of chemical and physical approaches may limit application of these methods for forming acellular structures suitable for wound healing.

[0005] Different strategies are used to heal superficial, deep, and chronic wounds. For example, Thomas Bayer et al. presented a patent on “Dry composition wound dressings and adhesives” (AU2011287215B2). Thomas Bayer et al. described patches including dry gelatin compositions. In an exemplary invention, the patch included implantable surgical mesh, a cross-linkable protein matrix (gelatin) and a protein cross-linking enzyme in contact with the matrix for cross-linking the gelatin. Axel Dr. Thierauf et al. presented a patent on “Multilayered wound dressing, useful particularly for weeping wounds or those that require irrigation, comprises fleece, contacting the wound, and membrane of water-insoluble polymer” (DE102004063599A1). Axel Dr. Thierauf et al. formed a multilayered wound dressing including a fleece, in contact with the wound; a watertight membrane made of at least one water- insoluble polymer. These inventions prevent wound infections, however for healing wounds an ignition for cell proliferation is also required. Acellular structures can form a framework for cell proliferation and accelerating healing processes.

[0006] There is, therefore, a need for a method to form an acellular structure with no harmful side effects. There is further a need for a method to produce a biocompatible and effective wound dressing using acellular structures.SUMMARY OF THE DISCLOSURE

[0007] This summary is intended to provide an overview of the subject matter of this patent, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of this patent may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0008] According to one or more exemplary embodiments, the present disclosure is directed to a method of forming an acellular sample. In an exemplary embodiment, an exemplary method may include forming a submucosal-free tissue and decellularization of cells existed among an interconnected framework of an exemplary submucosal-free tissue by applying a cold atmospheric plasma to an exemplary submucosal-free tissue. In an exemplary embodiment, forming an exemplary submucosal-free tissue may include decellularization of a submucosal layer of a tissue sample by soaking an exemplary tissue sample in an enzyme solution. In anexemplary embodiment, an exemplary enzyme solution may have a concentration in a range of 0.005 w / w to 0.05 w / w (enzyme / water).

[0009] In an exemplary embodiment, an exemplary tissue sample may include at least one of a bone sample, a muscle sample, a skin sample, an intestine sample, a tendon sample, an amniotic membrane sample, a peritoneal membrane sample, a liver sample, a spleen sample, a pancreas sample, a cartilage sample, and combinations thereof.

[0010] In an exemplary embodiment, an exemplary enzyme may include trypsin. In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue may include applying an exemplary cold atmospheric plasma to an exemplary submucosal-free tissue for a time period in a range of 3 minutes to 10 minutes.

[0011] In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue may include applying an exemplary cold atmospheric plasma to an exemplary submucosal-free tissue with a voltage of an exemplary cold atmospheric plasma in a range of 5 V to 50 V.

[0012] In an exemplary embodiment, an exemplary cold atmospheric plasma may include a cold argon atmospheric plasma. In an exemplary embodiment, an exemplary cold atmospheric plasma may include a nanosecond-pulsed dielectric barrier discharge (DBD).

[0013] In an exemplary embodiment, an exemplary method may further include forming a wound dressing patch by pressing an exemplary acellular sample using a mechanical pressing device. In an exemplary embodiment, forming an exemplary wound dressing patch may include pressing an exemplary acellular sample to a thickness in a range of 0.01 mm to 1 mm using an exemplary mechanical pressing device.

[0014] In an exemplary embodiment, an exemplary method may further include forming a wound dressing gel. In an exemplary embodiment, forming an exemplary wound dressing gel may include grinding an exemplary acellular sample using a mechanical device and forming a gel by mixing a hyaluronic acid solution with an exemplary grinded acellular sample with a weight ratio of an exemplary hyaluronic acid solution to an exemplary grinded acellular sample in a range of 25: 1 to 5 : 1 (an exemplary hyaluronic acid solution: an exemplary grinded acellular sample).

[0015] In an exemplary embodiment, an exemplary grinded acellular sample may have an average particle size in a range of 4 pm to 60 pm. In an exemplary embodiment, an exemplaryhyaluronic acid solution in water may have a weight ratio in a range of 50: 1 to 5: 1 (hyaluronic acid: water).

[0016] According to one or more exemplary embodiments, the present disclosure is directed to a method of forming a wound dressing patch. In an exemplary embodiment, an exemplary method may include forming an acellular sample and pressing an exemplary acellular sample using a mechanical pressing device. In an exemplary embodiment, forming an exemplary acellular sample may include forming a submucosal-free tissue and decellularization of cells existed among an interconnected framework of an exemplary submucosal-free tissue. In an exemplary embodiment, forming an exemplary submucosal-free tissue may include decellularization of a submucosal layer of a tissue sample by soaking an exemplary tissue sample in an enzyme solution. In an exemplary embodiment, an exemplary enzyme solution may have a concentration in a range of 0.005 w / w to 0.05 w / w (enzyme / water). In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue may include applying a cold atmospheric plasma to an exemplary submucosal-free tissue.

[0017] In an exemplary embodiment, an exemplary cold atmospheric plasma may include a nanosecond-pulsed dielectric barrier discharge (DBD). In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary tissue sample may include applying an exemplary cold atmospheric plasma to an exemplary submucosal-free tissue with a voltage of an exemplary cold atmospheric plasma in a range of 5 V to 50 V for a time period in a range of 3 minutes to 10 minutes.

[0018] In an exemplary embodiment, an exemplary tissue sample may include at least one of a bone sample, a muscle sample, a skin sample, an intestine sample, a tendon sample, an amniotic membrane sample, a peritoneal membrane sample, a liver sample, a spleen sample, a pancreas sample, a cartilage sample, and combinations thereof.

