A decellularized matrix dressing preservation solution

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to a kind of acellular matrix dressing storage solution.The first aspect provides an acellular matrix dressing storage solution, which comprises glycerol, 1,2-hexanediol, dipropylene glycol, p-hydroxyacetophenone and water.In some embodiments of the first aspect, the weight percentage of each component is as follows: glycerol 1.5-2.5%;1,2-hexanediol 0.8-1.5%;dipropylene glycol 0.4-1.0%;p-hydroxyacetophenone 0.3-0.6%;and the balance is water.Each component used in the application is a commonly used ingredient in the field of cosmetics and pharmaceuticals, which has low toxicity and small irritation, and will not cause damage to the cell structure and biological activity of the acellular matrix dressing, and is suitable for the preservation of medical acellular matrix dressing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a decellularized matrix dressing preservation solution. Background Technology

[0002] Acellular matrix dressings, as a type of biological dressing, play an important role in the treatment of burns, trauma, and other wounds. They provide protection for the wound, reduce the risk of infection, and promote wound healing. However, acellular matrix dressings are susceptible to microbial contamination and moisture loss during storage, leading to a decline in their bioactivity and efficacy. Currently, while commercially available preservative solutions for acellular matrix dressings offer some preservation effects, some use highly irritating preservatives that may damage the cellular structure and bioactivity of the dressings. Furthermore, some preservative solutions lack sufficient moisturizing properties, failing to maintain the moisture content of the dressings for extended periods, thus affecting their effectiveness. Therefore, developing a preservative solution for acellular matrix dressings with excellent antiseptic, moisturizing, and safety properties is of significant practical importance. Summary of the Invention

[0003] Therefore, based on the above background, the present invention provides a decellularized matrix dressing preservation solution, which overcomes the shortcomings of existing preservation solutions in that they cannot simultaneously achieve dressing flexibility, antibacterial properties and biocompatibility.

[0004] To achieve the above objectives, in a first aspect, a decellularized matrix dressing preservation solution is provided, comprising glycerol, 1,2-hexanediol, dipropylene glycol, p-hydroxyacetophenone, and water.

[0005] In some embodiments of the first aspect, the weight percentages of the components are: glycerol 1.5-2.5%; 1,2-hexanediol 0.8-1.5%; dipropylene glycol 0.4-1.0%; p-hydroxyacetophenone 0.3-0.6%; and the balance is water. Specifically, the content of glycerol may be 1.5%, 2.0%, or 2.5%; the content of 1,2-hexanediol may be 0.8%, 1.0%, 1.2%, or 1.5%; the content of dipropylene glycol may be 0.4%, 0.6%, 0.8%, or 1.0%; and the content of p-hydroxyacetophenone may be 0.3%, 0.4%, 0.5%, or 0.6%.

[0006] In some embodiments of the first aspect, the weight percentages of the components are: glycerol 2%; 1,2-hexanediol 1%; dipropylene glycol 0.6%; p-hydroxyacetophenone 0.4%; and the balance being water.

[0007] In some embodiments of the first aspect, the weight ratio of 1,2-hexanediol to p-hydroxyacetophenone is (2:1) to (3:1); for example.

[0008] In some embodiments of the first aspect, the weight ratio of glycerol to dipropylene glycol is (3:1) to (5:1); for example.

[0009] In some embodiments of the first aspect, the weight ratio of 1,2-hexanediol to p-hydroxyacetophenone is 2.5:1.

[0010] In some embodiments of the first aspect, the weight ratio of glycerol to dipropylene glycol is 3.3:1.

[0011] Secondly, a method for preparing a decellularized matrix dressing preservation solution is provided, comprising the following steps: S1: Weigh out glycerin, 1,2-hexanediol, dipropylene glycol, p-hydroxyacetophenone and water according to the weight percentage; S2: Add glycerol, 1,2-hexanediol, dipropylene glycol and p-hydroxyacetophenone to water in sequence and stir until completely dissolved to obtain the decellularized matrix dressing preservation solution.

[0012] In some embodiments of the second aspect, in S1, 1.5-2.5% glycerol, 0.8-1.5% 1,2-hexanediol, 0.4-1.0% dipropylene glycol, 0.3-0.6% p-hydroxyacetophenone, and the balance being water are weighed by weight.

[0013] Thirdly, an application of a decellularized matrix dressing preservation solution in the preparation of wound repair medical devices is provided.

