Artificial dermis material and preparation method thereof

Abstract

The invention provides an artificial dermis material and a preparation method thereof.The preparation method comprises the following steps that pig dermis tissue is treated with high-concentration alkali liquor, and porous dermis tissue is obtained; sequentially performing degreasing treatment and drying treatment on the porous dermal tissue to obtain a porous acellular dermal matrix; adding the micronized pig dermal matrix, gelatin and honey into water, and uniformly mixing to obtain a composite adhesive liquid; and laying the composite adhesive liquid on one side of a porous acellular dermal matrix, and sequentially carrying out freezing fixation and vacuum drying to obtain the artificial dermal material. Therefore, the obtained artificial dermis material is closer to a physiological structure, the upper layer of dermis can provide mechanical support and ventilation functions, and the lower layer of dermis can quickly absorb tissue penetrating fluid to form a weakly acidic gel protective layer by utilizing high osmotic pressure of honey at the initial stage of wound formation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to an artificial dermis material and a preparation method thereof. BACKGROUND

[0002] Skin, as the largest tissue organ of human body, has multiple functions such as barrier, immunity, perception, body temperature regulation, metabolism and absorption. Autologous transplantation after skin injury is the gold standard for clinical treatment, but the shortage of autologous skin source limits its application in the treatment of patients with large-area skin defect.

[0003] Based on the development of biomedical engineering and regenerative medicine, artificial dermis with good biocompatibility and functionality can effectively solve the problem of autologous skin source shortage and provide a new choice for the treatment of wound injury. The wound healing process includes three overlapping and dynamic stages of inflammation, proliferation and tissue reconstruction, involving inflammation regulation, vascularization, bacterial infection, oxidative stress and other complex microenvironments. However, the existing artificial dermis has relatively single function, which is difficult to absorb tissue permeate, resist bacteria, resist inflammation and resist oxidation during the inflammation period of wound, and accelerate vascularization, promote tissue regeneration and prevent scar formation during the proliferation period.

[0004] Therefore, there is an urgent need to provide an artificial dermis material and a preparation method thereof. SUMMARY

[0005] The artificial dermis material and the preparation method thereof provided by the embodiments of the present application can solve the problem that the traditional artificial skin cannot simultaneously prevent adhesion, regulate inflammation, promote wound healing and prevent scar formation.

[0006] In a first aspect, the present application provides a preparation method of an artificial dermis material, which comprises the following steps: (1) treating porcine dermal tissue with high-concentration alkali solution to obtain porous dermal tissue; (2) sequentially performing defatting treatment, drying treatment and cutting treatment on the porous dermal tissue to obtain porous acellular dermal matrix; (3) mixing microparticulate porcine dermal matrix, gelatin and honey in water to obtain a composite adhesive liquid, wherein the microparticulate porcine dermal matrix is obtained by grinding the porcine dermal tissue after pretreatment; (4) laying the composite adhesive liquid on one side of the porous acellular dermal matrix, and then sequentially performing freezing fixation and vacuum drying to obtain the artificial dermis material.

[0007] Preferably, in step (1), the thickness of the porcine dermal tissue is 0.2-2 mm, and the high-concentration alkali solution is a sodium hydroxide solution with a concentration of 2-4 mol / L.

[0008] Preferably, the porcine dermal tissue is immersed in a high-concentration alkali solution for oscillation treatment; wherein the oscillation time is 1-4 h, and the rotation speed is 60-120 rpm / min.

[0009] Preferably, in step (2), the defatting solution used in the defatting treatment is at least one of isopropyl alcohol, methanol, ethanol or acetone.

[0010] More preferably, the concentration of the defatting solution is 50-100 wt%, and the defatting treatment is performed under ultrasonic conditions for 0.5-2 h.

[0011] Preferably, in step (2), the drying treatment includes freeze-drying treatment and vacuum drying treatment; wherein the freeze-drying treatment is performed at a temperature of -20 to -80 ℃ for 1-4 h; and the vacuum drying treatment is performed at a temperature of -10 to -50 ℃ for 24-72 h.

[0012] More preferably, the thickness of the porous acellular dermal matrix is 0.05-0.5 mm.

[0013] Preferably, after the oscillation treatment and the defatting treatment, a step of washing with physiological saline or PBS buffer is further included.

[0014] Preferably, in step (3), the pretreatment includes sequentially immersing the porcine dermal tissue in an alkali solution and a defatting solution for immunogen removal and defatting treatment, and then cutting, freezing in liquid nitrogen and grinding to obtain the microparticulate porcine dermal matrix; preferably, the particle size of the microparticulate porcine dermal matrix is less than 300 microns.

[0015] Preferably, in step (3), in terms of mass percentage, the content of the microparticulate porcine dermal matrix in the composite adhesive solution is 1-5%, the content of the gelatin is 5-10%, the content of the honey is 5-10%, and the content of the water is 75-89%.

[0016] Preferably, in step (3), after obtaining the microparticulate porcine dermal matrix, a step of sequentially using a first modification solution and a second modification solution to functionalize the microparticulate porcine dermal matrix is further included.

[0017] Preferably, the first modification solution is a methanol solution containing asiaticoside.

[0018] More preferably, the concentration of asiaticoside in the first modification solution is 100-500 µg / mL.

[0019] Preferably, the second modification solution is a mixed solution of a tannic acid solution and a metal ion solution; wherein the metal ion solution is one or more of a magnesium ion solution, a zinc ion solution or a copper ion solution.