[0019] According to one or more exemplary embodiments, the present disclosure is directed to a method of forming a wound dressing gel. In an exemplary embodiment, an exemplary method may include forming an acellular sample, grinding an exemplary acellular sample using a mechanical device, and forming a gel by mixing a hyaluronic acid solution with an exemplary grinded acellular sample with a weight ratio of an exemplary hyaluronic acid solution to an exemplary grinded acellular sample in a range of 25:1 to 5:1 (an exemplary hyaluronic acid solution: an exemplary grinded acellular sample). In an exemplary embodiment, forming anacellular sample may include forming a submucosal-free tissue and decellularization of cells existed among an interconnected framework of an exemplary submucosal-free tissue. In an exemplary embodiment, forming an exemplary submucosal-free tissue may include decellularization of a submucosal layer of a tissue sample. In an exemplary embodiment, decellularization of an exemplary submucosal layer of an exemplary tissue sample may include soaking an exemplary tissue sample in an enzyme solution with a concentration of an exemplary enzyme solution in a range of 0.005 w / w to 0.05 w / w (enzyme / water). In an exemplary embodiment, decellularization of exemplary cells existed among an interconnected framework of an exemplary submucosal-free tissue may include applying a cold atmospheric plasma to an exemplary submucosal-free tissue.

[0020] In an exemplary embodiment, an exemplary grinded acellular sample may have an average particle size in a range of 4 pm to 60 pm. In an exemplary embodiment, an exemplary cold atmospheric plasma may include a nanosecond-pulsed dielectric barrier discharge (DBD).

[0021] In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue may include applying an exemplary cold atmospheric plasma to an exemplary submucosal-free tissue with a voltage of the cold atmospheric plasma in a range of 5 V to 50 V for a time period in a range of 3 minutes to 10 minutes.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawing figures depict one or more implementations in accord with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements.

[0023] FIG. 1A illustrates a flowchart of a method of producing an acellular sample, consistent with one or more exemplary embodiments of the present disclosure;

[0024] FIG. IB illustrates a flowchart of a method of producing a wound dressing patch, consistent with one or more exemplary embodiments of the present disclosure;

[0025] FIG. 1C illustrates a flowchart of a method of producing a wound dressing gel, consistent with one or more exemplary embodiments of the present disclosure;

[0026] FIG. 2 illustrates a schematic presentation of a tissue sample of an intestine used for preparing an exemplary wound dressing patch and wound dressing gel, consistent with one or more exemplary embodiments of the present disclosure;

[0027] FIG. 3 illustrates a fluorescent image of 4',6-diamidino-2-phenylindole (DAPI) staining of an exemplary acellular sample, consistent with one or more exemplary embodiments of the present disclosure;

[0028] FIG. 4 illustrates stained images of an exemplary acellular sample using hematoxylin eosin, consistent with one or more exemplary embodiments of the present disclosure;

[0029] FIG. 5 illustrates stained images of an exemplary acellular sample using Masson's trichrome, consistent with one or more exemplary embodiments of the present disclosure;

[0030] FIG. 6 illustrates scanning electron microscope (SEM) images of an exemplary acellular sample, consistent with one or more exemplary embodiments of the present disclosure;

[0031] FIG. 7 illustrates photomicrograph images of an experimental group treated with an exemplary produced acellular sample and an experimental control group treated with Masson's trichrome staining, consistent with one or more exemplary embodiments of the present disclosure;

[0032] FIG. 8 illustrates photomicrograph images of an experimental group treated with an exemplary produced acellular sample stained with Hematoxylin and Eosin (H&E) at 5 days and 21 days, consistent with one or more exemplary embodiments of the present disclosure;

[0033] FIG. 9 illustrates transmission electron microscopy (TEM) images of burned tissue, control sample, and acellular treated sample, consistent with one or more exemplary embodiments of the present disclosure;

[0034] FIG. 10 illustrates photomicrograph images of an exemplary acellular sample with immunohistochemical staining to examine Vimentin protein, consistent with one or more exemplary embodiments of the present disclosure;

[0035] FIG. 11 illustrates photomicrograph images for Desmin protein examination by immunohistochemistry staining, consistent with one or more exemplary embodiments of the present disclosure; and

[0036] FIG. 12 illustrates photomicrograph images of an exemplary acellular sample with immunohistochemical staining for controlling CD31 marker, consistent with one or more exemplary embodiments of the present disclosure.DESCRIPTION OF EMBODIMENTS

[0038] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0039] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following discussion. In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high- level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0040] Wound and tissue healing may be a programmed, overlapping, dynamic, and natural four-step biological process in body. Wound and tissue healing process may include homeostasis, inflammation, proliferation, and maturation steps. An exemplary maturation step may require a precise balance between apoptosis of existing cells and production of new cells. In an exemplary embodiment, apoptosis may refer to a programmed death of cells. An exemplary apoptosis may refer to a cell death in multicellular organisms. During apoptosis,cellular macromolecules may turn into smaller pieces in a controlled manner. Finally exemplary smaller pieces of exemplary cellular macromolecules may collapse into smaller intact pieces that may be removed by phagocytosis without damaging surrounding cells or causing inflammation. Wound healing may be a complex process that may include several cellular and biochemical reactions. Exemplary cellular and biochemical reactions may include inflammation, vascularization, and collagen deposition. Abnormal accumulation of collagen in wound area may lead to creation of hypotrophic scars.

[0041] In an exemplary embodiment, an acellular sample may be used for accelerating cell proliferation in wound regions. In an exemplary embodiment, an exemplary acellular sample may penetrate into injured tissue and help body to restore damaged tissue. In an exemplary embodiment, an exemplary acellular sample may prevent abnormal collagen accumulation which may be responsible for hypotrophic scars. In an exemplary embodiment, an exemplary acellular sample may be biocompatible.

[0042] The present disclosure is directed to exemplary embodiments of a method for producing an exemplary acellular sample. In an exemplary embodiment, an exemplary acellular sample may include a framework of tissue without cells. In an exemplary embodiment, an exemplary acellular sample may be used for forming a wound dressing. In an exemplary embodiment, an exemplary wound dressing may include at least one of a wound dressing patch, a wound dressing gel, and combinations thereof. In an exemplary embodiment, using an exemplary acellular sample as a wound dressing component may help cells to proliferate in an already prepared framework.