[0014] In some embodiments of the third aspect, the medical device is a burn dressing, an ulcer repair membrane, or a tissue-engineered scaffold.

[0015] Compared with the prior art, in one embodiment of the present invention, at least one of the following beneficial effects is achieved by organically combining glycerol, 1,2-hexanediol, dipropylene glycol and p-hydroxyacetophenone: 1. Excellent preservative properties: p-Hydroxyacetophenone is a safe and effective preservative that inhibits the growth and reproduction of microorganisms, ensuring that the decellularized matrix dressing is not contaminated by microorganisms during preservation. Simultaneously, 1,2-hexanediol also has a certain antibacterial effect, working synergistically with p-hydroxyacetophenone to enhance the preservative effect of the preservation solution.

[0016] 5) Excellent moisturizing properties: Glycerin and dipropylene glycol are both good moisturizers that can absorb moisture from the air, prevent moisture loss from the fish skin dressing, maintain the moisture state of the decellularized matrix dressing for a long time, and preserve its biological activity.

[0017] 6) High safety: All components used in this invention are commonly used in the cosmetics and pharmaceutical fields. They have low toxicity and low irritation and will not damage the cell structure and biological activity of the decellularized matrix dressing. They are suitable for the preservation of medical decellularized matrix dressings.

[0018] 7) Good stability: The components have good compatibility, and the prepared preservation solution has high stability. No layering or precipitation will occur during storage and use.

[0019] Terminology Definition The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0020] The term "ATCC" refers to the United States Culture Collection. The term "ATCC" and the number that follows it together indicate a strain of microorganism deposited at the United States Culture Collection with that number.

[0021] The term "OECD" refers to the Organization for Economic Cooperation and Development. Attached Figure Description

[0022] Figure 1 Images showing the operation process of Experiment Example 4; Figure 2 This is a photograph taken after the skin irritation experiment in Experiment Example 4; Figure 3 This is a scene of preparing a water bath after the blood dilution for Experiment Example 5 has been prepared. Figure 4 This is a schematic diagram of the venous blood drawing scenario in Experiment Example 5; Figure 5 This is a schematic diagram of blood dilution in Experiment Example 5 (Note: 1mL venous blood + 4mL physiological saline = diluted blood). Figure 6 This is a schematic diagram of the grouping of the whole blood system in Experiment 5 (where, left: positive control 1, middle: negative control 1, right: experimental group 1). Figure 7 This is a schematic diagram of the grouping of the red blood cell suspension system in Experiment 5 (where, left: experimental group 2, middle: negative control 2, right: positive control 2). Figure 8 These are experimental records of the preservation solution and physiological saline in Experiment Example 5; (from left to right, the first photo is before the experiment, the second is the preservation solution, and the third is the physiological saline). Figure 9 The absorbance test record for Experiment 5 is shown below (where Experiment 1 shows the absorbance test results of the whole blood system in Example 1, and Experiment 2 shows the absorbance test results of the red blood cell suspension group in Example 1). Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to its embodiments; it should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0024] [Preservation solution and preparation of decellularized matrix dressing according to specific embodiments of the present invention] Weigh the corresponding substances according to the weight percentages recorded in Table 1; add the above components to purified water to prepare a solution, stir until completely dissolved; filter sterilize using a 0.22 μm filter membrane, dispense and store in the dark to obtain Examples 1-3 and Comparative Examples 1-7. The composition of Examples 1-3 and Comparative Examples 1-7 is shown in Table 1.

[0025] Table 1. Composition of the Examples and Comparative Examples (in weight percentage wt%)

[0026] [Antibacterial performance related experiments] Experiment Example 1: Antibacterial Rate Experiment under Single Bacterial Solution Conditions To demonstrate that the dressing preservation solution of this application can give the preserved dressing a certain degree of antibacterial properties and prevent the dressing from spoiling, this experiment was conducted.

[0027] Tested bacterial strains: Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), and Candida albicans (ATCC 10231).

[0028] Test method: Refer to ISO 20776 "Test of antimicrobial efficacy", the specific steps are as follows: 1) Preparation of bacterial suspension: The test bacterial strains were cultured at 37℃ for 24 hours and adjusted to a McFarland turbidity of 0.5 (approximately 1.5 × 10⁻⁶). 8 (CFU / mL).

[0029] 2) Mixed samples: Take the preservation solution and bacterial suspension of each example or comparative example and mix them at a volume ratio of 1:9, and place them in a sterile test tube.

