TWI252113B - Artificial skin graft and preparation method thereof - Google Patents

Abstract

Disclosed herein are an artificial skin graft for use as a temporary and permanent coverage of wounded skin and a preparation method thereof, in which the artificial skin graft is manufactured from the cross-linking of a sheet of acellular mammalian dermis with a flattening element such as a silicon sheet.

Description

1252113 IX. DESCRIPTION OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to artificial skin grafts for temporary and permanent coverage of wounded skin. (artificial skin graft) and a method of preparing the same, wherein the artificial skin graft is a sheet of acellular mammalian dermis and a flattening element such as a stone film It is manufactured by cross-linking. 10 [Prior Art] Background of the Invention Animal skin has functions of resisting foreign pollutants, reducing ultraviolet rays, absorbing and respirating effects, regulating body temperature, and regulating moisture to avoid dehydration and sensation. Animal skin is complex in structure and can be basically divided into three layers: 15 (1) Epidermis (ePidermis), which is the outermost layer of the skin, containing five layers of neatly arranged cells, from the outermost layer to the lowest layer, respectively. (cuticle), transparent layer, granular layer, with acanthosis and basement membrane (BM); (2) dermis, which is closely attached to the basement membrane of the epidermal layer, and Table 20 cortex and dermis The junctions are ridged and staggered. The dermis layer is divided into a mastoid layer and a mesh layer from the outside. The dermis is rich in collagen fibers, elastic fibers, basal material, etc., and provides skin elasticity and ductility. If the dermis is damaged, it is very easy to have scars; and (3) subcutis, which is connected to the reticular layer of the dermis, and 1252113 consists of loose fibrous webs and fat cells, and has sweat glands, Hair bulbs, blood vessels, lymphatic vessels and nerves. The fat cells in the subcutaneous tissue layer store energy and act as a cushion and are also a large warehouse for absorbing foreign drugs. 5 If an animal has a skin injury, it will cause its body function to lose balance, which will cause serious imbalance of organs and even death. Burns and trauma can damage the epidermal and dermal layers of the skin and even cause full thickness skin damage. When treating wounds that cannot be healed, it is often necessary to permanently seal the wound by autologous skin grafting. For open infectious wounds, the success rate of skin grafting with thin split-thickness skin graft (thin STSG) is high. However, due to the thinner dermis layer of the thin-skinned skin graft, the wound eventually forms a hypertrophic or contraction scar, resulting in unattractive healing, 15 even forming a contracture that affects reconstruction. The function of the skin afterwards. In addition, for large burn wounds, the available parts are limited. Or, in some patients, the wound regeneration ability is weak (for example, patients with venous ulcers, acne patients, and foot ulcers), and it is more difficult to take skin from such patients for autologous skin transplantation. 2 Because of the limitations of autologous skin grafting in these respects, various biological dressings have been proposed and shown to be complete as split-thickness skin graft (STSG) ( Temporary and permanent substitutes of the epidermis and the dermis) or parts (skin or dermis) 0 1252113 The so-called "biological dressing" refers to a dressing containing biological properties, and the "biological properties" generally refer to the dressing that is in contact with the wound. In the case where a surface portion contains collagen. Biological dressings differ from other synthetic dressings in that they utilize the excellent fusion of fibrin-elastin binding power (FEBP) of the wound. Phenomenon (inc〇rp〇rati〇n), and thereby achieve the following functions: · reduce the loss of water and protein and evaporation of heat, maintain the humidity and temperature of the wound, reduce the pain of the wound, promote the proliferation of normal granulation tissue of the wound, Inhibits the proliferation of wound bacteria, promotes the healing of shallow second wounds, promotes smoothing of the wound and temporarily coats exposed vital tissues (such as nerves, blood vessels, tendons, etc.) and covers the outer surface of the autologous skin graft.俾 to improve the transplant success rate and so on. The characteristics and prerequisites of the biological dressing include: a rapid and long-lasting adhesion to the wound, which enables the gel 15 protein in the biological dressing to form a tight fusion with the wound to achieve a temporary closure of the wound. ; non-antigenic (ncm-antigenic); non-toxic, non-toxic to the animal or human body to cause wound necrosis; sterile (stenle), will not cause serious infection of animals or human body and life-threatening Viability, active biological dressings have a better adhesion to wound tissue, especially when the wound requires a long period of coverage. For example, if a skin graft is active, it can obtain blood flow nutrients from the scar, and thus the moon b maintains a longer survival and coverage on the wound. These characteristics determine the extent to which the wound is acceptable for the skin graft. In addition, it is basically "active, and "aseptic," conflicting, but the active treatment of the 1252113 material involves first surface cleaning or bactericide for surface elimination, and Generally, bacteria and mold culture, and products that are aseptically grown for 72 hours can be used for clinical use. Since 1966, the use of pig skin as a biological dressing has become a common mode of treatment for wounds. The pig skin is similar in anatomical structure to the human skin, and the source is inevitable. The pig skin is not widely available in the allograft skin. It is a widely used temporary dressing. The method for manufacturing a pig skin biological dressing comprises: shaving a portion to be peeled and sterilizing according to a surgical principle, and cutting at least 猪·〇〇8_〇·〇3〇 thick pigskin from the sterilized portion and Disinfected in a sodium hypochloride solution and then sealed in a sterile package. A small piece of pig skin before encapsulation was taken out as a test sample' and sent to general bacteria and mold culture, and the corresponding packaged product of the test sample that was aseptically grown for 72 hours was used for clinical use as a temporary dressing for wounds. 15 The disadvantage of pig skin is that it requires costly radiation disinfection and freeze-drying after removing the cells to reduce its antigenicity and inhibit bacterial growth. Furthermore, due to the different species of pigs and humans, pig skin belonging to xenograft can only be temporarily applied to the wound, and will gradually become necrotic due to the formation of rejection after about one week (with large area burning 20 The rejection of a wounded patient can be delayed due to inhibition of immune function), and must be removed and replaced with another pig skin to continue to protect the wound.