[0020] More preferably, the concentration of the tannic acid solution is 10 mmol / L, and the concentration of the metal ion solution is 0.1-30 mmol / L.

[0021] Preferably, the mass-volume ratio of the micro-particleized porcine dermal matrix to the first modification solution is 1: (2-5).

[0022] Preferably, the mass-volume ratio of the micro-particleized porcine dermal matrix to the tannic acid solution is 1: (5-10), and the volume ratio of the tannic acid solution to the metal ion solution is 1:1.

[0023] Preferably, the temperature of the mixed reaction is 25-30℃, and the time is 20-24h.

[0024] More preferably, after the reaction is completed, the reaction product is further subjected to the steps of freeze fixation and vacuum drying in sequence.

[0025] Preferably, in step (4), the laying thickness of the composite adhesive mucilage is 1-5mm.

[0026] Preferably, the temperature of the freeze fixation is -20~-80℃, and the time is 1~4h, and the temperature of the vacuum drying is -10~-50℃, and the time is 24~72h.

[0027] In the second aspect, the application further provides an artificial dermal material prepared by the preparation method of any one of the above-mentioned first aspect.

[0028] Compared with the prior art, the application has at least the following beneficial effects: (1) In the application, the porcine dermal tissue is first treated with a high-concentration lye, which not only effectively plays a role in decellularization and virus inactivation, but also has a certain corrosion effect on the porcine dermal tissue, so as to further expand the natural pore structure of the porcine dermal tissue, so that the surface of the porous decellularized dermal matrix formed has obvious pore structures and good air permeability. Then, the micro-particleized porcine dermal matrix is prepared from the porcine dermal tissue, and the composite adhesive mucilage is prepared by mixing the micro-particleized porcine dermal matrix with honey and gelatin; finally, the porous decellularized porcine dermal matrix is used as an upper layer of artificial dermis, the upper layer provides mechanical support and air permeability, and the composite adhesive mucilage formed by the micro-particleized porcine dermal matrix, gelatin and honey is used as a lower layer of artificial dermis and is adhesively compounded with the upper layer to obtain an artificial dermis material. The upper layer pore structure can simulate the natural dermal layer, and the lower layer micro-particleized matrix composite honey and gelatin can simulate the reticular layer, which is closer to the physiological structure. During use, the upper layer of dermis can provide mechanical support and air permeability, and the lower layer of dermis can form a weakly acidic protective layer by rapidly absorbing tissue permeate liquid in the early stage of wound formation due to the high osmotic pressure of honey.

[0029] (2) In some preferred embodiments of the present application, the microparticulate porcine dermal matrix is first modified by a first modification solution containing asiaticoside and a second modification solution containing tannic acid solution and metal ion solution, and then the modified microparticulate porcine dermal matrix, gelatin and honey are mixed to obtain a weakly acidic gel layer, which can effectively inhibit bacterial infection during the inflammatory period. Meanwhile, the microparticulate porcine dermal matrix has a functional surface layer constructed by tannic acid and magnesium ion complexing, which is responsive to acidic environment and can gradually release tannic acid and metal ions under the condition of weakly acidic wound surface, thereby playing a multiple role of antibacterial, anti-inflammatory, antioxidant and promoting angiogenesis, so as to accelerate the transition from the inflammatory period to the proliferation period of the wound. With the gradual absorption of the honey gel layer and the continuous release of the surface complex, the asiaticoside pre-loaded in the interior of the matrix is released step by step, further regulating the collagen synthesis process in the proliferation period and remodeling period, and effectively preventing the formation of scar tissue. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0031] Figure 1 is a macrostructure diagram of the porous decellularized dermal matrix provided by embodiment 1 of the present application; Figure 2 is a scanning electron microscope diagram of the functionalized modified microparticulate decellularized matrix provided by embodiment 1 of the present application; Figure 3 is a DNA content data diagram of the porcine dermal tissue before and after treatment with an alkali solution provided by embodiment 1 of the present application; Figure 4 is a fat content data diagram of the porcine dermal tissue before and after defatting treatment with a defatting solution provided by embodiment 1 of the present application; Figure 5 is an antioxidant data diagram of the microparticulate decellularized matrix before and after surface functionalization treatment in the present application and embodiment 2; Figure 6 is an inflammation factor (TNF-α) expression data diagram of the microparticulate decellularized matrix before and after surface functionalization treatment provided by embodiment 1 and embodiment 2 of the present application; Figure 7 is a vascular endothelial growth factor (VEGF) expression data diagram of the microparticulate decellularized matrix before and after surface functionalization treatment provided by embodiment 1 and embodiment 2 of the present application; Figure 8Figure 1 is a graph of cell toxicity data for artificial skin prepared according to Example 1 of the present application. DETAILED DESCRIPTION

[0032] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0033] The present application provides a preparation method of artificial skin material, which comprises the following steps: (1) treating porcine dermal tissue with high-concentration lye to obtain porous dermal tissue; (2) sequentially performing defatting treatment, drying treatment and cutting treatment on the porous dermal tissue to obtain porous acellular dermal matrix; (3) mixing microparticulate porcine dermal matrix, gelatin and honey in water to obtain composite adhesive liquid, wherein the microparticulate porcine dermal matrix is obtained by grinding porcine dermal tissue after pretreatment; (4) laying the composite adhesive liquid on one side of the porous acellular dermal matrix, and then performing freezing fixation and vacuum drying to obtain the artificial skin material.