[0030] 3) Incubation treatment: Incubate the bacterial suspension from step 1) and the mixed sample obtained from step 2) in a 37°C incubator for 24 hours.

[0031] 4) Plate count: Take 100 μL of the mixture, spread it evenly on TSA medium, incubate at 37°C for 24 hours, and then calculate the colony forming units (CFU).

[0032] 5) Antibacterial rate calculation: The antibacterial rate is calculated according to the formula = [(control group CFU - experimental group CFU) / control group CFU] × 100%, where the bacterial suspension in step 1) is the control group and the mixed sample obtained in step 2) is the experimental group.

[0033] The test results are shown in Tables 2-11: Table 2. Antibacterial rate of different bacterial species in Example 1 (in %)

[0034] Table 3. Antibacterial rate of different bacterial species in Example 2 (in %)

[0035] Table 4. Antibacterial rates of different bacterial species in Example 3 (in %)

[0036] Table 5. Antibacterial rates of Comparative Example 1 against different bacterial species (in %)

[0037] Table 6. Antibacterial rates of Comparative Example 2 against different bacterial species (in %)

[0038] Table 7. Antibacterial rates of different bacterial species in Comparative Example 3 (in %)

[0039] Table 8. Antibacterial rates of different bacterial species in Comparative Example 4 (in %)

[0040] Table 9. Antibacterial rates of different bacterial species in Comparative Example 5 (in %)

[0041] Table 10. Antibacterial rates of 6 different bacterial species in Comparative Examples (in %)

[0042] Table 11. Antibacterial rates of different bacterial species in Comparative Example 7 (in %)

[0043] Results Summary: The preservation solutions of Examples 1-3 all exhibited inhibition rates exceeding 95% against Staphylococcus aureus, Escherichia coli, and Candida albicans, indicating significant antibacterial effects. Among them, the inhibition rate against Staphylococcus aureus was the highest (average 99.1%), meeting the requirements of ISO 20776 for highly effective antibacterial agents. The antibacterial rates of Comparative Examples 1-4 were extremely low, far lower than those of Examples 1-3, indicating that low-concentration glycerol alone has no effective antibacterial effect. In Comparative Examples 9-11, some component values ​​were not within the range of "glycerol 1.5%-2.5%, 1,2-hexanediol 0.8%-1.5%, dipropylene glycol 0.4%-1.0%, p-hydroxyacetophenone 0.3%-0.6%". Although their performance was better than that of Comparative Examples 1-4 with single components, it was not as good as that of Examples 1-3, proving that "glycerol 1.5%-2.5%, 1,2-hexanediol 0.8%-1.5%, dipropylene glycol 0.4%-1.0%, p-hydroxyacetophenone 0.3%-0.6%" is the optimal range. In the examples, the composite system of glycerol + 1,2-hexanediol + dipropylene glycol + p-hydroxyacetophenone achieved synergistic antibacterial effects among the components, which is the core of achieving highly efficient antibacterial activity.

[0044] [Safety-related experiments] In the above experiments, the antibacterial properties of Examples 1-3 have been demonstrated through comparison with the comparative examples. Considering that the comparative examples do not possess antibacterial properties comparable to Examples 1-3 and cannot solve the technical problem of this invention, the safety of Examples 1-3 was verified in the following safety-related experiments. The "compound formulation" mentioned below refers to a formulation of "glycerol 1.5%-2.5%, 1,2-hexanediol 0.8%-1.5%, dipropylene glycol 0.4%-1.0%, and p-hydroxyacetophenone 0.3%-0.6%".

[0045] Experiment Example 2: Microbial Safety Testing and Results 1) Purpose of detection To verify the microbial safety of the decellularized matrix dressing preservation solution of this invention (i.e., the dressing preservation solution itself is sterile), the Youkang Biotechnology Broad-Spectrum Microbial Comprehensive Detection Kit (model: UK-Micro-6, specification: 48 tests / kit) was used. This kit enables one-stop detection of total bacterial count, total fungal and yeast count, and specific pathogenic microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium tetani), simplifying the operation process and standardizing testing standards. It provides efficient and accurate detection data for product safety evaluation and industrial production quality control. This kit has passed ISO 13485 quality management system certification and is widely used in medical devices, biological agents, and other fields. It is suitable for detecting preservation solutions containing preservatives, effectively eliminating matrix interference, and the stability and specificity of the test results meet industry standards.

