Preparation method and application of bionic skin dressing based on sipunculus nudus body wall

Abstract

The invention relates to the technical field of biological medicine, and discloses a preparation method and application of a sipunculus nudus body wall bionic skin dressing. Sipunculus nudus is selected as a matrix of the bionic skin dressing, and the bionic skin dressing is prepared by treating and processing the Sipunculus nudus. The bionic skin dressing is formed by regularly staggering protein fibers, has a uniform porous structure, increases the contact surface area of the dressing and a wound, and has relatively strong mechanical properties; compared with a traditional wound dressing, the wound dressing has high air permeability, the temperature of a wound can be reduced through evaporation of water, and the pain feeling is reduced; meanwhile, good adhesiveness, oxidation resistance and other functions are achieved, and rapid healing of wounds can be promoted; the cell toxicity is relatively low, and the biocompatibility is good; the hemolysis rate of the national safety standard is met, blood can be rapidly enriched to form blood coagulation fulcrums, and the method has good application prospects in the fields of skin tissue repair, skin regeneration, skin organs and the like.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to a preparation method and application of a bionic skin dressing based on the body wall of Siegespermum truncatum. Background Art

[0002] According to incomplete statistics from the World Health Organization (WHO), millions of people around the world suffer skin trauma every year due to cuts, abrasions, wear, tear, punctures and other reasons, and need medication and dressing treatment. In the treatment of large-area trauma, skin transplantation is one of the options, but the source of skin transplant donors is limited and the skin function will be damaged after transplantation. Therefore, post-traumatic treatment is an urgent problem that needs to be solved.

[0003] The skin is not only the largest organ in the human body, protecting the internal tissues and organs from external physical factors, but also the first line of defense of the human immune system. It can secrete some antibacterial substances to form a natural antibacterial environment, reducing the chance of pathogens colonizing and infecting the skin surface. However, the skin is relatively fragile and easily damaged. After the skin is damaged, external pathogens can easily invade the human body and cause local inflammatory reactions, such as redness, swelling, pain, fever, etc. If the wound infection is not controlled in time, the infection will spread to deeper tissues and organs, causing more serious diseases. Wound healing is a natural physiological reaction of the human body, which is usually divided into four main stages: coagulation and inflammation, new tissue formation, epithelial cell regeneration, and scar formation. The entire healing process may take several weeks or months. A healthy body and proper treatment will increase the speed of wound healing and reduce the occurrence of complications.

[0004] Dressing is a material that covers the wound surface to protect the wound and promote repair. It can reduce the secondary impact of physical stimulation on the wound, absorb blood and exudate from the wound to keep the wound and surrounding tissues dry and clean, reduce the risk of wound infection, promote wound healing, provide comfort and protection, and reduce pain and discomfort. The existing wound dressings are mainly used for bandaging and covering wounds. In the existing related literature patents, the modified synthesis method is usually used to improve the biocompatibility of the material or increase the rate at which the dressing promotes wound healing. The generally used form is hydrogel (CN115869459B) or fiber material (CN106120327B) and the like. Hydrogel dressings or fiber materials are mostly made of natural polymer materials, which are interconnected by hydrogen bonding, ionic crosslinking, covalent crosslinking, etc. to form a three-dimensional network structure, and have poor biocompatibility; many materials do not contain active ingredients and have weak interactions with organisms; the structure and biological properties of wound dressings synthesized by polymer materials are different from those of human skin, and the guiding effect on skin repair is weak; and the preparation process of wound dressings such as hydrogels and fiber materials is complicated, some new dressings have special methods of use, and the choice of materials may cause allergies or discomfort, etc. Therefore, it is of great significance to construct a bionic skin dressing with a natural three-dimensional network structure, high biocompatibility, high similarity to the skin structure, and active ingredients.

[0005] Siegeworms, also known as naked siegeworms, commonly known as "sandworms" and "sand intestines", belong to the phylum Siegeworms, class Siegeworms, order Siegeworms, and are marine invertebrates. They are one of the biological resources with important value in marine fishery resources. The nutrients of siegeworms include polysaccharides, peptides, proteins, minerals, fatty acids and trace elements, etc., and have biological activities such as antioxidant, blood pressure lowering, immune regulation, antibacterial and healing promotion, and bone formation promotion. Siegeworms contain more than 80% protein, which plays an important role in muscle and tissue growth and repair.