[0034] In the embodiments of the present application, the porcine dermal tissue is first treated with high-concentration lye, which not only effectively plays a role in decellularization and virus inactivation, but also has a certain corrosion effect on the porcine dermal tissue, so as to further expand the natural pore structure of the porcine dermal tissue, so that the porous acellular dermal matrix formed has obvious pore structure and good air permeability. Then, the microparticulate porcine dermal matrix is prepared from the porcine dermal tissue, and the microparticulate porcine dermal matrix is mixed with honey and gelatin to prepare a composite adhesive liquid. Finally, the porous acellular porcine dermal matrix is used as an upper layer of artificial skin, the upper layer of dermis provides mechanical support and air permeability, and the composite adhesive liquid formed by the microparticulate porcine dermal matrix, gelatin and honey is used as a lower layer of dermis to be adhesively compounded with the upper layer of dermis to obtain the artificial skin material. The pore structure of the upper layer can simulate the natural dermis layer, and the microparticulate matrix compounded with honey and gelatin in the lower layer can simulate the reticular layer, which is closer to the physiological structure. During use, the upper layer of dermis can provide mechanical support and air permeability, and the lower layer of dermis can rapidly absorb tissue permeate to form an acidic protective layer in the early stage of wound formation due to the high osmotic pressure of honey.

[0035] According to some preferred embodiments, in step (1), the thickness of the porcine dermis tissue is 0.2-2 mm (for example, it can be 0.2 mm, 0.5 mm, 0.8 mm, 1 mm, 1.5 mm, 1.8 mm or 2 mm), and the high-concentration alkali solution is a sodium hydroxide solution with a concentration of 2-4 mol / L (for example, it can be 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L or 4 mol / L), preferably 2-3 mol / L.

[0036] According to some preferred embodiments, the porcine dermis tissue is immersed in the high-concentration alkali solution for oscillation treatment; wherein the oscillation time is 1-4 h (for example, it can be 1 h, 2 h, 3 h or 4 h), and the rotation speed is 60-120 rpm / min (for example, it can be 60 rpm / min, 80 rpm / min, 100 rpm / min or 120 rpm / min).

[0037] In the embodiments of the present application, first, fresh porcine skin tissue is taken, and the epidermis layer and the fat layer are removed using an electric peeling machine to obtain porcine dermis tissue with a certain thickness. Then, the porcine dermis tissue is placed in the above-mentioned high-concentration sodium hydroxide solution for oscillation treatment at room temperature. During the treatment, the sodium hydroxide solution with the above-mentioned concentration can not only remove the immunogenic substances in the porcine dermis tissue, but also corrode the porcine dermis tissue due to the high concentration of sodium hydroxide, thereby expanding the natural pores and forming a porous structure, eliminating the need for a punching process, and forming an artificial dermis upper layer with air permeability. If the concentration of the sodium hydroxide solution used is too high, not only will the mechanical properties of the porcine dermis tissue decrease, but also the collagen may be excessively degraded.

[0038] According to some preferred embodiments, in step (2), the defatting solution used in the defatting treatment is at least one of isopropyl alcohol, methanol, ethanol or acetone; preferably, the concentration of the defatting solution is 50-100 wt% (for example, it can be 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt% or 100 wt%), and the defatting treatment is carried out under ultrasonic conditions for 0.5-2 h (for example, it can be 0.5 h, 1 h or 2 h).

[0039] In the embodiments of the present application, by further placing the porous dermis tissue in a defatting solution and assisting with ultrasonic treatment, and by controlling the concentration of the defatting solution, the immunogenic substances in the porous dermis tissue can be further removed while ensuring the integrity of the structure of the porous dermis tissue. Tests show that the fat content in the porous dermis tissue in the embodiments of the present application can be effectively controlled to be below 1%, meeting the requirements for fat residue in animal-derived medical devices.

[0040] It should be noted that in the embodiment of the present application, considering the different effects of different types of alcohol solutions on the removal of fat in porous dermis and the different safety, different concentrations of different types of defatting solutions are selected, when isopropyl alcohol solution and methanol are used, the concentration of the solution is 50-100 wt%, when ethanol or acetone solution is used, the concentration of the solution is 60-80 wt%, and in the embodiment of the present application, the porous dermis is preferably further treated with ethanol solution.

[0041] According to some preferred embodiments, in step (2), the drying treatment comprises freeze-drying treatment and vacuum drying treatment; wherein the temperature of the freeze-drying treatment is -20~-80℃ (for example, it can be -20℃, -50℃ or -80℃), and the time is 1~4h (for example, it can be 1h, 2h, 3h or 4h); the temperature of the vacuum drying treatment is -10~-50℃ (for example, it can be -10℃, -20℃, -30℃, -40℃ or -50℃), and the time is 24~72h (for example, it can be 24h, 30h, 48h, 60h or 72h); the thickness of the porous acellular dermal matrix is 0.05-0.5mm (for example, it can be 0.05mm, 0.1mm, 0.2mm, 0.3mm, 0.4mm or 0.5mm); after the oscillation treatment and the defatting treatment, a step of washing with physiological saline or PBS buffer is further included.

[0042] In the embodiment of the present application, after the high-concentration alkali solution and the defatting solution are respectively used to treat the porcine dermis, the treated porcine dermis is washed with physiological saline or PBS buffer, so that the residual alkali solution or defatting solution on the surface of the porcine dermis can be removed, thereby avoiding adverse effects on the subsequent treatment process. The defatted porous dermis is sequentially subjected to freezing fixation and vacuum drying, and then is cut to the above-mentioned thickness using a skin peeling machine, so as to obtain the porous acellular dermal matrix.