[0046] 2) Testing materials a) Samples to be tested: The prepared decellularized matrix dressing preservation solution (Examples 1-3) was sampled in 10 mL portions. All sampling was performed in a sterile laminar flow hood to avoid external microbial contamination. b) Test kit: Youkang Bio's Broad-Spectrum Microbial Comprehensive Detection Kit (Model: UK-Micro-6, Batch No.: 20240512) includes a complete set of detection components; c) Preprocessing components: Specialized matrix balancing solution (pH 7.2±0.2, which can neutralize the antibacterial components in the preservation solution and eliminate detection interference), and 0.45μm pore size sample purification column (used to filter particulate impurities in the sample and avoid clogging the reaction pores). d) Multi-channel detection board: The 96-well polystyrene well contains 6 independent reaction wells, corresponding to the detection channels for total bacterial count, fungi and yeast, Staphylococcus aureus, Pseudomonas aeruginosa, and Clostridium tetani, respectively. Each channel is pre-coated with a specific reaction substrate. e) Reagents: TTC chromogenic reagent (for total bacterial count detection; live bacteria can be reduced to red formazan), rosin stain (for fungal and yeast detection; colonies turn red), Staphylococcus aureus antibody latex reagent (generates agglutination reaction upon binding with target bacteria), Pseudomonas aeruginosa oxidase substrate (turns blue-green upon contact with Pseudomonas aeruginosa oxidase), Clostridium tetani fluorescent probe (reacts with target bacterial enzymes under anaerobic conditions to produce fluorescence). f) Quality control products: Positive control mixed bacterial suspension (containing Escherichia coli ATCC 25922, Candida albicans ATCC 10231, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Clostridium tetani ATCC 19406, concentration 10) 3 CFU / mL), negative control solution (sterile phosphate buffer, used to exclude interference from the reagents themselves); g) Auxiliary materials: Anaerobic culture bags (containing methylene blue indicator to quickly create an anaerobic environment and ensure normal growth of Clostridium tetani), stop solution (1 mol / L HCl, used to terminate the enzymatic reaction and fix the detection signal), and colony counting grid plates (used for quantitative counting of bacterial and fungal colonies).

[0047] h) Auxiliary instruments: A Level 2 biosafety cabinet (model: BSC-1300IIA2, ensuring aseptic operation), a Youkang Bio-compatible microbial detection incubator (model: UK-360, with anaerobic culture module, temperature control accuracy ±0.5℃), pipettes (0.1-10mL, accuracy ±0.5%, for precise pipetting), a Youkang Bio fluorescence reader (model: UK-FL800, compatible with reagent kit fluorescence detection channel, excitation wavelength 490nm, emission wavelength 520nm), and sterile centrifuge tubes (15mL, polypropylene, centrifugally resistant).

[0048] 3) Detection methods a) Sample pretreatment Inside a biosafety level 2 cabinet, take 5 mL of the preservation solution sample from each example or comparative example, add 5 mL of the matrix equilibration solution provided with the kit, and vortex at 1500 rpm for 1 min to mix thoroughly, thus preparing a 1:2 dilution. Slowly transfer the dilution to a sample purification column, centrifuge at 4000 rpm for 3 min, and collect the supernatant after centrifugation for later use. This step can remove 99% of the antibacterial components and particulate impurities in the preservation solution, avoiding interference with subsequent detection reactions.

[0049] b) Sample addition and incubation i. Take out the multichannel detection plate from the kit and add 0.2 mL of the supernatant to each well corresponding to the total bacterial count, fungi and yeast, Staphylococcus aureus, Pseudomonas aeruginosa, and Clostridium tetani reaction. Perform three parallel replicates for each sample. At the same time, set up a quality control group: add 0.2 mL of positive control mixed bacterial solution to the positive control well and add 0.2 mL of negative control solution to the negative control well. Perform three parallel replicates for each group.