[0006] The body wall of the star worm is formed by regular interlacing of protein fiber filaments, has a uniform porous mesh structure, has strong mechanical properties, and has similar functions to mammalian skin; the uniform porous structure makes it have high air permeability, can reduce the wound temperature through the evaporation of water, and reduce pain; at the same time, it has good adhesion, antioxidant and other functions, which can promote rapid wound healing; it has low cytotoxicity and good biocompatibility; and it has a hemolysis rate that meets national safety standards, and can quickly enrich blood to form a coagulation fulcrum; the natural three-dimensional mesh structure can make cells well integrated with the body wall and promote cell growth; it has good application prospects in the fields of skin tissue repair, skin regeneration, and skin organoid construction. The present invention combines the good biological properties of the star worm body wall to prepare the star worm body wall bionic skin dressing, and there is currently no wound dressing prepared based on this raw material. Summary of the invention

[0007] The purpose of the present invention is to provide a wound dressing based on the bionic skin dressing of the body wall of Siegespilot, based on the special spatial structure and bioactive substances of the natural body wall of marine organisms, so as to solve the problems in the skin repair process. In view of the shortcomings of current wound dressings such as poor biocompatibility, weak interaction with organisms, and poor guiding repair effect, the present invention provides a bionic skin dressing of the body wall of Siegespilot, which solves the problems of traditional dressings such as complex three-dimensional structure construction process, poor air permeability, lack of bioactive ingredients, certain cytotoxicity, and cumbersome use methods.

[0008] The present invention utilizes the advantages of the natural three-dimensional network structure of the body wall of the Siegespilot, good adhesion, good air permeability, good ductility, good water absorption and swelling and degradability, good hemolysis and coagulation, good biocompatibility, good cell adhesion, and promotion of cell growth, and adopts methods such as activated carbon depigmentation, bioenzyme degreasing, ultrasonic endotoxin removal, and supercritical carbon dioxide drying to prepare the Siegespilot body wall bionic skin dressing. It has broad application prospects in the research fields of promoting wound healing, medical anti-inflammatory dressings, cell culture scaffolds, skin tissue regeneration and repair, and skin organoid construction.

[0009] In order to achieve these purposes and other advantages of the present invention, a method for preparing a Sipunculus body wall bionic skin dressing is provided, comprising the following steps:

[0010] S1. Wash the dried starfish with deionized water to remove surface impurities; soak the treated sample in deionized water to allow the dried starfish to fully absorb water and swell;

[0011] S2. Remove the surface fibers of the sample processed in S1 to obtain a flat and smooth material;

[0012] S3. Remove the residual pigments in the body wall of the sample treated in S2 by using activated carbon depigmentation method;

[0013] S4. The sample treated in S3 is degreased by enzyme degreasing method to remove lipids from the body wall;

[0014] S5. The sample treated in S4 is subjected to ultrasonic endotoxin removal to remove residual endotoxin in the body wall;

[0015] S6. Wash the sample treated in S5 with deionized water and PBS; fix the sample with glutaraldehyde;

[0016] S7. Dry the sample processed in S6 by supercritical carbon dioxide drying method; obtain the material for use, and name it as Siemprel body wall bionic skin dressing.

[0017] Optionally, the specific steps of step S1 are: washing the dried starfish purchased from Beihai, Guangxi with deionized water for 2 to 3 times until the solution is colorless and transparent; soaking the washed starfish in deionized water for 30 minutes, changing the water, and repeating 2 to 3 times.

[0018] Optionally, the specific steps of step S2 are: fully soak the Sipunculus body wall material, remove the protruding fibers on the surface with the help of a scraper or other tools, and soak in 0.5% to 2% papain for 2 to 6 hours until the Sipunculus dressing has a thickness of 0.3 to 0.5 mm.

[0019] Optionally, the specific steps of step S3 are: prepare 0.3-0.5g of activated carbon powder, put 10g of solid tissue containing pigment into a suitable container, add an appropriate amount of deionized water to make a solution. Add the prepared activated carbon powder, stir evenly, stir at a moderate speed of about 30-50 revolutions per minute, and continue stirring for 30-60 minutes to allow the activated carbon to fully contact the pigment. After the stirring is completed, use filter paper to filter, separate the body wall and activated carbon, and you can get the body wall of the siphonophore with reduced pigment.