Biobrane is a synthetic biological dressing whose outer layer is made of nylon and lycopene, while the inner layer contains collagen extracted from pig skin. The collagen in the inner layer of Bi〇brane fuses to the wound' while the outer layer of nylon-shixi gum polymer 8 1252113 isolates the wound from the outside world. Biobrane is malleable, soft, non-toxic and sterile. In our previous experiments, it was found that when Bi〇brane was applied to the cleaned back wound of rats for 3 weeks, it still maintained a adhesion of 207 gm/cm2 and did not produce 5 during this experiment. Wound infection or antigen-antibody reaction (HJ·Wang ei α/. (1992), J. Ρ/απ R〇c, 1:1, 48_51) 0 Based on the need to obtain a large area of skin for transplantation, in 1970 At the end of the period, a large amount of cultured epidermis was successfully developed from a small piece of excised epidermis by tissue culture of normal human epidermal keratinocytes (keratin SyteS), and the cultured epidermis was self-organized. The cultUred epidermal autograft (CEA) has been successfully used on burn wounds as a thin epidermal covering to rebuild a wide range of skin defects (jG Rheinwald, H. Green (1975)? Cell, 6:331; H. Green et α/. (1979), Proc. Natl Acad. Sci. USA, 76:5665-5668; GG Ill 15 Gallico β/·(1984), TV· £>zg7· J. Me<i· , 311: 448-454). However, in subsequent studies, it was found that the epidermal surface generated by a flat dermal-epidermal conjunction without the presence of rete ridges is fragile and continues to blisters, thus The cultured epidermal autograft (CEA) has poor adhesion to the full-thickness wound, and the "acceptance rate" of CEA is therefore not good. It is formed only when it is transplanted into the connective tissue containing the basement membrane. The structure of the reticular ridge so that it can be attached to the wound (DT Woodley W α/. (1988), 259: 2566-2571 ! SR Herzog et a. (1988), J. Trauma^ 28:195-198 N. Carver et al. (1993)? Br. J. Plast. Surg., 1252113 46:384-392). In addition, because the dermis layer of the thin-skin graft is thin, the wound eventually forms a hypertrophic or contraction scar, which causes unsightly healing, and even forms a contracture that affects the function of the reconstructed skin. . 5 Therefore, scientists and skin remodeling therapists over the years have attempted to design a skin grafting pattern using a dermal template containing a basement membrane (BM), ie before transplanting a cultured epidermal autograft (CEA). First, or simultaneously transplant a man-made, synthetic, homologous or heterogeneous dermis containing a basement membrane (BM), so that CEA can grow smoothly, in addition to promoting wound healing, it can reduce the phenomenon of wound contracture or hyperplasia, and then achieve no The best effect of scar healing on scars. In recent years, several dermal substitutes have been proposed to support thin-skinned skin grafts and CEA, including: collagen-glycosaminoglycan dermal membrane (ST Boyce, JF 15 Hansbrough) (1988), Surgery, 103:421-431; ST Boyce et al (1988), J. Biomed. Mater Res., 22:939-957; JF Hansbrough ei a/. (1989), 262:2125-2130; And ST Boyce da/· (1990), corpse/zarmaco/·, 3:136), fibrin (V_ Ronfard et al. (1991), Burns^ 17:181-184; B. Hafemann 20 ( 1994), Bwr-like, 20:168-172; and AD Jr. Jabs da/. (1992), ir., 89:268_271), allogeneic dermis (CB Cuono et al (1986), Lancet, 1:1123-1124; CB Cuono et al (1987), Plast. Reconstr. Surg., 80:626-635; RC Langdon et al (1988), J. Invest. Dermatol, 91:478-485; 10 1252113 I Mckay et al (1994), Burns, 20:S19-S22; WL Hickerson et al. (1994), 20:S52-S56; g&WASchiozereia/ (1994), 20:503-507), from Autodermis (HA Navsaria da/· (1994), Xian my 20: S57-S06) and true xenogeneic skin 5 (xenodermis) (B Hafemann (1994.), 20: 168-172) and the like. In 1981, Burke and Yannas et al. invented a two-layer artificial skin with epidermis and dermis called "Integra" (JF Burke W α/· (1981), Ann. Surg., 194:413; DM Heimbach et al. (1988), Ann. Surg., 208:313; RL Sheridan et al (1994), Euk J. 10 corpse/(xst· iSWg., 17:91; and JF Burke (1987), Japawe% Jbwmd 17 (6): 431-438), in which the epidermis is a crepe film, and as the dermis is a structure having a high porosity and is composed of bovine collagen and shark cartilage. Derived from the glycosaminoglycan (glycosaminoglycan 15 (chondroitin-6-sulfate)] cross-linked extract (TD Chou Wa/· (2001), 108 (2 ): 378-384) After 10 years of invention, Dr. Heimback conducted joint research and human experiments in several medical centers in the United States, proving that the dermis of "Integra" artificial skin can be transformed through the process of biodegradation. For the recipient's 2 〇 dermis. The clinical use of "Integra" artificial skin is: transplant it On the wound, after the dermis is combined with the wound and healed, the sputum film is torn off and an autologous skin graft is applied to the dermis (TD Chou as α/.丨2001\ Plast Reconstr. Surg.f 108 (2): 378-384; J. Kopp et al (2003)?

Swrgery 29 (6): 653-657; and C.S. Chu a/. 1252113 (2002), Journal of Trauma-Injury Infection & Critical Care, 52 (1) · 122-129). The development of Integra artificial skin provides physicians with new options in treating patients' wounds. However, due to the long development period of this finished product, its price is expensive. An integral artificial skin 5 with an area of 30x10 cm2 is sold at NT$60,000 in Taiwan. This high cost is not affordable for general burn patients. of. In addition, since Integra artificial skin does not have a basement membrane (BM), it is rare to see that CEA has successfully grown strongly when CEA is transplanted onto it. EP 1 375 647 A1 discloses an artificial dermis made of plasma which is coagulated by the addition of platelets in the presence of platelets, and fibroblasts or other dermal cells are embedded in the artificial dermis. Further, keratinocytes can be sown on the surface of the artificial dermis, so that the 5 HAI artificial dermis is particularly suitable for the treatment of large-area burns, chronic skin ulcers 'or 'through the use of genetically altered cells, the artificial dermis can be used as 15 A tool for gene therapy. Sources of allogeneic skin grafts include human cadaver or fetal amniotic membranes. The disadvantage of using skin grafts from humans is limited access (11111 (1 & ¥ heart & 13丨1), high cost, risk of infectious diseases and immune rejection (M. Loss //· (2000), Burns, 26 (7): 644-20 52). This rejection is caused by endothelial cells, fibroblasts, and height.