[0043] According to some preferred embodiments, in step (3), the pretreatment comprises sequentially immersing the porcine dermis in an alkali solution and a defatting solution for immunogen removal and defatting treatment, and then sequentially cutting, freezing in liquid nitrogen and grinding to obtain the micronized porcine dermis matrix; preferably, the particle size of the micronized porcine dermis matrix is less than 300 microns (for example, it can be 300 microns, 250 microns, 200 microns, 180 microns, 150 microns, 120 microns or 100 microns).

[0044] In the embodiment of the present application, in the pretreatment, the same defatting solution and drying treatment parameters in steps (1) and (2) are adopted, and the difference is that the alkali solution is sodium hydroxide solution or potassium hydroxide solution, and the concentration of the alkali solution is 0.1-1 mol / L. The alkali solution with the concentration can effectively remove the cell nucleus of the porcine dermis, prevent immune rejection, and effectively maintain the integrity of the collagen tissue structure and morphology in the porcine dermis. After the defatting treatment, the freeze-dried acellular dermis is cut into small pieces of 1 cm*1 cm, treated with liquid nitrogen for 5 min, and ground by using a knife-type mixing grinder for 5-10 min, the rotation speed of the knife-type grinding instrument is 6000-10000 revolutions / min, and the ground sample is filtered by using a filter screen to obtain microparticulate porcine dermis matrix with a diameter of less than 300 microns. The microparticulate porcine dermis matrix is bridged by gelatin and honey matrix, the microparticulate porcine dermis matrix with the particle size can ensure that the number of bridging points and the bonding area are optimal, and the microparticulate porcine dermis matrix with the particle size can form a microchannel network to promote cell migration and endothelial cell structure formation.

[0045] According to some preferred embodiments, in step (3), the content of the microparticulate porcine dermis matrix in the composite adhesive liquid is 1-5% (for example, can be 1%, 2%, 3%, 4% or 5%) by mass percentage, the content of the gelatin is 5-10% (for example, can be 5%, 6%, 7%, 8%, 9% or 10%), the content of the honey is 5-10% (for example, can be 5%, 6%, 7%, 8%, 9% or 10%), and the content of the water is 75-89% (for example, can be 75%, 78%, 80%, 82%, 85% or 89%).

[0046] In the embodiment of the present application, the microparticulate porcine dermis matrix is mixed with gelatin and honey to form a stable network structure, the gelatin thus provides certain mechanical support as a structural framework to ensure that the subdermal layer formed by the composite adhesive liquid has certain mechanical strength; the honey has a high osmotic pressure and is weakly acidic, has antibacterial and anti-inflammatory effects, and the high osmotic pressure can effectively absorb tissue permeate to form a weakly acidic gel layer. At the same time, the addition of honey can endow the subdermal layer with certain flexibility; the microparticulate porcine dermis matrix can simulate the natural dermal reticular layer structure to provide channels for cell migration and vascular ingrowth, promote fibroblast adhesion and spreading, and guide directional cell migration.

[0047] The contents of gelatin, honey and micro-particle pig dermal matrix are reasonably controlled, so that the sub-dermis formed by the composite adhesive liquid has good mechanical strength, flexibility and biological performance. It is found through experiments that if the content of gelatin is too low, it is not conducive to ensuring that the sub-dermis formed by the composite adhesive liquid has good mechanical properties, and if the content of gelatin is too high, the mechanical brittleness of the sub-dermis will increase, which is also not conducive to ensuring good mechanical properties. The appropriate content of honey not only gives the composite adhesive liquid good flexibility, but also forms a weakly acidic gel layer, which helps to inhibit bacterial proliferation and also has anti-inflammatory effect. If the content of honey is too low, it is not conducive to ensuring that the sub-dermis formed by the composite adhesive liquid has good flexibility and antibacterial and anti-inflammatory properties, and if the content of honey is too high, its structure and morphology will be difficult to effectively maintain. Further, if the content of micro-particle pig dermal matrix is too low, the guidance of cell migration will be insufficient, and if the content of micro-particle pig dermal matrix is too high, the micro-particle pig dermal matrix in the composite adhesive liquid will easily settle and cause stratification.

[0048] It should be noted that the honey used in the embodiments of the present application is a medicinal grade honey.

[0049] According to some preferred embodiments, after obtaining the micro-particle pig dermal matrix in step (3), the method further comprises the steps of sequentially using a first modification solution and a second modification solution to functionalize and modify the micro-particle pig dermal matrix. The first modification solution is a methanol solution containing asiaticoside, the concentration of asiaticoside in the first modification solution is 100-500 µg / mL (for example, it can be 100 µg / mL, 200 µg / mL, 300 µg / mL, 400 µg / mL or 500 µg / mL), and the second modification solution is a mixed solution of a tannic acid solution and a metal ion solution. The metal ion solution is one or more of a magnesium ion solution, a zinc ion solution or a copper ion solution. The concentration of the tannic acid solution is 10 mmol / L, and the concentration of the metal ion solution is 0.1-30 mmol / L (for example, it can be 0.1 mmol / L, 0.5 mmol / L, 1 mmol / L, 5 mmol / L, 10 mmol / L, 20 mmol / L or 30 mmol / L).