[0020] Optionally, the specific steps of step S4 are: prepare a lipase buffer (phosphate buffer, pH=7.4) into an enzyme solution with a concentration of 0.1-1%. Place the treated Sipunculus body wall in a container and add the enzyme solution, the volume of which is generally 2-4 times the volume of the sample. Then react at 37°C while stirring appropriately at a stirring speed of 80-150 rpm and a reaction time of 0.5-2 hours. After the reaction is completed, separate the liquid portion from the body wall by filtration to obtain a defatted sample.

[0021] Optionally, the specific steps of step S5 are: placing the body wall of Sipunculus spheniscus in a container containing phosphate buffer and placing it in an ultrasonic cleaner. Setting the ultrasonic power to 200-220W and the treatment time to 15-30 minutes, so that part of the endotoxin is separated from the body wall and enters the buffer, and then removing the liquid containing endotoxin by replacing the buffer.

[0022] Optionally, the specific steps of step S6 are: the treated sample is rinsed with 0.1-0.2M PBS with a pH of 7.4, and the process is repeated 2-3 times. The rinsed material is sterilized under ultraviolet light for 1-3 hours on both sides.

[0023] Optionally, the specific steps of step S7 are: soak the sterilized Siegespidae body wall in a 2-5% glutaraldehyde solution and fix it at 4°C for 4-6 hours. Transfer the fixed Siegespidae body wall to ethanol and soak it for 2-4 hours to ensure that the organic solvent inside the sample is fully replaced. Perform supercritical carbon dioxide drying, then take out the sample to obtain the Siegespidae body wall bionic skin dressing.

[0024] Another object of the present invention is to protect the Sipunculus body wall bionic skin dressing prepared by the above method.

[0025] The third purpose of the present invention is to request protection for the application of the above-mentioned Siempuri body wall bionic skin dressing, especially in the preparation of wound dressings, promotion of skin tissue repair and regeneration, organoids and other materials.

[0026] In the technical solution of the present invention, a bionic skin dressing of the body wall of the star worm is proposed, comprising the body wall of the star worm and the extract contained therein. The body wall of the star worm has the advantages of a natural three-dimensional network structure, good adhesion, good air permeability, good ductility, good water absorption and swelling and degradability, good hemolytic and coagulation properties, good biocompatibility, good cell adhesion, supporting three-dimensional cell culture, promoting cell growth, anti-inflammatory and promoting healing, etc.

[0027] Compared with the prior art, the present invention has the following beneficial effects: The bionic skin dressing based on the body wall of Siempuri spp. prepared by the present invention is formed by regularly interlaced protein fiber filaments and has a uniform porous structure. It increases the contact surface area between the dressing and the wound and has strong mechanical properties. Compared with traditional wound dressings, the wound dressing prepared by the present invention has higher air permeability, can reduce the wound temperature by evaporation of water, and reduce pain. It also has good adhesion, antioxidant and other functions, and can promote rapid wound healing. It has lower cytotoxicity and good biocompatibility. It also meets the national safety standard for hemolysis rate, can quickly enrich blood to form a coagulation fulcrum, and has good application prospects in the fields of skin tissue repair, skin regeneration, skin organoids, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the described drawings are only some embodiments recorded in the invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0029] Figure 1 These are optical and scanning electron microscope images of the Sipunculus body wall bionic skin dressing of the embodiment;

[0030] Figure 2 This is a graph of the air permeability of the bionic skin dressing of the Sipunculus body wall in the embodiment;

[0031] Figure 3 This is a graph of the antioxidant activity of the Sipunculus body wall bionic skin dressing obtained in the embodiment;

[0032] Figure 4 is a blood compatibility diagram obtained from the bionic skin dressing of the Sipunculus body wall in the embodiment;

[0033] Figure 5 This is a coagulability diagram obtained from the bionic skin dressing of the Sipunculus body wall in the embodiment;

[0034] Figure 6 Schematic diagram of the effect of the bionic skin dressing of Siempuri body wall on the activity of RAW264.7 cells in the embodiment;

[0035] Figure 7 This is the growth of RAW264.7 cells in Example 1 on the Sipunculus body wall bionic skin dressing.

[0036] The purpose and features of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The drugs and reagents used in the examples include: Sipuncula spp. (purchased from Beihai, Guangxi); ABTS; potassium persulfate; DPPH; anhydrous ethanol; ferrous sulfate; salicylic acid; 30% H2O2; MTT; DMSO; sodium dihydrogen phosphate; disodium hydrogen phosphate; sodium hydroxide; anhydrous calcium chloride; DMEM; fetal bovine serum; double antibody; 25% glutaraldehyde; activated carbon powder; lipase; deionized water, etc.