The cleavage ability of keratinocytes is triggered by Langerhans cells. Cuono et al. describe a two-stage technique for combining dermal and CEA with allografts. Initially, they transplanted allogeneic skin to the resected burn wound. After four weeks they removed the epidermis of allogeneic skin and transplanted CEA 12 1252113 to the dermis, which improved the “acceptance rate” to 80-85% ( CB Cuono et al (1986), Lancet, 1:1123-1124; CB Cuono et al (1987), eg rg., 80: 626-635; and RC Langdon da/. (1988), J. Dermaio/·, 91: 478-485). 5 The most accepted alternative to date appears to be an acellular dermal matrix that removes cells involved in immune responses in allogeneic skin grafts through acellularization techniques. The acellular allodermal graft can be used to support the growth of autologous split-thickness skin (ASTS) grafts, and histological analysis reveals infiltration of fibroblasts, new Angiogenesis, new epidermal formation, and no recurrence (D. Wainwright α/. (1994), J. Venge Care / 7, 17: 124_136; and SA Livesey et al (1995), TYansplantation, 60 ·Λ-9). 15 Kangesu et al. showed that de-epidermalised autodermis can significantly improve the growth of cultured keratinocytes (T_ Kangesu α/. (1993), 5r. J. P/ aW. 46:401-409). In addition, Chu et al. found that silver-nylon dressing and direct current can avoid infection of the fistula and promote the reticulated autologous epithelial dermal complex skin graft (autoepidermal). -allodermal composite graft) and reduces the contracture of the wound (CS· Chu d β/· (1995), J. D. 39: 273-278). However, none of the above mentioned documents or patents mentions whether the use of these 13 1252113 grafts does not cause wound contracture or hyperplasia at all, thus achieving a smooth and scar-free wound healing. In our previous studies, it was found that the fusion of pig skin and wound formation (incorp〇rati〇n) can be more than 5 14 days when the wound is thoroughly cleaned to reduce sensation. In addition, after several pathological studies, it was found that after the epidermal cells of the epidermal layer of the pig skin were rejected, the residual low-repellent dermis layer could be accepted and retained by the human body through biodegradation. Using this experience and hypothesis, it has been reported in our 1997 paper that porcine dermis with keratinocytes removed can be used to promote the expansion and reticular cleavage of 10-segmented skin grafts in rats (autol 〇gOUS spiit-thickness skin graft, ASTS) growth. The acdlular xenodermal graft shows a skin healing effect similar to that of the acellular allodermal graft, both of which promote the growth of autologous split skin and delay the sputum of the wound. Shrink. The results of tissue section 15 showed that pig heterologous dermal collagen was replaced by collagen in rats by fibrosis or biodegradation (HJ Wang et al. (1997), Journal of Trauma-Injury Infection &CW"(3)/ Care, 42(2): 177-182). However, in this paper study, a mesh-cut autologous split-skin graft is used, which reduces the rate of 20 wound contractures and leaves an unsightly mesh at the healing wound. Hypertrophic scars. Thus, in the art, there is still an urgent need for an artificial skin graft that promotes stable growth of CEA, promotes wound healing, and reduces wound contracture or hyperplasia, in order to achieve scar-free wound healing. 1252113 Required. SUMMARY OF THE INVENTION Accordingly, in a first aspect, the present invention provides an artificial skin graft comprising: (a) acellular mammalian dermis having a predetermined thickness (acellular mammalian) a dermis having a first surface and a second surface, wherein the first surface 10 and the damaged skin are disposed when the artificial skin graft is placed into a damaged skin area of a body to be treated Regional contact; and (b) - a flattening element that intersects the second surface of the acellular mammalian dermis. The artificial skin graft according to the present invention can be packaged in a storage bag containing a preservative to form an aseptic package for skin grafting. In a second aspect, the invention provides a method for making an artificial skin graft comprising the steps of: (i) freezing a piece of mammalian skin containing a skin layer and a dermis layer from a mammal; (ii) thawing the mammalian skin of step (i); 20 (iii) removing the epidermal layer of the thawed mammalian skin by an acellularization technique, whereby Mammalian dermis that does not contain epithelial nests is formed; (iv) colds the mammalian dermis obtained in step (iii); dried east; and (v) freezes the resulting flattened element and step (iv) One of the dry feeding 15 1252113 milk animal dermis faces the surface of the epidermal layer. The above and other objects, features, and advantages of the present invention will become apparent upon reference to the accompanying claims claim DESCRIPTION OF THE DRAWINGS Figure 1 is a silicone-acellular porcine dermis (SAPD) according to the present invention; Figure 2 shows SAPD in the first week (left) and the second week (right) after transplantation. The wound condition of the group; 0 Figure 3 shows the results of pathological study of HE staining of the wound sections of the SAPD group during the first week of transplantation (magnification ιοοχ) and the second week (lower magnification of 400X); The results of Masson's staining on the wound tissue of the SAPD group rats at the fifth week (magnification ι〇〇χ) are shown; 5 Figure 5 is a view showing the SAPD group during the six weeks after transplantation.

A comparison of wound contractures in the Biobrane group, where p < 〇〇 5 indicates significant; Figure 6 shows the growth of rat wounds after one week of autologous thin skin grafting; : Figure 7 shows autologous thin skin grafting After week, the autografted mouse skin in the wound of the rat survived well; Figure 8 shows the pathological section of Masson's staining of autologously transplanted skin after six weeks of autologous thin skin transplantation; Figure 9 shows that autologous thin skin grafting was performed. Wound healing in rat wounds at 7th week 16 1252113 (left) and 9th (right); Figure 10 shows wound healing at 1 week after wound healing in autologous thin skin grafts Figure 11 shows a cultured human epithelial cell (a sheet 〇f cultured human epithelial cells) obtained from the Taiwan Institute of Zoology; Figure 12 shows a nude mouse cut out on the back with a 2x2 cm2 wound. Figure 13 shows a nude mouse wound receiving a silicone-free porcine dermal graft (SApD) on day 14 after transplantation; Figure 14 shows when transplanted with a xenogeneic epithelial cell graft 10 (CEX) 14th After the day, Growth of cultured cells transplanted on SAPD nude mouse wounds; Figure 15 shows growth of cultured cells transplanted into SAPD group wounds 28 days after transplantation--Heterogeneic Epithelial Cell Graft (CEX) Case; 15 20 Figure 16 shows the results of tissue section staining of the SAPD group wounds with mouse anti-human cytokeratin antibody on day 28 after transplantation of a xenografted epithelial cell transplant (CEX), Where the arrow points to the rete ridges; ffi 17 shows the CEX growth on the wound in the SAPD group 42 days after transplantation of a xenografted epithelial cell transplant (CEX). Figure 18 shows The results of immunohistochemical staining of the sApD group were performed with anti-type 4 collagen* white antibody after transplantation for 42 days after a heterologous epithelial cell migration (C) (CEX), wherein the upper magnification was 100X, The magnification below is 4 years old, while the arrow is drunk on the ground base 17 1252113