[0050] Specifically, first, the microparticulate porcine dermal matrix is added into a methanol solution containing asiaticoside for stirring loading, so that the asiaticoside is loaded inside; wherein, the mass-volume ratio of the microparticulate porcine dermal matrix to the asiaticoside methanol solution is 1: (2-5) (for example, it can be 1:2, 1:3, 1:4 or 1:5); the stirring loading time is 4-24h (for example, it can be 4h, 5h, 10h, 12h, 18h or 24h), and the stirring speed is 60-120rpm / min (for example, it can be 60rpm / min, 80rpm / min, 100rpm / min or 120rpm / min). Then, the mixed solution after stirring loading is subjected to centrifugal treatment to obtain the microparticulate porcine dermal matrix loaded with asiaticoside, and then the microparticulate porcine dermal matrix is subjected to functional modification using a mixed solution containing a tannic acid solution and a metal ion solution. After the reaction is completed, the reaction product is sequentially subjected to freeze fixation and vacuum drying to obtain the functionalized microparticulate porcine dermal matrix; wherein, the mass-volume ratio of the microparticulate porcine dermal matrix to the tannic acid solution is 1: (5-10) (for example, it can be 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10), and the volume ratio of the tannic acid solution to the metal ion solution is 1:1; the temperature of the mixed reaction is 25-30℃ (for example, it can be 25℃, 28℃ or 30℃), and the time is 20-24h (for example, it can be 20h, 22h or 24h).

[0051] In view of the fact that asiaticoside can regulate collagen synthesis and prevent scar formation in the proliferation and remodeling stages, and magnesium ions and zinc ions have good anti-inflammatory and pro-angiogenic abilities and high biological safety, in order to realize dynamic regulation and functional synergy in different stages of the wound healing process, in the embodiments of the present application, the microparticulate decellularized porcine dermal matrix loaded with asiaticoside is first subjected to a complexation reaction with tannic acid and metal ions to construct an acid-sensitive metal polyphenol complex layer on the surface, so that the asiaticoside is encapsulated inside the particles. In the early stage of wound healing, the honey forms a weakly acidic gel environment after high-osmotic absorption of tissue fluid, triggering the gradual release of tannic acid and metal ions from the complex layer, thereby playing the roles of antibacterial, anti-inflammatory, antioxidant and pro-angiogenic, and accelerating the transition from the inflammatory stage to the proliferation stage. With the continuous degradation of the complex layer, the asiaticoside inside is gradually released to regulate collagen metabolism and inhibit scar formation in the later healing stage. In this way, through the time-sequential controlled release of the tannic acid-metal ion complex and the asiaticoside, effective regulation of the whole wound healing process is realized, and the dual functions of promoting tissue repair and inhibiting scar formation are achieved.

[0052] According to some preferred embodiments, in step (4), the laying thickness of the composite mucilage is 1-5mm (for example, it can be 1mm, 2mm, 3mm, 4mm or 5mm).

[0053] In the embodiment of the present application, the obtained composite adhesive liquid is laid on one side of the porous acellular dermal matrix layer by spin coating or blade coating, and then is frozen and vacuum dried to obtain the artificial dermis material; in the process of spin coating or blade coating, the thickness of the composite adhesive liquid is controlled to ensure that the artificial dermis material has good air permeability, liquid permeability, and good antibacterial, anti-inflammatory and pro-angiogenic properties; if the thickness of the composite adhesive liquid is too low, the functional components will be insufficiently loaded and the mechanical strength will be low; and if the thickness of the composite adhesive liquid is too high, the functional components cannot be filled and attached to the defect site.

[0054] According to some preferred embodiments, after the reaction is completed, the reaction product is further subjected to the steps of freezing and vacuum drying. The freezing temperature is-20~-80℃ (for example, it can be-20℃, -50℃ or-80℃), and the freezing time is 1~4h (for example, it can be 1h, 2h, 3h or 4h); the vacuum drying temperature is-10~-50℃ (for example, it can be-10℃, -20℃, -30℃, -40℃ or-50℃), and the vacuum drying time is 24~72h (for example, it can be 24h, 30h, 48h, 60h or 72h).

[0055] The embodiment of the present application also provides an artificial dermis material prepared by the preparation method of any one of the first aspect.

[0056] In the embodiment of the present application, the artificial dermis material comprises a dermis upper layer and a dermis lower layer, the dermis lower layer is composed of surface-functionalized microparticulate porcine dermal matrix, gelatin and honey, and the dermis upper layer is a porous acellular porcine dermal matrix layer; the dermis upper layer provides mechanical support and air permeability, and the dermis lower layer uses the high osmotic pressure of honey to rapidly absorb tissue exudate to form an acidic protective layer in the early stage of wound formation. First, the microparticulate porcine dermal matrix is modified by a first modified solution containing asiaticoside and a second modified solution containing tannic acid solution and metal ion solution, and then the modified microparticulate porcine dermal matrix, gelatin and honey are uniformly mixed to obtain a weakly acidic gel layer; the gel layer can effectively inhibit bacterial infection in the inflammation period, and at the same time, the microparticulate porcine dermal matrix constructs a functional surface layer by complexing tannic acid with magnesium ions, which is responsive to acidic environment and can gradually release tannic acid and metal ions under the weakly acidic conditions of the wound, thereby synergistically playing the multiple roles of antibacterial, anti-inflammatory, antioxidant and pro-angiogenic, so as to accelerate the transition from the inflammation period to the proliferation period of the wound. With the gradual absorption of the honey gel layer and the continuous release of the surface complex, the asiaticoside pre-loaded in the matrix is gradually released, further regulating the collagen synthesis process in the proliferation period and the remodeling period, and effectively preventing the formation of scar tissue.

[0057] In order to more clearly illustrate the technical solutions and advantages of the present application, the following will describe in detail a kind of artificial dermis material and its preparation method through several examples.