[0039] The test equipment used in the examples is detailed as follows:

[0040] In order to enable those skilled in the art to better understand the inventive technology of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0041] like Figure 1-7 As shown, an embodiment of the present invention provides a technical solution: a bionic skin dressing of the body wall of a siphonophore, which is formed by regularly interlacing protein fibers, has a uniform porous structure, has high air permeability, has good adhesion, anti-oxidation and other functions, has low cytotoxicity, good biocompatibility; and has a hemolysis rate that meets national safety standards, etc.

[0042] A method for preparing a Sipunculus body wall bionic skin dressing comprises the following steps:

[0043] S1. Wash the Sipunculus with deionized water to remove surface impurities; soak the treated sample in deionized water to allow the Sipunculus to dry and swell;

[0044] S2. Remove the surface fibers of the sample processed in S1 to obtain a flat and smooth material;

[0045] S3. Remove the residual pigments in the body wall of the sample treated in S2 by using activated carbon depigmentation method;

[0046] S4. The sample treated in S3 is degreased by enzyme degreasing method to remove lipids from the body wall;

[0047] S5. The sample treated in S4 is subjected to ultrasonic endotoxin removal to remove residual endotoxin in the body wall;

[0048] S6. Wash the sample treated in S5 with deionized water and PBS; fix the sample with glutaraldehyde;

[0049] S7. Dry the sample processed in S6 by supercritical carbon dioxide drying method; obtain the material for use, and name it as Siemprel body wall bionic skin dressing.

[0050] Example 1

[0051] Preparation of bionic skin dressing of Sipunculus sphenotype

[0052] The star worms purchased from Beihai, Guangxi were washed with deionized water for 2 to 3 times until the solution was colorless and transparent; the washed star worms were soaked in deionized water for 30 minutes, the water was changed, and repeated 2 to 3 times. After fully soaking, the protruding fibers on the surface of the star worm body wall material were removed with the help of a scraper or other tools until the thickness of the star worm dressing was 0.5 mm. Prepare 0.3 to 0.5 g of activated carbon powder, put 10 g of solid tissue containing pigment into a suitable container, add an appropriate amount of deionized water to make a solution. Add the prepared activated carbon powder to it, stir evenly, stir at a moderate speed of about 30-50 revolutions per minute, and continue stirring for 30 to 60 minutes to allow the activated carbon to fully contact the pigment. After the stirring is completed, use filter paper to filter and separate the body wall and activated carbon to obtain the star worm body wall with reduced pigment. Lipase is prepared into a 0.5% concentration enzyme solution with a buffer solution (phosphate buffer, pH = 7.4). Place the treated body wall of the star worm in a container and add enzyme solution. The volume of the enzyme solution is generally 3 times the volume of the sample. Then react at 37°C while stirring appropriately. The stirring speed is 100 rpm and the reaction time is between 1 hour. After the reaction is completed, the liquid part is separated from the body wall by filtration to obtain a defatted sample. Place the body wall of the star worm in a container containing phosphate buffer and put it into an ultrasonic cleaner. Set the ultrasonic power to 220W and the treatment time to 20 minutes to allow part of the endotoxin to detach from the body wall and enter the buffer. Then remove the liquid containing endotoxin by replacing the buffer. Rinse the treated sample with 0.1M PBS with a pH of 7.4, repeat 2 to 3 times, and sterilize the rinsed material under ultraviolet light for 1 hour on both sides. Soak the sterilized body wall of the star worm in 2.5% glutaraldehyde solution and fix it for several hours at 4°C for 4 hours. The fixed Siegespidae body wall was transferred to ethanol and soaked for 2 hours to ensure that the organic solvent inside the sample was fully replaced. The sample was dried with supercritical carbon dioxide and then taken out to obtain the Siegespidae body wall bionic skin dressing.

[0053] Example 2

[0054] Observation of the bionic skin dressing of Sipunculus sphenodontus under optical microscope and scanning electron microscope

[0055] The prepared Sipunculus body wall bionic skin dressing was observed under an optical microscope; the dried sample was sprayed with gold and photographed under a scanning electron microscope. The results are shown in the attached figure. Figure 1 As shown (Figure A shows the body wall morphology of Siegespilot under optical microscope; Figure B shows the body wall morphology of Siegespilot under scanning electron microscope at 45×).

[0056] Effect verification: refer to the attached Figure 1 , Example 2 can be seen to have a clear and uniform fiber network structure under both an optical microscope and a scanning electron microscope, and the structure is complete.