[0043] In an exemplary embodiment, an exemplary acellular sample may be produces by forming a submucosal-free tissue and decellularization of cells existed among an interconnected framework of an exemplary submucosal-free tissue. In an exemplary embodiment, an exemplary submucosal-free tissue may refer to a tissue sample without submucosal layer. An exemplary submucosal layer may existed beneath an exemplary tissue sample in contact with interior body organs of a living creature. In an exemplary embodiment, an exemplary living creature may include at least one of a human, an animal, and combinations thereof. In an exemplary embodiment, an exemplary tissue sample may include at least one of a bone sample, a muscle sample, a skin sample, an intestine sample, a tendon sample, an amniotic membrane sample, a peritoneal membrane sample, a liver sample, a spleen sample, a pancreas sample, a cartilage sample, and combinations thereof.

[0044] In an exemplary embodiment, an exemplary submucosal-free tissue may be formed by decellularization of an exemplary submucosal layer of an exemplary tissue sample. In an exemplary embodiment, an exemplary submucosal layer may be eliminated by soaking an exemplary tissue sample in an enzyme solution. In an exemplary embodiment, an exemplary tissue sample may be soaked in an exemplary enzyme solution. In an exemplary embodiment, as used herein soaking may refer to fully drowning an exemplary tissue sample in an exemplary enzyme solution. In an exemplary embodiment, an exemplary mixture of an exemplary tissue sample may be soaked in an exemplary enzyme solution for a time period in a range of 30 minutes to 90 minutes. In an exemplary embodiment, an exemplary mixture of an exemplary tissue sample may be soaked in an exemplary enzyme solution at a temperature in a range of 15°C to 35°C. In an exemplary embodiment, an exemplary enzyme may include trypsin. In an exemplary embodiment, an exemplary trypsin solution may include trypsin in water with a weight ratio in a range of 0.005 w / w to 0.05 w / w (enzyme / water).

[0045] In an exemplary embodiment, after forming an exemplary submucosal-free tissue, cells existed among an interconnected framework of an exemplary submucosal-free tissue may be eliminated. In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue may be performed by applying a cold atmospheric plasma to an exemplary submucosal-free tissue. In an exemplary embodiment, an exemplary acellular sample may form after eliminating an exemplary submucosal layer and exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue. In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue may include applying an exemplary cold atmospheric plasma to an exemplary submucosal-free tissue for a time period in a range of 3 minutes to 10 minutes. In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue may include applying an exemplary cold atmospheric plasma to an exemplary submucosal-free tissue with a voltage of an exemplary cold atmospheric plasma in a range of 5 V to 50 V. In an exemplary embodiment, an exemplary cold atmospheric plasma may include a cold argon atmospheric plasma. In an exemplary embodiment, argon gas may be purged toward an exemplary submucosal-free tissue. In an exemplary embodiment, an exemplary argon gas may be purged toward an exemplary submucosal-free tissue with a flow rate in a range of 1 L / min to 10 L / min.In an exemplary embodiment, a distance between an exemplary submucosal-free tissue and an exemplary cold atmospheric plasma may be in a range of 1 cm to 10 cm. In an exemplary embodiment, using argon in an exemplary cold atmospheric plasma may produce higher amount of radicals in comparison with an exemplary cold atmospheric plasma without argon. In an exemplary embodiment, an exemplary radical may include at least one of reactive oxygen species, reactive nitrogen species, and combinations thereof. In an exemplary embodiment, an exemplary cold atmospheric plasma may include a nanosecond-pulsed dielectric barrier discharge (DBD). In an exemplary embodiment, an exemplary DBD may be an emerging technique for ionizing volatile molecules directly from mixtures. In an exemplary embodiment, plasma produced by an exemplary device using DBD may be trilled from a cylindrical electrode. In an exemplary embodiment, inner part of an exemplary cylindrical electrode may be covered with copper. In an exemplary embodiment, an exemplary cylindrical DBD device may include two copper electrodes. In an exemplary embodiment, exemplary two copper electrodes may generate cold atmospheric plasma by applying AC high voltage through a power supply. In an exemplary embodiment, an argon working gas may be purged while applying AC high voltage through a power supply.

[0046] In an exemplary embodiment, an exemplary acellular sample may be used as a wound dressing. In an exemplary embodiment, an exemplary wound dressing may include at least one of a wound dressing patch, a wound dressing gel, and combinations thereof. In an exemplary embodiment, forming an exemplary wound dressing patch may include pressing an exemplary acellular sample using a mechanical pressing device. In an exemplary embodiment, an exemplary mechanical pressing device may include two pressing plates in which an exemplary acellular sample may be placed between exemplary two pressing plates. In an exemplary embodiment, an exemplary acellular sample may be pressed by an applied force to both external surfaces of exemplary two pressing plates. In an exemplary embodiment, forming an exemplary wound dressing patch may include pressing an exemplary acellular sample to a thickness in a range of 0.01 mm to 1 mm using an exemplary mechanical pressing device. In an exemplary embodiment, a size of an exemplary wound dressing patch may proportional to a size of an exemplary wound.