[0058] Example 1: (1) Take fresh pig skin tissue respectively, remove the epidermis layer and fat layer using an electric peeling machine, and obtain two pig dermis layer tissues (thickness is 1 mm); place the two pig dermis layer tissues in high-concentration alkali solution (sodium hydroxide) with a concentration of 3 mol / L and alkali solution (sodium hydroxide) with a concentration of 0.5 mol / L respectively, and oscillate them on a shaking bed at a speed of 100 r / min for 1 h for treatment, and then wash them with physiological saline for 2-3 times until neutral, to obtain porous dermis tissue and dermis tissue; (2) Soak the porous dermis tissue and dermis tissue in a degreasing solution (ethanol solution) with a concentration of 80 wt% respectively, and ultrasonic degreasing for 1 h, then wash the degreasing solution with physiological saline, and then wash away the physiological saline with purified water, then freeze and fix the degreasing treated porous dermis tissue and dermis tissue at -20℃ for 2 h, and then vacuum dry at -10℃ for 36 h, cut the dried porous dermis tissue and dermis tissue to 0.5 mm using a peeling machine, to obtain porous decellularized dermis matrix; then cut the cut dermis tissue into small pieces with a size of 1 cm*1 cm, freeze treat using liquid nitrogen for 5 min, and grind using a knife-type mixing grinder for 8 min, the rotation speed of the knife-type grinder is 8000 r / min, filter the ground sample using a filter screen to obtain particles with a diameter less than 300 μm (100-200 μm), to obtain micronized pig dermis matrix; (3) Add the micronized pig dermis matrix to a methanol solution of asiaticoside with a concentration of 200 µg / mL according to a ratio of 1 g:2 mL, stir for 12 h at 100 rpm / min, and then centrifuge to obtain the micronized pig dermis matrix loaded with asiaticoside; then, add a tannic acid solution with a concentration of 10 mmol / L according to a ratio of 1 g:5 mL, stir for 10 min, then add a metal ion solution (zinc chloride solution with a concentration of 0.3 mmol / L) according to a ratio of 1:1, continue to stir for 24 h at room temperature for complexation reaction, and then wash with purified water for 3 times, to obtain the functionalized and modified micronized pig dermis matrix; add 5 wt% of the functionalized and modified micronized pig dermis matrix, 8 wt% of gelatin and 8 wt% of honey into 79 wt% of water, stir and mix at 40℃, to obtain a composite adhesive liquid. (4) Pave the composite adhesive liquid on one side of the porous decellularized dermis matrix by spin coating, then freeze and fix at -40℃ for 2 h, and then vacuum dry at -10℃ for 36 h, to obtain an artificial dermis material; wherein, the coating thickness of the composite adhesive liquid is 3 mm.

[0059] AsFigures 1-2 As shown, the upper layer of artificial dermis prepared in Example 1 is subjected to corrosion treatment with high concentration of sodium hydroxide, and a uniform and clear porous structure is visible on the surface. The microparticulate decellularized porcine dermal matrix after functional modification is visible with particles formed by tannin / metal ion complexation.

[0060] Example 2: Example 2 is basically the same as Example 1, except that in step (3), the microparticulate porcine dermal matrix is not subjected to functional modification with asiaticoside methanol solution, tannin solution and metal ion solution, i.e., the microparticulate porcine dermal matrix, gelatin and honey are directly mixed in water to obtain a composite adhesive liquid.

[0061] Example 3: Example 3 is basically the same as Example 1, except that in step (1), two porcine dermal layer tissues (thickness of 1 mm each) are obtained by removing the epidermis and fat layers from fresh porcine skin tissues using an electric peeler. The two porcine dermal layer tissues are placed in an alkali solution (sodium hydroxide) with a concentration of 0.5 mol / L and subjected to treatment by oscillation at a speed of 100 r / min for 1 h on a shaker, followed by washing with physiological saline for 2-3 times until neutralization to obtain two dermal tissues.

[0062] Example 4: Example 4 is basically the same as Example 1, except that in step (3), 10 wt% of the functionalized microparticulate porcine dermal matrix, 8 wt% of gelatin and 8 wt% of honey are added to 74 wt% of water, and the mixture is stirred and mixed at 40°C to obtain a composite adhesive liquid.

[0063] Example 5: Example 5 is basically the same as Example 1, except that in step (3), 5 wt% of the functionalized microparticulate porcine dermal matrix, 8 wt% of gelatin and 15 wt% of honey are added to 72 wt% of water, and the mixture is stirred and mixed at 40°C to obtain a composite adhesive liquid.

[0064] Example 6: Example 6 is basically the same as Example 1, except that in step (4), the laying thickness of the composite adhesive liquid is 8 mm.

[0065] Comparative Example 1: Comparative Example 1 is basically the same as Example 1, except that in step (3), 5 wt% of the functionalized microparticulate porcine dermal matrix and 10 wt% of gelatin are added to 85 wt% of water, and the mixture is stirred and mixed at 40°C to obtain a composite adhesive liquid.

[0066] Comparative Example 2: The comparative example 2 is basically the same as the example, except that in step (3), 5wt% of the functionalized and modified micro-particle pig dermal matrix and 10wt% of honey are added to 85wt% of water, and stirring is performed at 40°C to obtain a composite adhesive liquid.