[0057] Example 3

[0058] Breathability of Sipunculus body wall bionic skin dressing

[0059] The water vapor permeability (WVTR) of the bionic skin dressing of the body wall of the star worm was tested according to the pharmaceutical industry standard YY / T047.2-2004 "Test methods for contact wound dressings Part 2: Water vapor permeability of breathable membrane dressings". Add sufficient deionized water to a vial with an inner diameter of 20 mm, cover the tube mouth with the prepared bionic skin dressing of the body wall of the star worm, leave some air in the bottle (5±1 mm), and seal the tube mouth wall and the bionic skin dressing of the body wall of the star worm with a sealing film, weigh it, and record the weight as W1. Then place the entire sample in a constant temperature incubator and weigh it again after 24 hours, which is recorded as W2. Repeat three times for each group of samples and take the average value. The results are as follows Figure 2 The calculation formula is as follows:

[0060] WVTR (g·m -2 ·d -1 )=24×(W1-W2) / A / tA is the area of ​​the centrifugal tube mouth, m 2 ; t represents the time interval, h; W1 and W2 are the initial weight of the sample and the weight of the sample after being placed in the incubator for 24 h, respectively.

[0061] Effect verification: refer to the attached Figure 2 Compared with traditional medical gauze, the bionic skin dressing of the star worm body wall has better air permeability, which can ensure that the wound temperature is lowered through the evaporation of water and reduce pain.

[0062] Example 4

[0063] Antioxidant activity of the bionic skin dressing made from the body wall of Sipunculus truncatula

[0064] The prepared Sipuncula body wall bionic skin dressing was treated to obtain Sipuncula wound dressing extract, i.e., liquid state. Then, ABTS free radical scavenging activity, DPPH free radical scavenging activity, and hydroxyl free radical scavenging activity were tested. The results are as follows: Figure 3 shown.

[0065] ABTS free radical scavenging activity: First, prepare 7mmol / L ABTS solution and 2.457mmol / L potassium persulfate solution, mix them in a volume ratio of 1:1, and react for 48 hours in a dark and low temperature environment to form an ABTS free radical solution. Dilute it with anhydrous ethanol to an absorbance of 0.700±0.02 at 734nm before use. Then mix the extract of the Siegespitella wound dressing with the ABTS solution, and set up a control blank group at the same time, and replace the extract of the Siegespitella body wall bionic skin dressing with an equal amount of deionized water. After mixing, place it in the dark to react for 6 minutes, and then measure and record the absorbance at a wavelength of 734nm to analyze the scavenging ability of the Siegespitella body wall bionic skin dressing on ABTS free radicals. The calculation formula is as follows:

[0066] ABTS scavenging capacity (%) = (A0-A1) / A0×100 A0: absorbance of blank group, A1: absorbance of Siempuri body wall biomimetic skin dressing extract treated group

[0067] DPPH free radical scavenging activity: DPPH was dissolved in anhydrous ethanol to prepare a standard solution of 0.04 mg / mL. Subsequently, the extract of the bionic skin dressing of the body wall of the star worm was mixed with the DPPH solution to form a reaction system; at the same time, a control group was set up, and the extract of the bionic skin dressing of the body wall of the star worm was replaced with an equal amount of anhydrous ethanol. In addition, a blank group was set up to correct the absorbance value, and deionized water and DPPH solution were mixed in a 1:1 ratio. After mixing, the reaction was allowed to stand for 30 minutes under light-proof conditions. Finally, the absorbance values ​​of each group of samples were measured at a wavelength of 517 nm to analyze the scavenging ability of the bionic skin dressing of the body wall of the star worm for DPPH free radicals. The calculation formula is as follows:

[0068] DPPH scavenging ability (%) = (1-(A1-A2) / A0) × 100 A0: blank group absorbance, A1: siphoned body wall biomimetic skin dressing extract group absorbance, A2: control group absorbance

[0069] Hydroxyl radical scavenging activity: FeSO4 was dissolved in deionized water to prepare a 2mmol / L solution, salicylic acid was dissolved in deionized water to prepare a 6mmol / L solution, and H2O2 was diluted to a 1mmol / L solution. Take 3mL 2mmol / LFeSO4 solution, 1mL 1mmol / LH2O2 solution and 3mL 6mmol / L salicylic acid solution, and then add the extract of the biomimetic skin dressing of the body wall of the star worm to mix. The control group used an equal volume of deionized water instead of the extract of the wound dressing of the star worm to perform the same steps. The blank group was designed to eliminate the influence of the extract of the biomimetic skin dressing of the body wall of the star worm itself on the absorbance. All mixed samples were placed in a constant temperature water bath at 37℃ for 15min, and then the absorbance of each group was measured at a wavelength of 510nm to analyze the scavenging ability of the biomimetic skin dressing of the body wall of the star worm on hydroxyl radicals. The calculation formula is as follows:

[0070] Hydroxyl radical scavenging ability (%) = (1-(A1-A2) / A0) × 100 A0: blank group absorbance, A1: siphoned body wall biomimetic skin dressing extract group absorbance, A2: control group absorbance

[0071] Effect verification: refer to the attached Figure 3 The bionic skin dressing of the body wall of Sienna has good antioxidant properties and can scavenge oxygen free radicals, which also proves that the bionic skin dressing of the body wall of Sienna has a certain effect in anti-inflammation.

[0072] Example 5

[0073] In vitro antibacterial activity of Sipunculus truncatula biomimetic skin dressing Escherichia coli and Staphylococcus aureus were activated in LB liquid medium. The activated bacterial solution was diluted with sterile saline to an OD value of 0.1 at 600nm. 50μL of saline was added to a 96-well plate as a blank control and 50μL of 50mg·mL -1 The extract of the Sipunculus body wall biomimetic skin dressing was used as the sample group. 50 μL LB medium and 50 μL diluted bacterial solution were added to each group, mixed, and the absorbance was measured at 600 nm, recorded as the 0h OD value, and then placed in a 37℃ incubator for 8h and 24h to measure the OD value. The results are shown in Figure 4 As shown. Calculate the inhibition rate respectively:

[0074] Antibacterial rate (%) = ((A N空 -A 0空 )-(A N样 -A 0样 )) / A 0空 ×100 AN空 is the OD value of the blank control after N h, A 0空 is the OD value of the blank control at 0h, A N样 is the OD value of the Sipunculus wound dressing group after Nh, A 0样 is the OD value of the Sipunculus wound dressing group at 0 h, where N = 8, 24, unit: h

[0075] Effect verification: Referring to Appendix 1, the Siegesporium body wall bionic skin dressing has an inhibitory effect on both Escherichia coli and Staphylococcus aureus, and has a certain long-lasting effect on Escherichia coli.

[0076] Table 1 shows the in vitro antibacterial activity of the Sipunculus body wall bionic skin dressing obtained in Example; “-” means no antibacterial effect

[0077] Example 6

[0078] Blood compatibility of Sipunculus sphenotype bionic skin dressing

[0079] Take fresh blood, centrifuge at 7000rpm for 5min, and collect red blood cells. Wash the red blood cells with PBS and prepare a 20% red blood cell suspension. Then add distilled water, PBS, and Siempuri biomimetic skin dressing, and gently mix. Incubate at 37℃ for 4h, centrifuge at 7000rpm for 5min, collect the supernatant, and use an enzyme marker to detect the absorbance of the solution at 540nm. The results are as follows: Figure 4 Calculate the hemolysis rate:

[0080] Hemolysis rate (%) = (A2-A0) / (A1-A0) × 100 A0 is the absorbance after adding PBS; A1 is the absorbance after adding distilled water; A2 is the absorbance after adding Sipuncula wound dressing extract

[0081] Effect verification: refer to the attached Figure 4 After the red blood cells were incubated with deionized water for 1 hour, the sample solution turned red and hemolysis occurred in the red blood cells; after the red blood cells were incubated with PBS for 1 hour, the sample supernatant turned light yellow, indicating that there was no hemolysis; the supernatant of the Sipunculus body wall bionic skin dressing was lighter in color, indicating that a small amount of hemolysis occurred, but the hemolysis rate was lower than the critical safety range of the hemolysis rate of 5%.

[0082] Example 7

[0083] Coagulation properties of the Sipunculus body wall bionic skin dressing The bionic skin dressing of the Sipunculus body wall was cut into discs with a diameter of 14 mm, placed in a six-well plate and incubated at 37°C for 10 minutes. Then, 100 μL of blood was dripped on the sample, followed by 20 μL of 0.2 mol / L CaCl2 solution. The six-well plate containing the sample and blood was incubated at 37°C for 20 minutes. After incubation, 10 mL of deionized water was added to the six-well plate and incubated at 37°C for another 5 minutes. No sample was placed in the blank control group, and the other experimental conditions were the same. Finally, the absorbance value of the sample solution at 540 nm was tested using an enzyme marker, and the results are as follows: Figure 5 As shown. Calculate the blood coagulation index (BCI) based on the absorbance value of the sample:

[0084] BIC(%)=Is / Iw×100 Is is the absorbance value of the experimental sample group; Iw is the absorbance value of the blank control sample group

[0085] Effect verification: refer to the attached Figure 5 After adding blood to the sample, the blood was completely absorbed by the sample, indicating that the Siempuri body wall bionic skin dressing has good coagulation properties.