[0047] In an exemplary embodiment, an exemplary wound dressing gel may be produced by grinding an exemplary acellular sample using a mechanical device. In an exemplary embodiment, grinding an exemplary acellular sample may include soaking an exemplaryacellular sample in polyethylene glycol solution. In an exemplary embodiment, as used herein “soaking” may refer to fully drowning an exemplary acellular sample in an exemplary polyethylene glycol solution. In an exemplary embodiment, an exemplary acellular sample may be soaked in an exemplary polyethylene glycol solution for a time period in a range of 5 minutes to 30 minutes. In an exemplary embodiment, an exemplary polyethylene glycol solution may have a concentration in a range of 5 mg / L to 25 mg / L in water. In an exemplary embodiment, grinding an exemplary acellular sample may further include removing an exemplary acellular sample from an exemplary polyethylene glycol solution. In an exemplary embodiment, liquid nitrogen may be poured over an exemplary acellular sample. In an exemplary embodiment, an exemplary acellular sample may be grinded using a mechanical device while pouring an exemplary liquid nitrogen over an exemplary acellular sample. In an exemplary embodiment, an exemplary mechanical device may include at least one of a cylindrical grinder, centerless grinders, internal grinders, and combinations thereof. In another exemplary embodiment, an exemplary mechanical device may include a mortar. In an exemplary embodiment, an exemplary grinded acellular sample may have an average particle size in a range of 4 pm to 60 pm. In an exemplary embodiment, forming an exemplary wound dressing gel may include forming a gel after grinding an exemplary acellular sample. In an exemplary embodiment, an exemplary gel may be formed by adding a hyaluronic acid solution to an exemplary grinded acellular sample with a weight ratio of an exemplary hyaluronic acid solution to an exemplary grinded acellular sample in a range of 25:1 to 5:1 (hyaluronic acid solution: grinded acellular sample). In an exemplary embodiment, an exemplary hyaluronic acid solution in water may have a weight ratio in a range of 50: 1 to 5: 1 (hyaluronic acid: water).

[0048] In an exemplary embodiment, an exemplary wound dressing patch may be used for healing deep wounds. In an exemplary embodiment, “deep wound” may refer to a wound that penetrates into interior tissue layers of body. In an exemplar embodiment, an exemplary deep wound may take longer period of time for healing in comparison with superficial wounds. In an exemplary embodiment, “superficial wound” may refer to injuries that only skin may be damaged and underlying tissues are intact. In an exemplary embodiment, an exemplary wound dressing gel may be used for healing superficial wounds.

[0049] FIG. 1A illustrates a flowchart of a method 100 of producing an acellular sample, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 100 may include a step 102 of forming a submucosal-free tissue bydecellularization of a submucosal layer of a tissue sample, and step 104 of decellularization of cells existed among an interconnected framework of the submucosal-free tissue.

[0050] In further detail with respect to step 102, step 102 of forming a submucosal-free tissue may include decellularization of a submucosal layer of a tissue sample by soaking an exemplary tissue sample in an enzyme solution. In an exemplary embodiment, an exemplary tissue sample may be fully drown in an exemplary enzyme solution. As use herein soaking an exemplary tissue sample in an exemplary enzyme solution may refer to fully drowning an exemplary tissue sample in an exemplary enzyme solution. In an exemplary embodiment, an exemplary tissue sample may be soaked in an exemplary enzyme solution for a time period in a range of 30 minutes to 90 minutes. In an exemplary embodiment, an exemplary tissue sample may be soaked in an exemplary enzyme solution at a temperature in a range of 15°C to 35°C. In an exemplary embodiment, an exemplary tissue sample may include at least one of a bone sample, a muscle sample, a skin sample, an intestine sample, a tendon sample, an amniotic membrane sample, a peritoneal membrane sample, a liver sample, a spleen sample, a pancreas sample, a cartilage sample, and combinations thereof. In an exemplary embodiment, an exemplary enzyme may include trypsin. In an exemplary embodiment, an exemplary enzyme solution in water may have a concentration in a range of 0.005 w / w to 0.05 w / w (enzyme / water).

[0051] In further detail with respect to step 104, step 104 of decellularization of cells existed among an interconnected framework of an exemplary submucosal-free tissue may include applying a cold atmospheric plasma to an exemplary submucosal-free tissue. In an exemplary embodiment, an exemplary cold atmospheric plasma may be applied to an exemplary submucosal-free tissue for a time period in a range of 3 minutes to 10 minutes. In an exemplary embodiment, decellularization of exemplary cells existed among an exemplary interconnected framework of an exemplary submucosal-free tissue may include applying an exemplary cold atmospheric plasma to an exemplary submucosal-free tissue with a voltage of an exemplary cold atmospheric plasma in a range of 5 V to 50 V. In an exemplary embodiment, an exemplary cold atmospheric plasma may include a cold argon atmospheric plasma. In an exemplary embodiment, an exemplary cold atmospheric plasma may include a nanosecond-pulsed dielectric barrier discharge (DBD). In an exemplary embodiment, an exemplary DBD may be an emerging technique for ionizing volatile molecules directly from mixtures. In an exemplary embodiment, plasma produced by an exemplary device using DBD may be trilled from acylindrical electrode. In an exemplary embodiment, inner part of an exemplary cylindrical electrode may be covered with brass.

[0052] In an exemplary embodiment, cold atmospheric plasma may be a partially ionized gas including ions, electrons, ultraviolet photons and reactive neutrals. In an exemplary embodiment, exemplary reactive neutrals may include radicals, excited and ground-state molecules. In an exemplary embodiment, an exemplary cold atmospheric plasma may be a technology that may use ionized gas (plasma) to generate reactive oxygen and nitrogen species. In an exemplary embodiment, an exemplary cold atmospheric argon plasma may produce partially ionized argon gas including ions, electrons, ultraviolet photons and reactive neutrals. In an exemplary embodiment, an exemplary cold atmospheric argon plasma may produce at least one of a partially ionized argon gas, a partially ionized oxygen gas, a partially ionized nitrogen gas, and combinations thereof. In an exemplary embodiment, an exemplary cold atmospheric plasma may operate at a temperature in a range of 30°C to 50°C. In an exemplary embodiment, an exemplary cold atmospheric plasma may reduce bacterial count within a wound. In an exemplary embodiment, an exemplary cold atmospheric plasma may promote wound healing. In an exemplary embodiment, an exemplary cold atmospheric plasma may have a flow rate of argon in a range of 1 L / min to 10 L / min. In an exemplary embodiment, an exemplary cold atmospheric plasma may use argon as a source of plasma. In an exemplary embodiment, an exemplary cold atmospheric plasma may be applied to an exemplary submucosal-free tissue with a distance between an exemplary submucosal-free tissue and an exemplary cold atmospheric plasma in a range of 1 cm to 10 cm. In an exemplary embodiment, an exemplary submucosal-free tissue may have a perimeter in a range of 2 cm to 15 cm.