[0050] ii. Perform group culture according to the conditions specified in the kit: iii. Total bacterial count reaction wells: Place the test plate in a constant temperature incubator at 36℃±1℃ and incubate aerobically for 6 hours. Observe every 2 hours during this period to avoid over-incubation and overlapping of colonies. iv. Fungal and yeast reaction wells: Transfer to a constant temperature incubator at 25℃±1℃ and incubate aerobically for 20 hours. This temperature can promote the growth of fungi and yeasts while inhibiting the reproduction of some miscellaneous bacteria. v. Staphylococcus aureus reaction wells: First, incubate in a constant temperature incubator at 36℃±1℃ for 4 hours. After removing the incubator, add 0.05mL of Staphylococcus aureus antibody latex reagent to each well, gently shake to mix, and let stand at room temperature for 30 minutes to wait for the agglutination reaction to occur. vi. Pseudomonas aeruginosa reaction wells: Place in a constant temperature incubator at 36℃±1℃ and culture aerobically for 8 hours to ensure that Pseudomonas aeruginosa fully multiplies and produces sufficient oxidase. vii. Tetanus bacteria reaction wells: Add 0.02 mL of tetanus fluorescent probe to each well, mix gently, place the corresponding area of ​​the detection plate into an anaerobic culture bag, seal the bag opening (the oxygen concentration inside the bag will drop to < 0.1% within 30 min, and the methylene blue indicator will change from blue to colorless, indicating that the anaerobic environment has been successfully constructed), and then incubate in a constant temperature incubator at 37℃±1℃ for 10 h.

[0051] c) Result Interpretation i. Interpretation of total bacterial count: Remove the cultured bacterial count reaction wells and cover them with the colony counting grid plate provided with the kit. Visually count the red colonies (formazan formed by the reduction of TTC by live bacteria is red). Calculate the final result according to "Total bacterial count (CFU / mL) = Colony count × 2" (since the sample has been diluted 1:2). If no red colonies are observed, the colony count is considered to be < 1 CFU / well, corresponding to a total bacterial count < 2 CFU / mL.

[0052] ii. Interpretation of fungi and yeast: Observe the reaction wells for fungi and yeast, count the red colonies (fungal colonies are fluffy, yeast colonies are round), and calculate the total number according to the dilution factor. If there are no red colonies, the total number is judged to be < 2 CFU / mL.

[0053] iii. Interpretation of Staphylococcus aureus: Visually observe whether white agglomerated particles appear in the reaction wells. If obvious agglomerated particles are present, the result is considered positive (detected); if the solution remains homogeneous and transparent without agglomeration, the result is considered negative (not detected).

[0054] iv. Interpretation of Pseudomonas aeruginosa: Observe the color change of the solution in the reaction well. If it changes from colorless to blue-green, it is considered positive (detected); if the solution remains colorless, it is considered negative (not detected).

[0055] v. Tetanus bacteria interpretation: Place the test plate into the Youkang Bio UK-FL800 fluorescence reader, set the excitation wavelength to 490nm and the emission wavelength to 520nm, and detect the fluorescence value of each reaction well; using the fluorescence value of the negative control well as a benchmark, if the fluorescence value of the sample well is ≥3 times the fluorescence value of the negative control, it is judged as positive (detected); if the fluorescence value is <3 times the fluorescence value of the negative control, it is judged as negative (not detected).

[0056] 4) Test results: as shown in Tables 12 and 13.

[0057] Table 12

[0058] Table 13 Comparison with Example 1

[0059] 5) Summary of Results a) Using the Youkang Biotechnology Broad-Spectrum Microbial Comprehensive Detection Kit (UK-Micro-6), the total bacterial count and total fungal and yeast count of the preservation solution of the decellularized matrix dressing of this invention were ≤2 CFU / mL, both far below the requirement of "total bacterial count ≤10" in GB / T19973.1-2015 "Microbiological Methods for Sterilization of Medical Devices Part 1: Estimation of Total Microbial Count on the Product". 3 CFU / mL, total fungal count ≤10 2 The requirement of "CFU / mL" indicates that the microbial count meets safety standards.

[0060] b) No specific pathogenic microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa, or Clostridium tetani were detected in Examples 1-3 and all comparative examples. Furthermore, the quality control system of the kit was validated effectively without any detection interference, indicating that the preservation solutions of Examples 1-3 have no risk of pathogenic microbial contamination and have good microbial safety.

[0061] In summary, the microbial safety of the decellularized matrix dressing preservation solution of this invention meets the requirements for clinical application of medical devices.

[0062] Experimental Example 3: Pre-experimentation of skin irritation Test method: Based on OECD 406 (skin sensitization test) method.

[0063] 1) Animal model: Healthy domestic rabbits, 3 per group. Hair was shaved from both sides of the spine on the back (area ≥ 3cm × 3cm) to avoid damaging the epidermis. Figure 8 The left side of the middle section is covered with gauze soaked in preservation solution, and the right side is covered with gauze soaked in physiological saline (control).

[0064] 2) Exposure protocol: Semi-closed fixation, continuous application for 7 days.