Figure 19 shows the wound condition of the Integra group of nude mice on the third day after transplantation; Figure 20 shows the Integra dermal template observed by the transparent silicone film on the day of the integral nude mice in the Integra group; 5 Figure 21 The appearance of the dermis revealed by tearing off the silicone film on the 14th day after transplantation in the 1nte§ra group of nude mice; Figure 22 shows that the Integra group of nude mice were transplanted with a heterologous epithelial cell graft (CEX) 14th. Day Wound Situation; Figure 23 shows the wound condition of the Integra group of nude mice on day 28 after transplantation of a xenografted epithelial fine cell graft (CEX); Figure 24 shows that the Integra group of nude mice were transplanted with a heterologous The wound condition on day 42 after allogeneic epithelial cell transplantation (CEX), where the arrow points to the wound healing site; Figure 25 shows that the Integra group of nude mice were transplanted with a xenogeneic epithelial fine 15 cell graft (CEX) On the 42nd day afterwards, the tissue sections of the wound were taken for the results observed by Η Ε staining, where the arrow Α refers to the wound healed by second intention, and the arrow B refers to Different places Allogeneic epithelial cell graft (CEX) shedding (sl〇Ugh) and wound necrosis (necrosis); and 20 Figure 26 shows that the Integra group of nude mice were transplanted with a xenogeneic epithelial cell graft (CEX) on day 42 after The results of the tissue section of the wound taken and stained with a type 4 collagen antibody, wherein the arrow A refers to the wound that healed through the second stage, and the arrow B indicates the occurrence of the allogeneic Epithelial cell graft (CEX) shedding and wound necrosis. 1252113 [Embodiment 3] The detailed description of the preferred embodiment is for the treatment of a large area of severely damaged skin wounds. We have attempted to develop a porcine dermis template containing a basement membrane (BM) for use as a Prefabricated graft, which promotes the growth of cultured epidermal autograft (CEA), saves the lives of patients with severely damaged skin and provides high quality wounds Heal. Based on our previous research on porcine dermis, we have made 10 occurrences. If a flattening element such as ruthenium film is cross-linked with acellular mammalian dermis, the artificial skin graft formed can promote the stable growth of CEA. It promotes wound healing and reduces the phenomenon of wound contracture or hyperplasia, which can achieve wound healing without scars. Accordingly, the present invention provides an artificial skin graft comprising: 15 (a) - a cell-free mammalian dermis having a predetermined thickness having a first surface and a second surface, wherein the artificial skin graft The first surface is in contact with the damaged skin area when placed in one of the damaged skin areas to be treated; and (b) - the second surface of the acellular mammalian dermis Flattening element The artificial skin graft according to the present invention can be applied to a damaged skin wound of the dermis, such as a full thickness skin wound or a layered skin wound (partial- Thickness skin wound) According to the present invention, the acellular mammalian dermis is a skin derived from humans, 19 I252113 cows, horses, sheep, and the like. Preferably, the cell free breast milk animal skin is derived from human skin or pig skin. Preferably, the artificial, mammalian dermis contained in the artificial skin graft according to the present invention has a predetermined thickness of 〇·〇〇8-〇.〇3〇英吋. 5 Preferably, the cell-free mammalian dermis used to prepare the artificial skin graft of the present invention is substantially free of epithelial nests. With regard to the so-called "substantially no epithelial nest", according to the present invention, the acellular mammalian dermis can be derogated as: when a mammalian dermis is microscopically made with 2 high power fields (magnification: 40 Χ) When examined and confirmed to have less than one epithelial 10 nest (iv) idermal nesO, the mammalian dermal sheet is considered to be a cell-free mammalian dermis suitable for use in the present invention. Preferably, the acellular mammalian dermis contained in the artificial skin graft according to the present invention comprises a basement membrane at a second surface thereof. Preferably, the 15-cell mammalian dermis contained in the artificial skin graft according to the present invention is obtained by the following steps: (i) taking a skin layer and a dermis layer from a mammal. The mammalian skin is frozen for a period of time; (ii) the mammalian skin of step (i) is thawed; (iii) the epidermal membrane of the mammalian skin is removed by a cell-free technique (acellularizati〇n techniqUe) a layer whereby a mammalian dermis substantially free of epithelial nests is formed, the surface of one of the mammalian dermis facing the epidermal layer being the second surface; and (iv) the breastfeeding obtained in step (iii) Animal dermis is freeze dried. 20 1252113 In the step of a cytoplasmic dermis of the present invention (in the sputum:: pig, cow, horse, sheep, etc.) _ ritual animal skin sputum from humans, in a better cytoplasmic dermis of the present invention The step (〇 - - 二 中 ' is used to prepare the other one in the present invention =: the skin of the skin is 1 to human. The no: in a preferred embodiment of the invention 'is used 1 :: The mammalian skin in the step (1) of the animal dermis has been subjected to a better example. The mammalian skin in the step (i) used in the preparation of the fine animal dermis has been sterilized with a sodium hypochlorite bath for a period of time. In a preferred embodiment of the present invention, in the step (iii) used to prepare the fine-free 15-cell mammalian dermis, a solution containing a proteolytic enzyme is used for treatment. The thawed mammalian skin whereby the epidermal layer of the thawed mammalian skin is removed almost completely. In a more preferred embodiment of the invention, it is used to prepare the non-fine 20 cell lactation Animal dermis steps (out) The solution used to treat the unrolled mammalian skin contains a proteolytic enzyme selected from the group consisting of: tripsin, pepsin, dispase, and the like. In a/or better specific example, in step (iii) used to prepare the cell-free mammal 21 1252113 animal dermis, a trypsin-containing solution is used to treat the thawed mammal. In a preferred embodiment of the invention, the flattening element is crosslinked to the second surface of the acellular mammal, via a method selected from the group consisting of: heat treatment, Treatment using a chemical crosslinking agent [e.g., glutaraldehyde, carbodiimide, etc.] In a more preferred embodiment of the invention, the flattening element is delivered by heating Linked to the second surface of the acellular mammalian dermis. Preferably, the cross-linking is completed at a thermal cross-linking temperature range of 25 to 180 ° C. According to the invention, the flattening element is A film of silicone film, PU film, collagen egg a film, a chitosan film, a hyaluronic acid film, a gelatin film. In a preferred embodiment of the invention, the flattening element is a silicone film. 15 The present invention also provides a film A method for making an artificial skin graft comprising the steps of: (1) taking a piece of mammalian skin containing a skin layer and a dermis layer from a mammal; storing it for a period of time; (ii) taking the step (i) Mammalian skin relief; 20 (d) to remove the epidermal layer of the thawed mammalian skin by a llUlarization technique whereby a mammalian dermis substantially free of epithelial nests is formed (d) cross-linking the steps (ie, the resulting mammalian dermis cold green drying; and (V) ordering - flattening elements and the surface of the epidermis layer of one of the cold-green dry-feeding 22 1252113 milk animal dermis obtained in step (iv) . The materials and operating steps used in the method according to the invention have at least the technical features mentioned above. Alternatively, the artificial skin graft according to the present invention may be packaged in a storage bag containing a preservative to form a sterile package for skin grafting. Accordingly, the present invention also provides an aseptic package for skin transplantation comprising: (a) a storage bag for containing an artificial skin graft; (b) an artificial skin graft as described above, or An artificial skin graft prepared by the method of 10 described above; (c) and a preservative. In a preferred embodiment of the invention, the aseptic package comprises a preservative selected from the group consisting of ethanol, glycerin, and a combination thereof. In a more preferred embodiment of the invention, the preservative is ethanol. In another more preferred embodiment of the invention, the preservative is glycerin. In still another more preferred embodiment of the invention, the preservative is a mixture of ethanol and glycerin. Preferably, the mixing ratio of ethanol to glycerin falls within the range of 1 〇: 至 to 0:10. The invention will be further illustrated by the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting. In order to facilitate the description of the practice of the present invention, pig skins are used in the following examples for the preparation of acellular porcine dermis, and for patients requiring skin 23 1252113 transplantation, from human skin of