[0053] FIG. IB illustrates a flowchart of a method 110 of producing a wound dressing patch, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 110 may include a step 112 of forming an acellular sample by forming a submucosal-free tissue, a step 114 of decellularization of cells existed among an interconnected framework of the submucosal-free tissue, and a step 116 of pressing the acellular sample.

[0054] In further detail with respect to step 112 and step 114, step 112 of forming an acellular sample by forming a submucosal-free tissue by decellularization of a submucosal layer of a tissue sample and step 114 of decellularization of cells existed among an interconnected framework of the submucosal-free tissue may be performed similarly to steps 102 and 104 of method 100, respectively. In an exemplary embalmment, an exemplary acellular sample maybe produced according to method 100. In an exemplary embodiment, an exemplary acellular sample may be used in step 116 of method 110. In an exemplary embodiment, a wound dressing patch may be produced using an exemplary acellular sample produced according to method 100.

[0055] In further detail with respect to step 116, step 116 of pressing the acellular sample may include pressing an exemplary acellular sample using a mechanical pressing device. In an exemplary embodiment, an exemplary acellular sample may be pressed to a thickness in a range of 0.01 mm to 1 mm. In an exemplary embodiment, an exemplary mechanical pressing device may include two pressing plates in which an exemplary acellular sample may be placed between exemplary two pressing plates. In an exemplary embodiment, an exemplary acellular sample may be pressed by an applied force to both external surfaces of exemplary two pressing plates. In an exemplary embodiment, an exemplary acellular sample may be produced with a perimeter in a range of 2 cm to 15 cm. In an exemplary embodiment, an exemplary acellular sample may be an acellular structure of at least one of a bone sample, a muscle sample, a skin sample, an intestine sample, a tendon sample, an amniotic membrane sample, a peritoneal membrane sample, a liver sample, a spleen sample, a pancreas sample, a cartilage sample, and combinations thereof.

[0056] In an exemplary embodiment, an exemplary wound dressing patch may be placed on a wound for a time period in a range of 1 day to 30 days. In an exemplary embodiment, an exemplary wound dressing patch may be absorbed into body. In an exemplary embodiment, an exemplary acellular sample may form a framework for proliferation of cells in a wound site. In an exemplary embodiment, an exemplary wound dressing patch may be used for healing deep wounds with damage to tissues of body. In an exemplary embodiment, an exemplary wound dressing patch may be used for healing wounds of at least one of a human, an animal, and combinations thereof. In an exemplary embodiment, an exemplary wound dressing patch may be stored at a temperature in a range of 15°C to 40°C. In an exemplary embodiment, an exemplary wound dressing patch may be stored in a humidity in a range of 5% to 30%. In an exemplary embodiment, an exemplary wound dressing patch may be stored for a time period of maximum 3 years after production.

[0057] FIG. 1C illustrates a flowchart of a method 120 of producing a wound dressing gel, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 120 may include a step 122 of forming an acellular sample by forming asubmucosal-free tissue, a step 124 of decellularization of cells existed among an interconnected framework of the submucosal-free tissue, a step 126 of grinding the acellular sample, and a step 128 of forming a gel by mixing a hyaluronic acid solution with the grinded acellular sample.

[0058] In further detail with respect to step 122 and step 124, step 122 of forming an acellular sample by forming a submucosal-free tissue by decellularization of a submucosal layer of a tissue sample and step 124 of decellularization of cells existed among an interconnected framework of the submucosal-free tissue may be performed similarly to steps 102 and 104 of method 100, respectively. In an exemplary embalmment, an exemplary acellular sample may be produced according to method 100. In an exemplary embodiment, an exemplary acellular sample may be used in step 126 and 128 of method 120. In an exemplary embodiment, a wound dressing gel may be produced using an exemplary acellular sample produced according to method 100.

[0059] In further detail with respect to step 122, step 122 of grinding the acellular sample may include using a mechanical device to grind an exemplary acellular sample. In an exemplary embodiment, grinding an exemplary acellular sample may include soaking an exemplary acellular sample in polyethylene glycol solution. In an exemplary embodiment, as used herein “soaking” may refer to fully drowning an exemplary acellular sample in an exemplary polyethylene glycol solution. In an exemplary embodiment, an exemplary acellular sample may be soaked in an exemplary polyethylene glycol solution for a time period in a range of 5 minutes to 30 minutes. In an exemplary embodiment, an exemplary polyethylene glycol solution may have a concentration in a range of 5 mg / L to 25 mg / L in water. In an exemplary embodiment, grinding an exemplary acellular sample may further include removing an exemplary acellular sample from an exemplary polyethylene glycol solution. In an exemplary embodiment, liquid nitrogen may be poured over an exemplary acellular sample. In an exemplary embodiment, an exemplary acellular sample may be grinded using a mechanical device while pouring an exemplary liquid nitrogen over an exemplary acellular sample. In an exemplary embodiment, an exemplary mechanical device may include at least one of a cylindrical grinder, centerless grinders, internal grinders, and combinations thereof. In another exemplary embodiment, an exemplary mechanical device may include a mortar. In an exemplary embodiment, an exemplary grinded acellular sample may have an average particle size in a range of 4 pm to 60 pm. In an exemplary embodiment, an exemplary grinded acellularsample may be stored at a temperature in a range of 5 °C to 25 °C. In an exemplary embodiment, an exemplary grinded acellular sample may be stored in a humidity in a range of 20% to 60%. In an exemplary embodiment, an exemplary grinded acellular sample may be stored for a time period of maximum 3 years after production.