[0065] 3) Observation indicators: Erythema and eschar formation No erythema: 0 points; Very slight erythema, almost imperceptible: 1 point; Well-defined erythema: 2 points; Moderate to severe erythema: 3 points; Severe erythema progressing to eschar formation, making erythema grading impossible: 4 points Edema formation No edema: 0 points; Very slight edema, almost imperceptible: 1 point; Mild edema, with clearly defined boundaries due to obvious elevation of the area: 2 points; Moderate edema, with a bulge of about 1 mm: 3 points; Severe edema, with swelling exceeding 1 mm and extending beyond the exposed area: 4 points Test results are shown in Table 14.

[0066] Skin reaction score (mean, n=3) Table 14 Skin reaction scores for Examples 1-3 (results were consistent across the three groups)

[0067] Behavioral observation: No itching, ulceration, or abnormal behavior (such as irritability).

[0068] Conclusion: The preliminary experiment of the example showed no signs of sensitization, and the maximum skin score was <0.

[0069] Experiment Example 4 Safety Test—Skin Irritation Test Test method: Conducted in accordance with OECD Test Guideline 404 (Skin Irritation / Corrosion).

[0070] 1) Animal Model: Healthy domestic rabbits were selected, with 5 rabbits per group (half male and half female). Hair was shaved from both sides of the spine on the back (area ≥3cm × 3cm) to avoid damaging the epidermis. The rabbits were divided into an intact skin group and a damaged skin group. a) Broken skin group: After disinfection with 75% alcohol, make a "well" shaped wound with a sterile blade (only to the epidermis, without bleeding).

[0071] 2) Dosage and treatment: a) Gauze soaked in preservation solution (dosage 0.5ml / 2.5cm) 2 ), covering the skin surface.

[0072] b) Semi-closed dressing: Secure with non-irritating adhesive tape, change daily for 7 consecutive days.

[0073] 3) Observation indicators: a) Formation of erythema and eschar No erythema: 0 points; Very mild erythema (almost imperceptible): 1 point; Well-defined erythema: 2 points; Moderate to severe erythema: 3 points; Severe erythema (beef-red) to eschar formation (unable to grade the erythema): 4 points b) Edema formation No edema: 0 points; Very slight edema (almost imperceptible): 1 point; Mild edema (the area is clearly defined due to obvious bulging): 2 points; Moderate edema (bulging about 1 mm): 3 points; Severe edema (bulging more than 1 mm and extending beyond the exposed area): 4 points 4) Other: animal weight, behavioral abnormalities (such as restlessness, scratching), mortality rate.

[0074] 5) Control group: The symmetrical area was treated with saline gauze.

[0075] Test results: Figure 8 Erythema / edema scores (mean, n=5), see Table 15 Table 15 Skin irritation test scores for the examples (three groups in total, results were consistent).

[0076] Behavioral observation: No abnormal behavior (such as agitation, scratching, or convulsions). All animals maintained stable body weight (change rate <2%). Figure 1 For operation process photos, Figure 2 (Photo taken after a skin irritation test).

[0077] Conclusion: The skin irritation in the example experiment was mild and reversible, with a maximum erythema score of 0 (damaged skin group), which meets the safety requirements for medical devices.

[0078] Experiment 5: Hemolysis Safety Test and Results 1) Purpose of detection To verify the hemolytic safety of the dressing preservation solution of the present invention, the degree of damage to human red blood cells (hemolysis rate) was tested, and the biocompatibility of the preservation solution when it comes into contact with human tissue during dressing storage and use was evaluated, so as to provide data support for the clinical application and safety evaluation of the product.

[0079] 2) Testing materials a) Test sample: 10 mL of the dressing preservation solution prepared in each embodiment of the present invention; b) Experimental reagents: physiological saline (0.9% sodium chloride solution), distilled water (both sterilized at 121℃ and 103.4kPa for 20min); c) Blood sample: 2 mL of venous blood from a healthy person, after anticoagulation treatment, and the experiment should be completed within 2 hours after collection; d) Instruments and equipment: constant temperature water bath (model: HH-S6, temperature control accuracy ±0.5℃), centrifuge (model: TDL-5-A, maximum speed 8000rpm), ultraviolet spectrophotometer (model: UV-1800, wavelength range 200-1000nm), sterile centrifuge tubes (10mL, made of polypropylene), sterile pipettes (1mL, 5mL, graduation value 0.01mL).