legal origin. It will be a more suitable biological material. Example 1 Preparation of acellular Porcine dermis (APD) 5 A. Preparation of fresh frozen porcine skin ·· Purchase healthy pigs and take ketamine (ketamine) , 5 mg/kg) and pigs were anesthetized with pentothal sodium (10 mg/kg). After that, the pig's back was sheared and the back skin was washed with gasoline to remove the oily ulcer, which was then washed twice with povidone-iodine and finally the back skin was washed with alcohol. A pig skin sheet having a thickness of 0.012 inch was obtained by an electric Dermatome (Padget). The cut thin pig skin piece was placed in a penicillin-containing physiological saline solution (1〇6 units of penicillin/500 mi 15 physiological saline) for 15 minutes, and then washed with physiological saline. The rind was then immersed in an aqueous solution containing 0.05% sodium hypochloride for 5 minutes, followed by rinsing three times with saline under sterile conditions. The following treatments were carried out under aseptic conditions in a laminar flow hood (1 〇〇 class). The treated pig skin was spread on a 4 inch wide yarn stretcher containing penicillin (1 〇 6 units / 500 ml physiological saline) and 5% glycerin on a laminar flow hood. The yarn is then rolled into a roll to wrap the pigskin. Place the warp-knitted pigskin in a sterile antifreeze (_7〇〇c) plastic box and 24 1252113. After screwing the lid, place the plastic box into a sterile antifreeze (-70Ό) plastic bag. Then, the plastic bag is sealed and sealed. Thereafter, the plastic bag was placed in a freezer at -4 c overnight, and then transferred to a g-70 〇c freezer for storage. 5 Pig skin treated to this stage can be used for the application of general wounds, or for the preparation of pig dermis. At each job, representative pig skins were taken for general microbial (bacterial, viral, and mold) testing in such a way that other pig skins prepared in the same batch did not contain pathogenic bacteria and mold. B. Preparation of acellular porcine dermis (preparati〇n acellular porcine 10 dermis) ·· Preparation of acellular porcine dermis can refer to HJ Wang as a/. (1997), Journal of Trauma-Injury Infection & Critical Care, 42 ( 2): The method described in 177-82 is carried out. The frozen pig skin piece prepared in the procedure A was taken out at room temperature by aseptic technique on a sterile operation table of a one-layer fume hood, and the pig skin piece was placed in a 2.5 〇/〇 after thawing. The solution of trypsin (Life Τ6〇1ιη〇1〇§^ΤΜ, USA) lasted for about 2 hours, and then when the epidermis was observed to appear in the pig skin, the epidermis was removed with tweezers. . Continue to immerse the pig skin in a solution containing 1% trypsin and gently rub for 3 minutes. After 20, the pig skin pieces were taken out and washed three times with a physiological saline solution containing penicillin (106 units of penicillin/500 ml saline), whereby a cell-free pig dermis was prepared. The intact state of the porcine dermis is evaluated by electron microscopy and microscopic examination of various tissue staining methods to confirm the retention of the basement membrane complex 25 1252113, the maintenance of the normal collagen bundle and the streak pattern, and the cell composition of the epidermal layer. Removal of matter. As already mentioned, the acellular porcine dermis is defined here as a microscope with 20 high power fields (magnification of 4 〇χ), and it is confirmed that it has less than 1 epithelium. Pig skin of the epidermal nest. The thus prepared cell-free porcine dermis was stored in a freezer at -4 °C in the manner described in the uranium procedure A. Representative pig skins were taken at each job for general microbial (bacterial, viral, and mold) testing, in this way to confirm that the 10 batches of porcine dermis prepared in the same batch did not contain pathogenic bacteria and mold. Example 2. Preparation of crushed-cell-free porcine dermis (j§iiic〇n-aceiiuiar dermis, SAPD) A. Freeze-drying treatment of cell-free pig dermis··Acellular pigs prepared in Example 1 and refrigerated The dermal panels were immersed in 15 sterile deionized water for 30 minutes and then washed three times with sterile deionized water. Then, the side-free cell-free dermis was placed in a non-recorded steel pan, then placed in a cold-beam dryer (Freezone 12, Labconco, USA) for freeze-drying for 36 hours, then freeze-dried. The acellular porcine dermis is stored in a dry box (collector, Taiwan) for later use. 1Λ B. Preparation of silicone film: Medical stone gelatin (MDX-4210 A/B, Dow Corning, USA) was uniformly mixed in a ratio of A/B=95/5, and the resulting mixture was placed in one. The bubble is depressurized in a dry box for 15 to 20 minutes. The bubble-free medical stone glue obtained thereby is placed on a Teflon template, and the thickness of the film-forming device 26 1252113 _ive, BelgiUm) is adjusted to push the film, and then the paper is supplied to the box (oven ' The air bubble-free medical driving was dried for 3 hours, thereby obtaining a transparent film-like stone film. After the film was removed, the film was measured by a spiral micrometer (Mitutoyo, Japan) to have a thickness of about 5 to about 0.3 mm. The thus obtained Shishi gum film was stored in a dry box for use. Preparation of sputum sputum cell-free pig dermis ········································································ It is divided into 1 hour. The transparent film obtained in the above procedure b was placed on a Tielonglong plate, and then an appropriate amount of the mixture was applied with a brush and uniformly applied to the transparent stone film. Then, one of the cold-beam dry cell-free pig dermal pieces prepared in the above procedure B is attached to the surface of the epidermal layer to the surface layer of the epidermis, and the woven fabric is placed on the acellular pig dermis - an unsuitable plate is added. 15 pressure. After 4 hours of thermal crosslinking at 37 ° C, the formed Shiqi gum _ cell-free pig dermis was transferred to a flow hood and was evacuated at room temperature for 6 G hours. Obtained - a two-layered (four) _ cell-free sputum dermal dressing (see Figure 1). Preservation of second gel/cell-free pig dermis· 20 a·liquid preservation method A 75% (Wv) ethanol solution was prepared with ethanol (95% EtOH, Merck, USA) and sterile deionized water, followed by 75% ethanol: A preservation solution was prepared by the ratio of glycerol (Calbi〇chem, USA) = 9 : 1 (v/v). The Shixia gum _ cell-free pig dermis obtained from the uranium program C was placed in a sho 27 ^ 252113

The foil bag of the skin and the preservation solution is enclosed in a sterilization tube bag and stored in a bag, and the skin piece is immersed in a refrigerator at 5 ° C for use. - Take representative pig skins on each day of operation ^' to perform microbial, viral and mildew tests, and confirm that the other pig skin pieces prepared in the same batch do not contain pathogenic bacteria and bacteria. b. 7-ray irradiation ([ray radiationi〇n) preservation method... The Shishijiao/cell-free plaque leather film prepared by the procedure C is placed in a sterilized B-package and then sent to the original energy plant. Irradiation _6 〇 irradiation, the intensity of the irradiation is 25M Ray. After 5 hours of irradiation, the sterilization tube bag was taken out and placed in a drying oven and stored at room temperature. At each work, representative pig skins were taken for general microbial (bacterial, viral, and mold) testing, in this way to confirm that the same batch of the prepared porcine dermis without the pathogenic bacteria and mold. Example 3·Comparison of silicon-acellular porcine dermis with Biobrane in order to evaluate the gelatin-acellular porcine dermis according to the invention and the commercialized 2 〇 biological dressing Biobrane for the full layer The healing effect of a full-thickness wound is carried out in animal experiments. The Biobrane used in this example is from Bertek Pharmaceuticals INC, WV USA. Thirty-six male Sprague-Dawley rats [35 〇g weight, obtained from National Defense Medical Center 28 1252113 National Anaesthesia Medical Center] were anesthetized by intraperitoneal administration of pentothal (30 mg/kg). . Surgery and transplant operations are performed in a laminar flow hood. A full-thickness skin lesion of approximately 4x5 cm2 was cut out from the back of the rat. Rats were randomly divided into two groups (n=18 per group). Group 1 rats received a silicone-cellless porcine dermal (SAPD) graft according to the present invention, while Group 2 rats received a Biobrane graft. The skin graft wound is fixed by the tie line method (tie_over), and then the outer periphery is used to strengthen the fixation. The grafts in each rat were examined at 1, 2, 3, 4, 5, and 6 weeks after transplantation. 