[0086] Example 8

[0087] Effects of Sipunculus sphenotype bionic skin dressing on the proliferation activity of RAW264.7 cells

[0088] The prepared Sipunculus body wall biomimetic skin dressing was irradiated under ultraviolet light for 1 hour, soaked in complete culture medium for 24 hours, and then transferred to a 6-well plate. RAW264.7 cells were inoculated in DMEM high-glucose medium containing 10% FBS and 1% double antibody, and cultured in a cell culture incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were taken, and RAW264.7 cells were inoculated in a 6-well plate after counting with a cell counting plate, so that the number of cells in each well was about 5×10 3 The sample group was set up with 3 parallel wells, and the blank control group was also set up with 3 parallel wells. Incubate in the incubator for 24 hours, add MTT dye solution (MTT final concentration is 0.5 mg / mL) and continue to culture for 4 hours, discard the supernatant, add 150 μL DMSO solution to each well, shake on a shaker at 37°C for 15 minutes, take the supernatant and add it to a 96-well plate, and use an enzyme reader to detect the absorbance at 570 nm. The results are as follows Figure 6 As shown. Calculation of RAW264.7 cell proliferation activity:

[0089] Cell proliferation activity (%) = (A 样品 -A 空白 ) / (A 对照 -A 空白 )×100

[0090] Effect verification: refer to the attached Figure 6 The cytotoxicity was evaluated by detecting the cell viability of RAW264.7 cells after being cultured with the bionic skin dressing of the Siegespinus body wall. The results showed that the bionic skin dressing of the Siegespinus body wall had good cell compatibility and the relative activity value of the sample was much higher than that of the control group.

[0091] Example 9

[0092] Growth of RAW264.7 cells on the Sipunculus body wall biomimetic skin dressing

[0093] The structure of RAW264.7 cells growing on the bionic skin dressing of the Sipunculus body wall was observed using a scanning electron microscope. Before scanning electron microscopy observation, the cells and dressings were pretreated. The cells were washed 1 to 2 times with PBS, then fixed with 2.5% glutaraldehyde at room temperature for 1 hour, washed 2 to 3 times with deionized water, and dehydrated step by step with 40%, 50%, 60%, 70%, 80%, 90%, and 100% ethanol for 10 minutes each time, dried in a vacuum freeze dryer, and observed by gold spraying. The results are shown in Figure 7 shown.

[0094] Effect verification: refer to the attached Figure 7 The cells adhered and grew on the bionic skin dressing of the Siegespinus body wall in a three-dimensional spherical shape, maintaining the stability of cell morphology and function. This further shows that the three-dimensional structure of the bionic skin dressing of the Siegespinus body wall is conducive to cell adhesion and growth.

[0095] In summary, the bionic skin dressing of the body wall of the star worm we announced has the advantages of natural three-dimensional network structure, good adhesion, good air permeability, good ductility, good water absorption and swelling and degradability, good hemolysis and coagulation, good biocompatibility, good cell adhesion, support for three-dimensional cell culture, promotion of cell growth, anti-inflammatory and healing. Specifically, the bionic skin dressing of the body wall of the star worm in this invention is formed by regular interlacing of protein fiber filaments, has a uniform porous structure, increases the contact surface area between the dressing and the wound, and has strong mechanical properties; compared with traditional wound dressings, the wound dressing prepared by the present invention has higher air permeability, can reduce the temperature of the wound by evaporation of water, and reduce pain; at the same time, it has good adhesion, anti-oxidation and other functions, can promote rapid wound healing; has low cytotoxicity and good biocompatibility; and has a hemolysis rate that meets national safety standards, can quickly enrich blood to form a coagulation fulcrum, and has good application prospects in the fields of skin tissue repair, skin regeneration, and skin organoids.

[0096] The above shows and describes the basic technology and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above embodiments, and can be implemented in other specific forms without departing from the spirit or basic features of the invention. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any attached figure mark in the claims should not be regarded as limiting the claims involved.