[0060] In further detail with respect to step 124, step 124 of forming a gel may include mixing an exemplary hyaluronic acid solution with an exemplary grinded acellular sample in a mixer. In an exemplary embodiment, an exemplary hyaluronic acid solution and an exemplary grinded acellular sample may be mixed in an exemplary mixer with a stirring speed in a range of 100 rpm to 1000 rpm. In an exemplary embodiment, an exemplary hyaluronic acid solution and an exemplary grinded acellular sample may be mixed in an exemplary mixer for a time period in a range of 5 minutes to 60 minutes. In an exemplary embodiment, an exemplary hyaluronic acid solution and an exemplary grinded acellular sample may be mixed with a weight ratio of an exemplary hyaluronic acid solution to an exemplary grinded acellular sample in a range of 25:1 to 5:1 (hyaluronic acid solution: grinded acellular sample). In an exemplary embodiment, an exemplary hyaluronic acid solution in water may have a weight ratio in a range of 50:1 to 5:1 (hyaluronic acid: water). In an exemplary embodiment, an exemplary water may include at least one of a distilled water, deionized water, and combinations thereof. In an exemplary embodiment, an exemplary grinded acellular sample may have an average particle size in a range of 4 pm to 60 pm.

[0061] In an exemplary embodiment, an exemplary wound dressing gel may be applied on a wound in which an exemplary wound may be covered by an exemplary wound dressing gel. In an exemplary embodiment, an exemplary wound dressing gel may be used for healing superficial wounds. In an exemplary embodiment, an exemplary superficial wound may refer to a wound that only skin may be damaged and underneath tissues may remain intact. In an exemplary embodiment, an exemplary hyaluronic acid may help skin reconstruction. In an exemplary embodiment, an exemplary wound dressing gel may heal an exemplary wound after maximum 15 days. In an exemplary embodiment, an exemplary wound dressing gel may be used at maximum 3 years after production. In an exemplary embodiment, an exemplary wound dressing gel may be stored at a temperature in a range of 5 °C to 25 °C. In an exemplary embodiment, an exemplary wound dressing gel may be stored in a sterile environment.

[0062] Example 1: Producing an acellular sample using cold plasma

[0063] As described below, an acellular sample may be produced using cold plasma. An exemplary acellular sample may be similar to an exemplary acellular sample produced according to method 100. For producing an exemplary acellular sample, small intestines were freshly prepared from several healthy adult sheep (it is necessary to check the animals for infectious, skin and viral diseases by a veterinarian of a slaughterhouse having a license). After washing intestine samples with tap water at the slaughterhouse and transferring them to a laboratory in phosphate buffer saline (PBS) solution containing penicillin / streptomycin and amphotericin at 4°C, they were immersed in Hanks' saline solution. It is important that the temperature of the sample does not exceed 4°C at this stage. For this purpose, it is necessary to transfer the sample to a laminar-flow hood. In the next step, the intestine samples were removed from the Hanks' saline solution and were placed on a plastic surface, in which the surface under the mucosa faced downside. The samples were cut into 10 cm x 5 cm pieces. In the next step, the samples were placed in a petri dish in which the submucosal surface was facing downside and 50 ml of NaCl solution (I M) was added to the samples and the mixture was kept in an incubator for 24 hours at a temperature of 37°C. The next day, the medium was changed and incubated again for another 24 hours. In the next step, the mucous layer was removed mechanically by using forceps and a scalpel handle. In the next step, the mucous and muscular layers along with the serous layer were removed by a special mechanical method using wet gauze and a very thin knife. The rest of the NaCl solution was removed and a 1 cm slit was cut in a lower and left comer of the sample to make it easy to identify the upper and lower surface. The sample inside the petri dish was washed with 50 ml of Hanks solution. In the next step, the sample was placed at room temperature of about 20°C for 5 minutes and then the Petri dish was transferred under the hood and the washing process was repeated 2 more times for half an hour.

[0064] For preparing PBS solution with pH= 4.7, 2.8 g of sodium chloride, 0.285.201 g of potassium chloride, 3.5814 g of dibasic sodium phosphate, and 0.27216 g of monopotassium phosphate were dissolved in 1 L of deionized distilled water. In the next step, 0.1 M sodium hydroxide and hydrochloric acid solutions were used to adjust the pH.

[0065] For preparing Hanks solution with pH=7, 8 g of sodium chloride, 400 mg of potassium chloride, 140 mg of calcium chloride, 100 mg of magnesium sulfate, 100 mg of magnesium chloride, 60 mg of disodium phosphate, 60 mg of monopotassium phosphate, 1 g of glucoseand 350 mg of sodium bicarbonate were dissolved in 1 L of deionized distilled water. Sodium hydroxide and 0.1 M hydrochloric acid solutions were used to adjust the pH.

[0066] Fat in the submucosal layer was removed by placing the samples in acetic acid solution (0.01-0.05%) (vinegar) in several stages for 12 hours and washing with distilled water three times. To remove all types of cells from the submucosal layer, the samples were soaked with 0.005-0.01% trypsin solution. For the next step, the sample was washed with Hanks solution (PBS / Pen / Strep 2% three times for 15 minutes) and after that, the samples were exposed to cold atmospheric plasma with argon source for 3 minutes to 10 minutes. An exemplary cold atmospheric plasma included a Nanosecond-Pulsed dielectric plasma source barrier discharge (DBD) which was trilled from a cylindrical electrode in which inner part of the cylindrical electrode was covered with brass. The prepared acellular samples were sterilized at 4°C and kept away from light for further use.

[0067] FIG. 2 illustrates a schematic presentation of a tissue sample of an intestine used for preparing a wound dressing patch and a wound dressing gel, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG 2, an exemplary intestine sample has different parts of Duodenum part (212), Jejunum part (210), Heum part (208), Cecum part (206), colon part (204), and Anus part (202). For preparing an exemplary acellular sample used for forming an exemplary wound dressing patch and wound dressing gel, Jejunum part (210) of the intestine sample was used.