1〇 Parameters used for evaluation include: angiogenesis (uniform pink and white when pressure is known), evidence of contraction (skin size), and strong adhesion to the wound bed. To examine the difference between the experimental groups, the size of the wound was evaluated by the Student-t test. Results: 15 The wounds of all rats treated with 8 8 1 > 1) were observed to exhibit pink-transplanted dermis via a clear silicone film. Both the SAPD and Biobmne grafts adhered to the wound bed. Figure 2 shows the wound condition of the S A P D group at the first week (left) and the second week (right) after transplantation. It can be seen through the transparent (tetra) film that there are 2 pink transplanted dermis in the wound. Figure 3 shows the results of the pathological study of HE staining in the wound tissue sections of the SApD group in the first week of transplantation (upper magnification, 1〇〇χ) and the second week^, magnification: 400Χ). The most superficial crimson-like substance is the Shixi film, which adheres well to the porcine dermis arranged neatly below, while 29 1252113 and the fusion of SAPD with the wound (1)^丨〇11) is good, and there is no obvious in the dermis. Inflammation and rejection of cellular infiltration. Thus, sApC> has been accepted by the rats. Figure 4 shows the results of 5 colors of RMass〇n,s dyed in the SAPD group at the 5th week after transplantation (magnification 10〇x), in which the adhesion of the silicone film on the wound to the pig dermis is good, and the collagen fibers of the wound It is blue stained, neatly arranged, and has no obvious inflammation and rejection. Thus, SApD has been accepted by rats. The comparison of wound size is shown in Figure 5. At the 6th week after transplantation, the healing wounds of the SAPD group showed mild contracture (about 35%); however, the wounds of the Biobrane group showed moderate to severe contracture (about 95%). At the 4th week after transplanting, the wounds of the Biobrane group showed signs of infection and necrosis; however, the wounds of the SAPD group did not show rejection. At the 6th week after transplantation, part of the wound infection in the SAPD group was triggered by the displacement of the silicone film. From the results obtained above, as a long-term temporary covering of the wound, Lu's SAPD showed an effect that was longer lasting, superior, and not repellent than Bi〇brane. Therefore, SAPD is an excellent wound dressing that can be used to temporarily cover wounds for a long time. 20 Example 4: Effect of Silicone-Acellular Porcine Dermal (SAPD) as a template for an autologous thin-skin graft The anesthesia and surgical procedures of the experimental animals were carried out in accordance with the procedure disclosed in Example 3. SAPD was transplanted into the back wound of rats (I2) (private 5 cm2) De 7 owed 'at 30 1252113 The second week of SAPD's silicone film was removed and placed into a skin cut from the abdomen of the rat As an autologous thin-skin graft (1〇/1〇〇〇英吋), the skin graft wound was fixed by tie-over, and then the periphery was used to strengthen the fixation. At the first week after transplantation, the plaster of Paris and the suture were removed, and it was found that the growth of the wound was good (Fig. 6). The wound examination performed at the 6th week after transplantation found that the autografted mouse skin survived well (Fig. 7), and the hair follicles and sweat glands attached to the skin had grown, and the growth of the porcine dermis underneath was stable. There is inflammation of rejection (Figure 8). At the 7th, 9th, and 11th week after transplantation, it was observed that the wound no longer had a contracture, and it was able to maintain 65% of its original wound size (Figure 9 and Figure 10). It is thus apparent that SAPD is an excellent graft for wound reconstruction, and the acellular porcine dermis contained therein can be used as a dermal template for rebuilding a wound and reducing the wound of a wound. 〇 Example 5 • Comparison of the efficacy of sputum, cell-free porcine dermis (s apd) and Integra grafts as templates for xenografts of xenografts in order to evaluate the sputum _ cell-free porcine dermal (SAPD) and commercial pupa according to the present invention. This animal experiment was conducted with the effect of Integra on the healing of fuii_thickness wound. A, surgical method: 20 The experimental animals used in this example were 24 nude mice [BALB/cAnNCrl-Foxnlnu strain, which is athymic nude mice, weighing about 12 g] was obtained from The Integrat Grafts of the National Center for Laboratory Animals of the National Defence Medical College was purchased from integra Life Sciences Corp USA, and the cultured human allogeneic epithelial cell graft (cultured 31 1252113 epidermal xenograft, CEX) (Figure 11) was obtained from Taiwan Animal Science and Technology Research. All. Mice were anesthetized by intraperitoneal administration of pent〇thal (30 mg/kg), and the surface of the dorsal 5 of nude mice was disinfected with 10% aqueous betadine and then on the back of the mice. A full-thickness skin wound of approximately 2 x 2 cm2 was cut out (Figure 12). Mice were randomly divided into two groups (n each group: = ι2). The first group of mice received the silicone-free cell-free porcine dermal (SAPD) graft according to the present invention, while the second group of mice received the Integra graft. All the grafts were fixed with a tie-over tie-over, and then wrapped with plaster to strengthen the fixation. The round plastic frame was used to prevent the skin from growing around the wound. And the impact of interpretation. Surgery and transplantation were performed in a sterile laminar flow hood. Grafts were examined at 7, 14, 21, 28, 35 and 42 days after transplantation. Parameters used for evaluation included: angiogenesis (uniform pink and white when applied), evidence of contraction (skin size), and strong adhesion to the wound bed. To examine the differences between the experimental groups, the size of the wound was assessed by the Student-t test. This study can be found in the description of ^丄 Wang et al (1997)? Journal of Trauma-Injury Infection & Critical Care, 42 (2): 177-82. 2〇 On the 14th day after transplantation, the Shishi tablets were removed from the SAPD or Integra graft. Thereafter, a cultured human epithelium sheet, which is a cultured epithelium xenograft (CEX), was transplanted to the SAPD group or

On the plasty template of the Integra group, the skin graft 32 1252113 was fixed with a symmetrical line tie method (tie-〇ver), and the outer periphery was wrapped with plaster to strengthen the fixation. The survival rate and contraction rate of the transplanted CEX at the wound site were recorded on the basis of 7 days. The comparison of wound size is shown in the figure. RESULTS: 5 Figure 13 shows the wound of a nude mouse receiving a silicone-free porcine dermal graft (SAPD) on the 14th day after transplantation. It can be seen through the silicone film that the dermis located below is pink. Since the experiment is fixed with a circular frame, this prevents the skin from growing in from the periphery of the wound and affecting interpretation. Figure 14 shows the growth of cultured cells transplanted onto SAPD nude mouse wounds after transplantation of a heterologous epithelial cell transplant 10 (CEX) day 14, from which the cultured cells were grown. good. Fig. 15 shows the growth of cultured cells transplanted on the wound of the SAPD group after being transplanted with the allogeneic epithelial cell graft (CEX) on the 28th day, and it can be seen from the figure that the cultured cells grew well. 15 Figure 16 shows the results of tissue section staining of SAPD group wounds with mouse anti-human cytokeratin antibody on day 28 after transplantation of a xenografted epithelial cell (CEX), arrowhead Refers to rete ridges. It can be seen from the figure that the growth of the SAPD is human epithelial cells, not the nude mouse epithelial cells, and the growth of retic ridges is found, which shows that CEX can firmly grow on the collagen fibers of the porcine dermis. on. Figure 17 shows the CEX growth on the wound of the SAPD group after 42 days after transplantation of a xenografted epithelial cell transplant (CEX). It can be seen from the figure that CEX grows well and the wound is contracted, retaining only 33 1252113 42% of the original wound, but the stratum corneum has formed. Figure 18 shows the results observed by immunohistochemical staining of the SApD group with anti-type 4 collagen antibody after transplanted with -type allogeneic epithelial cell transplantation (CEX) day 42, with 5 100X placed above 'The magnification below is 4〇〇X, and the arrow points to the basement membrane. It can be seen from the figure that there is a base film (where the arrow points) between CEX and SAPD, thereby stabilizing the growth of CEX. Regarding the wound condition of the Integra group nude mice, the dermal collagen of the Integra graft showed a red blood cell infiltration on the first day after transplantation (Fig. 10 19). On the 14th day after transplantation, the dermal template of Integr0g plants (consisting of shark cartilage and bovine dermis) was observed via transparent crepe film, which showed a pinkish appearance, indicating good growth (Fig. 2〇); When the Shi Xi film of the Integra graft was torn off, it also showed that the dermal collagen showed a pink survival state ( FIG. 21 ). 