[0080] 3) Detection methods a) Sample and reagent preheating like Figure 3 As shown, take 10 mL of dressing preservation solution, 10 mL of physiological saline, and 10 mL of distilled water, place them in sterile centrifuge tubes, and preheat them in a 37°C constant temperature water bath for 1 hour. During this period, gently shake once every 15 minutes to ensure that the liquid temperature is uniform.

[0081] b) Blood sample processing Collect 2 mL of venous blood and divide it into two equal portions (1 mL each): For the first 1mL of venous blood: add 4mL of normal saline, gently invert the centrifuge tube 5 times to dilute the blood. Figure 5 As shown), place it in a 37℃ constant temperature water bath for preheating for 1 hour. Figure 3 The scene of a water bath after the blood has been diluted. Figure 4 (For venous blood drawing scenarios) The second 1 mL venous blood sample: placed in a centrifuge and centrifuged at 2500 rpm for 10 minutes. Figure 4 (For centrifugation operation scenario), after centrifugation, discard the supernatant, retain the red blood cell precipitate at the bottom, add physiological saline and repeatedly pipette to mix, adjust the red blood cell concentration, and finally prepare 5 mL of 2% red blood cell suspension, and preheat it in a 37℃ constant temperature water bath for 1 hour.

[0082] c) Experimental grouping and sample preparation The experimental group and the control group were configured according to the proportions shown in Table 16. Figure 6 Each group has 3 parallel samples to ensure that pipetting is performed in a sterile laminar flow hood. Table 16 Experimental Group Design

[0083] After each group has been prepared, gently invert the centrifuge tubes three times to ensure the liquid is thoroughly mixed. The order of placement is: "Positive control group 1 → Negative control group 1 → Experimental group 1" and "Experimental group 2 → Negative control group 2 → Positive control group 2". Figure 6 The scene is arranged in groups for the whole blood system. Figure 7(Scene of grouping and arranging a red blood cell suspension system).

[0084] Note: Figure 6 (Whole blood system grouping): Left: Positive control 1, Middle: Negative control 1, Right: Experimental group 1. Figure 7 (Red blood cell suspension system grouping): Left: Experimental group 2, Middle: Negative control 2, Right: Positive control 2 Isothermal reaction and centrifugation All grouped samples were placed in a 37°C water bath and incubated in the dark for 1 hour, gently shaking once every 20 minutes to ensure complete reaction. After incubation, the samples were removed and centrifuged at 2500 rpm for 10 minutes.

[0085] Centrifugation effect is visible Figure 8 .

[0086] Absorbance detection After centrifugation, remove the supernatant from each group of samples, use a sterile pipette to aspirate the supernatant, and inject it into the quartz cuvette of the UV spectrophotometer. Set the detection wavelength to 545 nm, zero the meter with blank distilled water, and measure the absorbance of each group of supernatant in sequence, and record the data.

[0087] 1) Calculate the hemolysis rate Formula: (Sample absorbance - Negative control absorbance) / (Positive control absorbance - Negative control absorbance) × 100% Results: The hemolysis rate was 0.3% in the whole blood group and 2.02% in the erythrocyte group. 2) Summary of Results a) The hemolysis rate of the dressing preservation solution of the present invention is 0.3% in whole blood system and 2.02% in red blood cell suspension system, both of which are far below the standard of "hemolysis rate < 5% is qualified for biosafety" in "Biological evaluation of medical devices Part 4: Selection of blood interaction test" (GB / T16886.4); b) The mean absorbance of the negative control group (physiological saline) was only 0.003, with no obvious hemolysis, indicating that physiological saline has no destructive effect on red blood cells; the mean absorbance of the positive control group (distilled water) was 1.338 (whole blood system) and 1.000 (red blood cell suspension system), respectively, showing complete hemolysis, which verified the effectiveness of the experimental system and eliminated detection error. c) In summary, the dressing preservation solution of the present invention has good hemolytic safety, can stably protect the performance of the dressing during the storage of fish skin dressing, and will not cause damage to the human body due to hemolysis even when it comes into contact with human tissue during subsequent use, thus fully meeting the biosafety requirements of medical devices.