[0068] Example 2; Forming a wound dressing patch and wound dressing gel using the acellular sample

[0069] For forming a wound dressing patch, the produced acellular samples were pressed using a pressing device. The wound dressing patch was used in treatment of deep wounds in mice and rabbits. For forming an exemplary wound dressing gel, an exemplary acellular sample was powdered by liquid nitrogen and polyethylene glycol in three steps and using mechanical pressure. For this purpose, in the first step, 1 g of polyethylene glycol with a molecular weight of 4000 was dissolved in 10 ml of water, in the second step, the prepared acellular sample was cut into 1 cm x 1 cm pieces and was immersed in the polyethylene glycol solution for one hour. In the third step, the acellular sample is removed from the solution containing polyethylene glycol and was mechanically ground into a powder in a porcelain mortar with the gradual addition of liquid nitrogen. 0.5 g of the grinded acellular sample was mixed with 5 ml of hyaluronic acid 1%. An exemplary wound dressing gel was used for superficial wounds or skinwounds of the 1st degree in the time intervals of 7 and 14 days. The results of laboratory and animal tests confirmed the high efficiency of the wound dressing gel in healing superficial wounds.

[0070] In order to ensure complete removal of cells (acellularization) and determine remains of cell nucleus, 4',6-diamidino-2-phenylindole (DAPI) staining (FIG. 3) and hematoxylin eosin and Masson's trichrome (FIG. 4) were used to check appearance of cells that were removed and preserved framework of tissue samples. FIG. 3 illustrates a fluorescent image 300 of DAPI staining of an exemplary acellular sample, consistent with one or more exemplary embodiments of the present disclosure. FIG. 4 illustrates stained images 400 of an exemplary acellular sample using hematoxylin eosin (H&E), consistent with one or more exemplary embodiments of the present disclosure. FIG. 5 illustrates stained images 400 of an exemplary acellular sample using Masson's trichrome, consistent with one or more exemplary embodiments of the present disclosure.

[0071] Scanning electron microscope analysis was used to observe acellular ovine small intestine submucosa (AOSIS) and examine the attachment of Mesenchymal stem cells (MSCs) on the acellular sample. FIG. 6 illustrates scanning electron microscope image 600 of an exemplary acellular sample, consistent with one or more exemplary embodiments of the present disclosure. FIG. 6 shows image 602 of scaffold of an exemplary acellular sample and image 604 of adhesion and proliferation of MSCs on the scaffold of an exemplary acellular sample after 24 hours.

[0072] The acellular sample obtained after wax processing and sectioning was stained by H&E and Trichrome Masson and examined with a light microscope. One of important factors in this section may be evaluation of effectiveness and efficiency of the acellular sample and their effectiveness in the wound healing process and producing collagen. FIG. 7 illustrates photomicrograph images 700 of the experimental group treated with the produced acellular sample (702) and the experimental control group treated with Masson's trichrome staining (704), consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 7 in the sample treated with the produced acellular sample (702), collagen fragments in the reticular dermis layer can be seen structurally in blue color. FIG. 8 illustrates photomicrograph images 800 of the experimental group treated with the produced acellular sample stained with H&E at 5 days (802) and 21 days (804), consistent with one or more exemplary embodiments of the present disclosure. After 5 days, the adhesion of the acellularsample and integration with the submucosa tissue can be clearly seen in FIG. 8. After 21 days, the repair and epithelialization of the epidermis can be clearly seen with the placement of the basal, spiny and horny layers in the epidermis layer of the skin. On the 21st day of repair, although the thickness of the epithelium is restored and is naturally completed, no traces of the produced acellular sample can be seen in the place of wound. In addition, in order to ensure the preservation of the structure of the obtained acellular sample, transmission electron microscopy (TEM) and three types of immunohistochemical staining were performed to check cytokine, desmin, and vimentin.

[0073] FIG. 9 illustrates transmission electron microscopy (TEM) images 900 of burned tissue (902), control sample (904), and acellular treated sample (906), consistent with one or more exemplary embodiments of the present disclosure. In the TEM images related to the acellular treated sample (906), the transverse and longitudinal sections of collagen fibers in the connective tissue of the reticular dermis layer along with euchromatin fibroblast cells can be seen, but in the control sample (904), collagen fibers cannot be seen, and the fibroblast cells and parenchyma of reticular tissue are heterochromatin or dense chromatin and relative chromatin margins in fibroblast cells.

[0074] FIG. 10 illustrates photomicrograph images 1000 of an exemplary acellular sample with immunohistochemical staining to examine Vimentin protein, consistent with one or more exemplary embodiments of the present disclosure. FIG. 10 shows photomicrograph images 1000 of control sample (1002) and acellular sample (1004). As shown in FIG. 10, Vimentin which is a brown protein can be seen in a large amount in the treatment group repaired with an exemplary acellular sample.

[0075] FIG. 11 illustrates photomicrograph images 1100 of Desmin protein examination by immunohistochemistry staining, consistent with one or more exemplary embodiments of the present disclosure. Photomicrograph images 1100 show that expression of Desmin in epithelium and epidermal layer of skin is significant in the treated group. FIG. 11 shows photomicrograph image of decellularized scaffolds with immunohistochemical staining to examine Desmin protein (1104) and control sample (1102).

[0076] FIG. 12 illustrates photomicrograph images 1200 of an exemplary acellular sample with immunohistochemical staining for controlling CD31 marker, consistent with one or more exemplary embodiments of the present disclosure. FIG. 12 shows photomicrograph images1200 for expression of CD31 marker in experimental group treated with an exemplary acellular sample (1204) and control sample (1202).

[0077] To investigate effect of an exemplary wound dressing patch and an exemplary wound dressing gel, two groups of mice were selected, in one group a deep burn was created to check the effect of the wound dressing patch compared to the control group, and in the second group a superficial burn was created to check the effect of the wound dressing gel. In the mouse treated with an exemplary wound dressing patch, compared to the control mouse without an exemplary wound dressing patch, after 21 days, the wound was almost healed and no infection was observed on the wound surface. The mouse with superficial bum and the mouse of control group were treated by an exemplary wound dressing gel. Results showed that the wound was completely repaired after 14 days. In superficial burns, since the wound is not deep, there is no need for healing substances to penetrate lower surface of the wound, and gelatin structure of an exemplary wound dressing gel causes an exemplary gel to be released faster at the burn site, and process of cell proliferation and migration, and ultimately repair of the damaged skin be facilitated.