15 When Integ^· nude mice were transplanted with a xenografted epithelial cell graft (CEX) for 14 days, the wound showed that CEX had necrosis and could not grow on the dermal template layer of the Integra graft, thus forming a wound ulcer (Fig. twenty two). In the Integra group, the wounds on the 28th day after transplantation of a heterologous epithelial cell transplant (CEX) showed that CEX was necrotic and could not grow 20 times longer than the dermal layer of the 1ntegra graft, thus forming wound ulcers. (Figure 23). On day 42 after transplantation of CEX, wounds in nude mice of the Integra group showed that CEX could not grow on the dermis layer of the Integra graft, and the wound system healed itself via the epithelium of nude mice (Fig. 24). The HE staining of the wound tissue sections performed at this time showed that the CEX transplanted on the wound showed shedding 34 1252113 necrosis, and most of the wounds were in the form of epithelialization and contraction through the surrounding normal skin. A wound healed by second intention is formed (Fig. 25). On the 42nd day after CEX transplantation, the wound tissue sections were stained with anti-type 4 collagen antibody. The results showed that the basement membrane of the Integra group had only a very weak self-expression of type 4 collagen eggs, and no newborn ridges were found. Formed (Figure 26). All documents and patents cited in this specification are hereby incorporated by reference in their entirety in the present disclosure. In the event of a conflict, the detailed description of the case (including definitions) will prevail. Although the present invention has been described with reference to the above <RTI ID=0.0>>>>>'''''''' 'A lot of modifications and changes can be made under the Shenjing Patent Fan Shen attached to the 1^ document. 35 1252113 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a gel-acellular porcine according to the present invention. Dermis, SAPD); Figure 2 shows the wound status of the 5 SAPD group at the first week (left) and the second week (right) after transplantation; Figure 3 shows the first week of transplantation (upper, magnification) Ioox) and the second week (below, magnification 400X), the wound sections of the SAPD group were subjected to the pathological study of HE staining; Figure 4 shows the results of 10 Masson's staining of the wound tissue of the SAPD group at the fifth week ( Magnification ι〇〇χ); Figure 5 is a display of the saPD group during the six weeks after transplantation

A comparison of wound contractures in the Biobrane group, where p < 〇〇 5 indicates significant; Figure 6 shows the growth of rat wounds after one week of autologous thin skin grafting; Figure 73⁄4, the member shows autologous After six weeks of thin skin grafting, autologous transplanted mouse skin in the wound of the rat survived well; Figure 8 shows the pathological section of MaSS〇n, s staining of autologous skin after six weeks of autologous thin skin transplantation; Fig. 9 shows that the wound healing of the wounds of the autologous thin-skin grafts at the 7th week (left) and the 9th week (right) is shown in Fig. 9; And wound healing of the wounds of the autologous thin-skin grafted rats at the 11th week; Figure 11 shows the cultured human upper 36 1252113 skin-thickness tablets obtained by the Youwan Bay Animal Research Institute ( Figure 12 shows a nude mouse cut out on a back with a 2x2 cm2 wound; Figure 13 shows a nude mouse wound receiving a silicone-cellless porcine dermal graft (SAPD) in a transplant After the 14th day; 5 Figure 14 shows when being transplanted on a heterogeneous Growth of cultured cells transplanted on SAPD nude mouse wounds after day 14 of cell transplantation (CEX); Figure 15 shows the 28th day after transplantation of a heterologous epithelial cell transplant (CEX) 10 growth of cultured cells transplanted on the SAPD group wound; Figure 16 shows that the mouse anti-human cytokeratin antibody was used for the SAPD group on the 28th day after transplantation of a heterologous epithelial cell graft (CEX). The results observed in the staining of the tissue section of the wound, where the arrow points to the rete ridges; 15 Figure 17 shows the SAPD after being transplanted for 42 days after a heterologous epithelial cell graft (CEX) CEX growth on the wounds. Figure 18 shows the immunohistochemical staining of the SAPD group with anti-type 4 collagen antibody after 42 days of transplantation of a heterologous epithelial cell graft (CEX). As a result, the upper magnification is 20 i〇〇x, and the lower magnification is 400X, and the arrow points to the basement membrane; Figure 19 shows the wound condition of the Integra group nude mice on the first day after transplantation; 20 display Integra group nude mice in the Integra group on the 14th day after transplantation, the wound was observed through the transparent Shishi film, the Integra dermis template; 37 1252113 Figure 21 shows the Integra group of nude mice on the 14th day after transplantation The appearance of the dermis revealed after tearing off; Figure 22 shows the wound condition of the Integra group of nude mice on day 14 after transplantation of a heterologous epithelial cell graft (CEX); 5 Figure 23 shows that the Integra group of nude mice were transplanted Wound condition on day 28 after a xenografted epithelial cell transplant (CEX); Figure 24 shows wound condition on day 42 after transplantation of a heterologous epithelial cell transplant (CEX) in nude mice in the Integra group, Refers to the wound healing site; 10 Figure 25 shows the results observed in the Integra group of nude mice on the 42nd day after transplantation of a heterologous epithelial cell graft (CEX) for tissue staining of the wound for HE staining, Where arrow A refers to the wound healed by second intention, and where arrow B points, there is a heterologous epithelial cell graft (CEX) shedding (sl〇ugh) 15 and the wound is broken. Necrosis; and Figure 26 shows that the Integra group of nude mice were harvested on day 42 after transplantation of a heterologous epithelial cell transplant (CEX), and the tissue sections of the wound were taken and stained with anti-type 4 collagen antibody. The observed results, where arrow A refers to the wound that healed through the second phase, and the arrow B points to the existing xenograft (CEX) exfoliation and wound necrosis. [Main component symbol description] φ 38

Claims (1)