[0067] Comparative example 3: (1) Fresh pigskin tissue is taken, and the epidermis layer and fat layer are removed using an electric peeler to obtain pig dermal layer tissue (thickness of 1mm); the pig dermal layer tissue is placed in an alkali solution (sodium hydroxide) with a concentration of 0.5mol / L, and is oscillated at a speed of 100r / min for 1h on a shaker for treatment, and then is washed with physiological saline for 2-3 times until neutral, to obtain dermal tissue; (2) The dermal tissue is soaked in a degreasing solution (ethanol solution) with a concentration of 80wt%, and is degreased under ultrasonic for 1h, and then is washed with physiological saline to remove the degreasing solution, and then is washed with purified water to remove the physiological saline, and then the degreased dermal tissue is frozen and fixed at -20°C for 2h, and then is vacuum dried at -10°C for 36h, and then the dried dermal tissue is cut to 0.5mm using a peeler, to obtain a porous decellularized dermal matrix; the cut dermal tissue is cut into small pieces with a size of 1cm*1cm, is treated with liquid nitrogen freezing for 5min, and is ground using a knife-type mixing grinder at a speed of 8000r / min for 8min, and then the ground sample is filtered using a filter screen to obtain a micro-particle pig dermal matrix with a diameter of less than 300μm (100-200μm); (3) The micro-particle pig dermal matrix is added to a methanol solution of asiaticoside with a concentration of 200μg / mL at a ratio of 1g:2mL, and is stirred at 100rpm / min for 12h, and then is centrifuged to obtain a micro-particle pig dermal matrix loaded with asiaticoside; then, a tannic acid solution with a concentration of 10mmol / L is added at a ratio of 1g:5mL, and is stirred for 10min, and then a metal ion solution (a zinc chloride solution with a concentration of 0.3mmol / L) is added at a ratio of 1:1, and is stirred at room temperature for 24h for complexation reaction, and then is washed with purified water for 3 times, to obtain a functionalized and modified micro-particle pig dermal matrix; 5wt% of the micro-particle pig dermal matrix, 8wt% of gelatin and 8wt% of honey are added to 77wt% of water, and are stirred at 40°C to obtain a composite adhesive liquid; the composite adhesive liquid is frozen and fixed at -40°C for 2h, and then is vacuum dried at -10°C for 36h, to obtain an artificial dermal material.

[0068] The artificial dermal materials prepared in the example and the comparative examples are tested for performance, and the test results are shown in Table 1 and Figures 3 to 8DNA residual amount was detected according to the method specified in YY / T 0606.25-2023 "Tissue Engineering Medical Products Part 25: Determination of DNA Residual Amount of Animal-derived Biomaterials - Fluorescent Staining Method"; and fat residual amount was detected according to the method for processing solid samples specified in the second method of GB / T 5009.6-2016 "National Food Safety Standard - Determination of Fat in Food".

[0069] Cytotoxicity test: The leaching solution was prepared according to the method specified in GB / T 16886.12-2023 "Medical Devices - Biological Evaluation - Part 12: Sample Preparation and Reference Materials". The complete culture medium (89vol% high-sugar medium / 10vol% fetal bovine serum / 1vol% double antibody) was used as the leaching medium, which was first saturated with the leaching medium, and then leached at a leaching ratio of 3cm 2 / mL, and leached at 37±1℃ for 72±2h. Mouse fibroblasts (L-929) were inoculated in a 96-well plate at 1×10 4 cells / well, and the cells were adherent cultured for 24h, the cell culture medium was removed, and the leaching solution was used for continuous culture for 24h. The cytotoxicity test was performed according to the operation instruction of CCK-8 kit (Shanghai Biyun Tian Biological Technology Co., Ltd.).

[0070] Vascular endothelial growth factor (VEGF) expression test: The leaching solution was prepared according to the method specified in GB / T 16886.12-2023 "Medical Devices - Biological Evaluation - Part 12: Sample Preparation and Reference Materials". The complete culture medium (89vol% high-sugar medium / 10vol% fetal bovine serum / 1vol% double antibody) was used as the leaching medium, which was first saturated with the leaching medium, and then leached at a leaching ratio of 3cm 2 / mL, and leached at 37±1℃ for 72±2h. Endothelial cells (HUVEC) were inoculated in a 6-well culture plate at 2×10 5 cells / well, and the cells were adherent cultured for 24h, the cell culture medium was removed, and the leaching solution was used for culture. After 24h of culture, the cell supernatant was collected, and the VEGF content was detected according to the operation instruction of VEGF ELISA kit (Shanghai Zhiyan Biological Technology Co., Ltd.).

[0071] Tumor necrosis factor (TNF-α) expression test: The leaching solution was prepared according to the method specified in GB / T 16886.12-2023 "Medical Devices - Biological Evaluation - Part 12: Sample Preparation and Reference Materials". The complete culture medium (89vol% high-sugar medium / 10vol% fetal bovine serum / 1vol% double antibody) was used as the leaching medium, which was first saturated with the leaching medium, and then leached at a leaching ratio of 3cm 2 / mL, 37±1℃ for 72±2h, and then the concentration of the extract was adjusted to 1×10 5 The mouse monocyte macrophage (RAW246.7) was inoculated in a 24-well culture plate at a concentration of 1×10

[0072] Antioxidant test: the surface functionalized microparticulate acellular porcine dermal matrix was mixed with 0.1 mmol / L DPPH (PHYGENE) solution at a ratio of 1g:5mL, and reacted at room temperature for 30min in the dark. The absorbance of the supernatant at 517nm was measured, and the antioxidant capacity was calculated according to the instructions. Mechanical property test: the test was performed according to the method specified in GB / T 528-2009 "Tensile stress-strain properties of vulcanized rubber".