[0078] Industrial Applicability

[0079] . The present disclosure may be a method of producing an acellular sample using cold plasma. An exemplary acellular sample may be used for producing a wound dressing patch and a wound dressing gel. An exemplary wound dressing patch and an exemplary wound dressing gel may be used in hospitals, treatment clinics, and research and diagnostic laboratories. It is possible to use an exemplary wound dressing patch and an exemplary wound dressing gel routinely to increase speed of wound healing in patients. An exemplary wound dressing patch and an exemplary wound dressing gel have been designed with the aim of minimizing existing challenges related to burns and injuries to skin, preventing loss of time and money, the possibility of recovery before it worsens, and need for no invasive surgeries.

[0080] While the foregoing has described what are considered to be the best mode and / or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0081] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0082] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0083] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0084] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0085] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in variousimplementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0086] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and / or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

What is claimed is:

1. A method of forming an acellular sample, comprising: forming a submucosal-free tissue by decellularization of a submucosal layer of a tissue sample by soaking the tissue sample in an enzyme solution with a concentration of the enzyme solution in a range of 0.005 w / w to 0.05 w / w (enzyme / water); and decellularization of cells existed among an interconnected framework of the submucosal-free tissue by applying a cold atmospheric plasma to the submucosal-free tissue.

2. The method of claim 1, wherein the tissue sample comprises at least one of a bone sample, a muscle sample, a skin sample, an intestine sample, a tendon sample, an amniotic membrane sample, a peritoneal membrane sample, a liver sample, a spleen sample, a pancreas sample, a cartilage sample, and combinations thereof.

3. The method of claim 1, wherein the enzyme comprises trypsin.

4. The method of claim 1, wherein decellularization of the cells existed among the interconnected framework of the submucosal-free tissue comprises applying the cold atmospheric plasma to the submucosal-free tissue for a time period in a range of 3 minutes to 10 minutes.

5. The method of claim 1, wherein decellularization of the cells existed among the interconnected framework of the submucosal-free tissue comprises applying the coldatmospheric plasma to the submucosal-free tissue with a voltage of the cold atmospheric plasma in a range of 5 V to 50 V.

6. The method of claim 1, wherein the cold atmospheric plasma comprises a cold argon atmospheric plasma.

7. The method of claim 1, wherein the cold atmospheric plasma comprises a nanosecond- pulsed dielectric barrier discharge (DBD).

8. The method of claim 1, further comprising forming a wound dressing patch by pressing the acellular sample using a mechanical pressing device.

9. The method of claim 8, wherein forming the wound dressing patch comprises pressing the acellular sample to a thickness in a range of 0.01 mm to 1 mm using the mechanical pressing device.

10. The method of claim 1, further comprising forming a wound dressing gel, forming the wound dressing gel comprising: grinding the acellular sample using a mechanical device; and forming a gel by mixing a hyaluronic acid solution with the grinded acellular sample with a weight ratio of the hyaluronic acid solution to the grinded acellular sample in a range of 25:1 to 5:1 (the hyaluronic acid solution: the grinded acellular sample).

11. The method of claim 10, wherein the grinded acellular sample has an average particle size in a range of 4 pm to 60 pm.

12. The method of claim 10, wherein the hyaluronic acid solution comprises hyaluronic acid dissolved in water with a weight ratio in a range of 50:1 to 5:1 (hyaluronic acid: water).

13. A method of forming a wound dressing patch, comprising: forming an acellular sample, comprising: forming a submucosal-free tissue by decellularization of a submucosal layer of a tissue sample by soaking the tissue sample in an enzyme solution with a concentration of the enzyme solution in a range of 0.005 w / w to 0.05 w / w (enzyme / water); and decellularization of cells existed among an interconnected framework of the submucosal-free tissue by applying a cold atmospheric plasma to the submucosal-free tissue; and pressing the acellular sample using a mechanical pressing device.

14. The method of claim 13, wherein the cold atmospheric plasma comprises a nanosecond- pulsed dielectric barrier discharge (DBD).

15. The method of claim 13, wherein decellularization of the cells existed among the interconnected framework of the tissue sample comprises applying the cold atmospheric plasma to the submucosal-free tissue with a voltage of the cold atmospheric plasma in a range of 5 V to 50 V for a time period in a range of 3 minutes to 10 minutes.

16. The method of claim 13 wherein the tissue sample comprises at least one of a bone sample, a muscle sample, a skin sample, an intestine sample, a tendon sample, an amniotic membrane sample, a peritoneal membrane sample, a liver sample, a spleen sample, a pancreas sample, a cartilage sample, and combinations thereof.

17. A method of forming a wound dressing gel, comprising: forming an acellular sample, comprising: forming a submucosal-free tissue by decellularization of a submucosal layer of a tissue sample by soaking the tissue sample in an enzyme solution with a concentration of the enzyme solution in a range of 0.005 w / w to 0.05 w / w (enzyme / water); and decellularization of cells existed among an interconnected framework of the submucosal-free tissue by applying a cold atmospheric plasma to the submucosal-free tissue; grinding the acellular sample using a mechanical device; and forming a gel by mixing a hyaluronic acid solution with the grinded acellular sample with a weight ratio of the hyaluronic acid solution to the grinded acellular sample in a range of 25:1 to 5:1 (the hyaluronic acid solution: the grinded acellular sample).

18. The method of claim 17, wherein the grinded acellular sample has an average particle size in a range of 4 pm to 60 pm.

19. The method of claim 17, wherein the cold atmospheric plasma comprises a nanosecond- pulsed dielectric barrier discharge (DBD).

20. The method of claim 17, wherein decellularization of the cells existed among the interconnected framework of the submucosal-free tissue comprises applying the cold atmospheric plasma to the submucosal-free tissue with a voltage of the cold atmospheric plasma in a range of 5 V to 50 V for a time period in a range of 3 minutes to 10 minutes.

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