1252113 X. Patent Application Range: 1 . An artificial skin graft comprising: (a) a cell-free mammalian dermis having a predetermined thickness having a first surface and a second surface, wherein the artificial The first surface is in contact with the damaged skin area when the skin graft 5 is placed into one of the damaged skin areas to be treated; and (b) - the second surface of the acellular mammalian dermis Cross-linked flattening elements. 2. The artificial skin graft of claim 1, wherein the non-fine mammalian dermis has a predetermined thickness of from 0.008 to 0.030 inches. 3. The artificial skin graft of claim 1, wherein the non-cell mammalian dermis is derived from the skin of a human, pig, cow, horse or sheep. 4. The artificial skin graft of claim 3, wherein the acellular mammalian dermis is derived from a human. 15. The artificial skin graft of claim 3, wherein the non-cell mammalian dermis is derived from a pig. 6. The artificial skin graft of claim 1, wherein the damaged skin area to be treated is a full-thickness skin wound or a layered skin scar. 20. 7. The artificial application of claim 1 A skin graft, wherein the acellular mammalian dermis is substantially free of epithelial nests and comprises a basement membrane at the second surface. 8. The artificial skin graft of claim 1, wherein the acellular mammalian dermis is obtained by the following steps: 39 1252113 (i) a skin-containing epidermis and dermis obtained from a mammal The layer of mammalian skin is frozen for a period of time; (ii) the mammalian skin of step (1) is thawed; (iii) the epidermal layer of the thawed mammalian skin is removed by an acellularization technique, Thus, a mammalian dermis substantially free of epithelial nests is formed, one surface of the mammalian dermis facing the epidermal layer being the second surface; and (iv) freezing the mammalian dermis obtained in step (iii) dry. 10. The artificial skin graft of claim 8, wherein the mammalian skin used in the step (i) for preparing the acellular mammalian dermis is from human, pig, cow, horse or sheep. skin. 10. The artificial skin graft of claim 9, wherein the mammal 15 in the step (1) used to prepare the acellular mammalian dermis is derived from human skin. 11. The artificial skin graft of claim 9, wherein the mammalian skin used in the step (1) for preparing the acellular mammalian dermis is derived from the skin of the pig. 12. The artificial skin graft of claim 8 wherein the mammalian skin used in the step (1) of preparing the acellular mammalian dermis has been depilated and sterilized. 13. The artificial skin graft of claim 8, wherein the mammalian skin used in the step (1) for preparing the acellular mammalian dermis has been sterilized with a sodium hypocarbonate solution for a period of time. 40 1252113. 14. The artificial skin graft of claim 8, wherein in the step (iii) used to prepare the acellular mammalian dermis, a solution containing a proteolytic enzyme is used to treat the thawed The mammalian skin whereby the epidermal layer of the thawed mammalian skin is completely removed by almost 5. 15. The artificial skin graft of claim 14, wherein in the step (iii) used to prepare the acellular mammalian dermis, the solution used to treat the thawed mammalian skin contains a selection Proteolytic enzymes from the following groups: trypsin, pepsin, dispersing 10 enzymes, and a combination of these. 16. The artificial skin graft of claim 15, wherein in the step (iii) used to prepare the acellular mammalian dermis, a trypsin-containing solution is used to treat the thawed mammal. skin. 17. The artificial skin graft of claim 1, wherein the flattening 15 element is crosslinked to the second surface of the acellular mammalian dermis via a treatment selected from the group consisting of: heating Treatment, chemical crosslinker treatment, and a combination of these. 18. The artificial skin graft of claim 17, wherein the applanation element is crosslinked to the second surface of the acellular lactation 20 animal dermis via a heat treatment. 19. The artificial skin graft of claim 17, wherein the flattening element is crosslinked to the second of the acellular mammalian dermis via a chemical crosslinker selected from the group consisting of: Surface: glutaraldehyde, and carbodiimide. 41 1252113 20. The artificial skin graft of claim 1, wherein the flattening element is a silicone film, a PU film, a collagen film, a chitin film, a glass acid film or a gelatin film. 21. An artificial skin graft as claimed in claim 1 which is further aseptically processed and then packaged in a storage bag. 22. The artificial skin graft of claim 21, wherein the preservation bag further comprises a preservative. 23. The artificial skin graft of claim 22, wherein the preservative is selected from the group consisting of ethanol, glycerin, and a combination of the above. 24. The artificial skin graft of claim 23, wherein the preservative is ethanol. 25. The artificial skin graft of claim 23, wherein the preservative is glycerin. 15. The artificial skin graft of claim 23, wherein the preservative is a mixture of ethanol and glycerol. 27. The artificial skin graft of claim 26, wherein the mixing ratio of ethanol to glycerin falls within the range of 10:0 to 0:10. 28. A method for making an artificial skin graft comprising the steps of: (i) freezing a piece of mammalian skin containing a skin layer and a dermis layer from a mammal for a period of time; (ii) (1) removing the mammalian skin of step (1); (iii) removing the solution by a fine-graining technique; the epidermal layer of the eastern mammalian skin 42 1252113, whereby a mammal that does not substantially contain an epithelial nest Animal dermis is formed; (iv) freeze-dried the mammalian dermis obtained in step (iii); and (v) a flattened element and the cold obtained in step (iv); One of the surfaces facing the skin layer is cross-linked. 29. The method of claim 28, wherein the mammalian skin used in step (i) is a skin derived from humans, pigs, cows, horses or sheep. 30. The method of claim 29, wherein the mammalian skin used in step (1) 10 is derived from human skin. 31. The method of claim 29, wherein the mammalian skin used in step (1) is derived from the skin of a pig. 32. The method of claim 28, wherein the mammalian skin used in step (i) is depilated and sterilized. The method of claim 28, wherein the mammalian skin used in step (1) has been sterilized with a sodium hypochlorite solution for a period of time. 34. The method of claim 28, wherein in step (iii), a solution containing a proteolytic enzyme is used to treat the skin of the thawed mammal 20, whereby the thawed mammalian skin is The skin layer is almost completely removed. 35. The method of claim 34, wherein the proteolytic enzyme is selected from the group consisting of trypsin, pepsin, dispase, and a combination thereof. 43 1252113 36. The method of claim 35, wherein a trypsin-containing solution is used in step (iii). 37. The method of claim 28, wherein the base film is provided at a surface of one of the mammalian dermis formed in step (iii) facing the epidermal layer. The method of claim 28, wherein the crosslinking of step (v) is carried out by a method selected from the group consisting of heat treatment, chemical cross-linking treatment, and a combination thereof. 39. The method of claim 38, wherein in step (v), the flattening element is crosslinked to the epidermal layer by using a heat treatment to one of the freeze-dried mammalian dermis s surface. 40. The method of claim 38, wherein in step (v), the flattening element is crosslinked to the freeze-dried via a chemical crosslinking agent selected from the group consisting of One of the mammalian dermis faces the surface of the epidermal layer: glutaraldehyde, and carbodiimide. The method of claim 28, wherein the flattening element used in the step (v) is a silicone film, a PU film, a collagen film, a chitin film, a glass acid film or a gelatin film. 42. The method of claim 28, wherein the mammalian dermis formed in step (v) and associated with the flattening element is further sterilized in a holding bag containing a preservative. 43. The method of claim 42, wherein the preservative is selected from the group consisting of ethanol, glycerin, and a combination thereof. 44. The method of claim 43, wherein the preservative is ethanol. 45. The method of claim 43, wherein the preservative is glycerin. 44. The method of claim 43, wherein the preservative is a mixture of ethanol and glycerol. 47. The method of claim 46, wherein the mixing ratio of ethanol to glycerin falls within the range of 10:0 to 0:10. 5 48. An aseptic package for skin grafting comprising: (a) a storage bag for containing an artificial skin graft; (b) - as in any of claims 1 to 27 An artificial skin graft, or an artificial skin graft prepared by the method of any one of claims 28 to 47; 10 (c) and a preservative. 49. The aseptic package for skin grafting of claim 48, wherein the preservative is selected from the group consisting of ethanol, glycerin, and a combination thereof. 50. The aseptic packaging for skin transplantation according to item 49 of the patent application, wherein the preservative is ethanol. 51. The aseptic package for skin grafting of claim 49, wherein the preservative is glycerin. 52. The aseptic package for skin grafting of claim 49, wherein the preservative is a mixture of ethanol and glycerin. 20 53. For aseptic packaging for skin grafting according to item 52 of the patent application, wherein the mixing ratio of ethanol to glycerin falls within the range of 10:0 to 0:10.