[0097] In addition, although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in the embodiments may also be appropriately adjusted to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A bionic skin dressing based on the body wall of Sipunculus spheniscidae, characterized in that: By processing the body wall of Siempuri worms, a body wall structure with regularly interlaced protein fibers and uniform porosity was prepared, resulting in a bionic skin dressing that can promote the repair of skin wounds.

2. The method for preparing a bionic skin dressing based on the body wall of Silurus according to claim 1, characterized in that: The following steps are involved: S1. Wash and remove impurities from the star worms with deionized water; soak the treated samples in deionized water to allow the star worms to fully absorb water and swell; S2. Remove the surface fibers of the sample processed in S1 to obtain a smooth body wall material; S3. Use activated carbon to remove the residual pigments on the body wall of the sample treated in S2; S4. removing the body wall lipids from the sample treated in S3 by a biological enzymatic hydrolysis method; S5. The sample treated in S4 is subjected to ultrasonic endotoxin removal to remove residual endotoxin in the body wall; S6. Fix and cross-link the sample treated in S5 with glutaraldehyde; S7. The sample processed by S6 is dried by supercritical carbon dioxide drying method to obtain a new bionic skin dressing.

3. The method for preparing a bionic skin dressing based on the body wall of Silurus according to claim 2, characterized in that: S1. Wash the Sipunculus worms with deionized water until the solution is colorless and transparent; soak the washed Sipunculus worms in deionized water, change the water, and repeat 2 to 3 times.

4. The method for preparing a bionic skin dressing based on the body wall of Silurus according to claim 2, characterized in that: S2: After the Siegespilot body wall material is fully soaked, the protruding fibers on the surface are removed, and the Siegespilot body wall material is soaked in 0.5% to 2% papain for 2 to 6 hours until the thickness of the Siegespilot dressing is 0.3 to 0.8 mm.

5. The method for preparing a bionic skin dressing based on the Sipunculus body wall according to claim 2, characterized in that: S3: Place 10 g of solid tissue containing pigment into a container, add an appropriate amount of deionized water to make a solution, add 0.3-0.5 g of activated carbon powder to every 10 g of solid tissue containing pigment, stir evenly, and stir at a moderate speed of 30-50 revolutions per minute. Continue stirring for 30-60 minutes to allow the activated carbon to fully contact the pigment. After the stirring is completed, filter to separate the body wall and the activated carbon to obtain the Siegesporium body wall with reduced pigment.

6. The method for preparing a bionic skin dressing based on the body wall of Silurus according to claim 2, characterized in that: S4 prepares lipase into an enzyme solution with a buffer solution of 0.1 to 1%, puts the treated Sipunculus body wall into a container, and adds the enzyme solution, the volume of which is 2 to 4 times the volume of the sample; then reacts at 37°C while stirring, the stirring speed is 80 to 150 rpm, and the reaction time is between 0.5 and 2 hours; after the reaction is completed, separates the liquid part from the body wall by filtering to obtain a defatted sample.

7. The method for preparing a bionic skin dressing based on the Sipunculus body wall according to claim 2, characterized in that: S5 places the body wall of Sipunculus square in a container containing phosphate buffer, puts it into an ultrasonic cleaner, sets the ultrasonic power at 200-220W, and processes for 15-30 minutes, so that part of the endotoxin is separated from the body wall and enters the buffer, and the liquid containing the endotoxin is removed.

8. The preparation method and application of the bionic skin dressing based on the Sipunculus body wall according to claim 2, characterized in that: The S6 treated samples were rinsed with 0.1-0.2M PBS, pH 7.4, and repeated 2-3 times. The rinsed materials were sterilized under ultraviolet light for 1-3 hours on both sides.

9. The preparation method and application of the Sipunculus body wall bionic skin dressing according to claim 2, characterized in that: S7: Soak the sterilized Siegespidae body wall in a 2-5% glutaraldehyde solution and fix it at 4°C for 2-6 hours. Transfer the fixed Siegespidae body wall to ethanol and soak it for 2-4 hours to ensure that the organic solvent inside the sample is fully replaced. Perform supercritical carbon dioxide drying, then take out the sample to obtain the Siegespidae body wall bionic skin dressing.

10. An application of the Sipunculus body wall bionic skin dressing according to claim 1, characterized in that: include: It is used in the preparation of wound dressings, promotion of skin tissue repair and regeneration, and organoid materials.

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