[0073] Table 1 As can be seen from Figure 3 , the treatment method in Example 1 can effectively remove DNA residues and has low immunogenicity. As shown in Figure 4 , the fat content in the porcine dermal tissue in Example 1 can be effectively controlled below 1%, and the fat material in the porcine dermal tissue can be effectively removed. As can be seen from Figure 5 , compared with Example 2, the antioxidant capacity of the microparticulate acellular dermal matrix after functionalization treatment in Example 1 is significantly enhanced, which can reduce the oxidative stress level of chronic diabetic wounds and is beneficial to wound healing. As can be seen from Figure 6 , compared with Example 2, the expression of inflammatory factor (TNF-α) of the microparticulate acellular dermal matrix after functionalization modification in Example 1 is significantly reduced under the stimulation of lipopolysaccharide (LPS), and the anti-inflammatory capacity is significantly enhanced. As Figure 7 shows, compared with the blank control group and the microparticulate acellular dermal matrix group, the microparticulate acellular dermal matrix after functionalization modification can significantly promote the expression of VEGF of endothelial cells, improve the pro-angiogenic capacity of the material, and help to promote wound healing. As Figure 8 shows, the cell survival rate of the artificial dermis prepared in Example 1 has no significant difference from the control group, and has no cytotoxicity.

[0074] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing an artificial dermal material, characterized by, The preparation method comprises the following steps: (1) treating porcine dermal tissue with high-concentration lye to obtain porous dermal tissue; (2) sequentially performing defatting treatment, drying treatment and cutting treatment on the porous dermal tissue to obtain porous acellular dermal matrix; (3) mixing microparticulate porcine dermal matrix, gelatin and honey in water to obtain a composite gluey liquid; wherein the microparticulate porcine dermal matrix is obtained by grinding porcine dermal tissue after pretreatment; (4) laying the composite gluey liquid on one side of the porous acellular dermal matrix, and then sequentially performing freezing fixation and vacuum drying to obtain the artificial dermal material.

2. The production method according to claim 1, characterized by, In step (1), the thickness of the porcine dermal tissue is 0.2-2 mm, and the high-concentration lye is a sodium hydroxide solution with a concentration of 2-4 mol / L; and / or The porcine dermal tissue is immersed in the high-concentration lye for oscillation treatment; wherein the oscillation time is 1-4 h, and the rotation speed is 60-120 rpm / min.

3. The preparation method according to claim 1, characterized in that, In step (2), the defatting solution used in the defatting treatment is at least one of isopropyl alcohol, methanol, ethanol or acetone; Preferably, the concentration of the defatting solution is 50-100 wt%, and the defatting treatment is performed under ultrasonic conditions for 0.5-2 h.

4. The method of claim 1, wherein, In step (2), the drying treatment comprises freeze-drying treatment and vacuum drying treatment; wherein the temperature of the freeze-drying treatment is -20--80 ℃, and the time is 1-4 h; the temperature of the vacuum drying treatment is -10--50 ℃, and the time is 24-72 h; The thickness of the porous acellular dermal matrix is 0.05-0.5 mm; Preferably, after the oscillation treatment and the defatting treatment, a step of cleaning with physiological saline or PBS buffer is further included.

5. The preparation method according to claim 1, characterized in that, In step (3), the pretreatment comprises sequentially performing immunogen removal and defatting treatment by immersing the porcine dermal tissue in lye and defatting solution, and then sequentially performing cutting, liquid nitrogen freezing and grinding to obtain the microparticulate porcine dermal matrix; preferably, the particle size of the microparticulate porcine dermal matrix is less than 300 microns.

6. The method of claim 1, wherein, In step (3), in terms of mass percentage, the content of the microparticulate porcine dermal matrix in the composite gluey liquid is 1-5%, the content of gelatin is 5-10%, the content of honey is 5-10%, and the content of water is 75-89%.

7. The preparation method according to claim 1, characterized in that, In step (3), after obtaining the microparticulate porcine dermal matrix, a step of sequentially performing functional modification on the microparticulate porcine dermal matrix with a first modification solution and a second modification solution is further included; The first modification solution is a methanol solution containing asiaticoside; preferably, the concentration of asiaticoside in the first modification solution is 100-500 µg / mL; Preferably, the second modification solution is a mixed solution of a tannic acid solution and a metal ion solution; wherein the metal ion solution is one or more of a magnesium ion solution, a zinc ion solution or a copper ion solution; The concentration of the tannic acid solution is 10 mmol / L, and the concentration of the metal ion solution is 0.1-30 mmol / L.

8. The preparation method according to claim 7, characterized in that, The mass-volume ratio of the micro-particle porcine dermal matrix to the first modified solution is 1:(2-5); The mass-volume ratio of the micro-particle porcine dermal matrix to the tannin acid solution is 1:(5-10), and the volume ratio of the tannin acid solution to the metal ion solution is 1:1; The temperature of the mixing reaction is 25-30℃, and the time is 20-24h; Preferably, after the reaction is completed, the reaction product is further subjected to the steps of freeze-fixing and vacuum drying in sequence.

9. The production method according to claim 1 or 8, characterized by, In step (4), the laying thickness of the composite adhesive liquid is 1-5mm; and / or The temperature of the freeze-fixing is -20~-80℃, and the time is 1~4h, and the temperature of the vacuum drying is -10~-50℃, and the time is 24~72h.

10. An artificial dermal material, characterized by, The preparation method is prepared by any one of claims 1-9.

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