[0088] The specific groupings for this experiment are shown in Table 17: Table 17

[0089] The experimental results of Examples 1-3 are shown in Table 18: Table 18

[0090] Conclusion of hemolysis safety test of dressing preservation solution: This experiment, conducted according to GB / T 16886.4-2017 Biological Evaluation of Medical Devices Part 4: Selection of Blood Interaction Tests, tested the hemolytic safety of the dressing preservation solution described in Examples 1-3 of this invention using whole blood and red blood cell suspension systems. The absorbance was measured at a wavelength of 545 nm using an ultraviolet spectrophotometer. Figure 9 Based on the data, the conclusions are as follows: No hemolysis occurred in any of the test groups, and the hemolysis rate was far below the threshold for no hemolysis (≤5%), proving that the preservation solution formulation involved in the embodiments of the present invention has good biocompatibility in blood contact scenarios and has no risk of hemolysis.

[0091] Experiment Example 6: Safety Testing – Cytotoxicity Detection 1. Test methods: The tests were conducted in accordance with OECD Guidelines for the Testing of Chemicals 423 / 425 and GB / T 16886.5-2017 Biological evaluation of medical devices - Part 5: In vitro cytotoxicity tests.

[0092] 2. Testing materials: a) Preservation solution to be tested: The decellularized matrix preservation solution prepared in the examples / comparative examples was sterilized by a 0.22 μm filter membrane before use; b) Extraction medium: sterile Dulbecco's Modified Eagle Medium (DMEM, containing 10% (v / v) fetal bovine serum). c) Cell line: Logarithmic growth phase L929 mouse fibroblasts; d) Detection reagents: MTT solution (5 mg / mL, prepared with sterile PBS and sterilized with a 0.22 μm filter membrane), DMSO.

[0093] 3. Testing Steps a) Preparation of preservation solution dilution: The sterilized preservation solution was mixed with DMEM medium at a volume ratio to prepare a concentration gradient of 100% (pure preservation solution), 50%, 25%, and 10% (stored at 4°C in the dark and used within 24 hours); a blank control group (pure DMEM medium) and a negative control group (sterile physiological saline + DMEM) were also set up.

[0094] b) Cell seeding and treatment After cell digestion, a single-cell suspension was prepared and the concentration was adjusted to 5 × 10⁻⁶. 4 cells / mL (Note: Originally 2×10) 4 The concentration is too low; OECD typically uses 5×10⁻⁶. 4 To ensure cell adhesion density, add 100 μL of suspension to each well of a 96-well plate and incubate at 37°C with 5% CO2 for 24 h until cell adhesion is ≥80%. Discard the original culture medium, add 100 μL of the corresponding concentration of preservation solution to each well of each experimental group, and add an equal volume of blank / negative culture medium to the control group. Incubate at 37℃ and 5% CO2 for 48 h.

[0095] c) Toxicity testing Discard the liquid in the wells, add 100 μL of MTT solution to each well, and incubate at 37°C for 4 h. Discard the MTT solution, add 150 μL of DMSO to each well, and shake for 10 min to fully dissolve the crystals; The OD value of each well was measured at 570 nm using an ELISA reader, and the cell viability was calculated.

[0096] 2. Experimental Arrangement For the preservation solutions of Examples 1-3, cytotoxicity tests were performed simultaneously according to the steps described above.

[0097] Test results:

[0098] Results Summary: This experiment, conducted according to GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests and the OECD Chemicals Testing Guidelines, used the MTT assay to detect the cytotoxicity of the decellularized matrix preservation solution described in Examples 1-3 of this invention to L929 mouse fibroblasts. The results showed that: Safety verification showed that the cell viability of all test groups (Examples 1-3 and Comparative Example 1) was ≥94.5%, which met the criteria for non-cytotoxicity (viability ≥80%). The preservation solution formulations involved in Examples 1-3 all had good biocompatibility and the safety risks were controllable.

[0099] Experiment Example 7 Safety Testing—Acute Toxicity Test Test method: Refer to OECD Chemicals Testing Guide 423.

[0100] a) Animal model: Healthy mice were selected, with 5 mice in each group (half male and half female). The experimental group was injected intraperitoneally with preservation solution, while the control group was injected with physiological saline. The mice were divided into three groups according to the injection dosage: 0.15 ml / g, 0.20 ml / g, and 0.25 ml / g.

[0101] b) Observation period: Records were kept immediately after injection, at 4 hours, 24 hours, 48 ​​hours, 72 hours, and on days 7 and 14. Weight, food intake, body temperature, and stool characteristics.

[0102] Abnormal behavior: seizures, difficulty breathing, or death.

[0103] c) Experimental results:

[0104] Survival rate: 100% (no deaths within 14 days).

[0105] Weight changes: The average weight decreased, but there was no significant difference compared with the control group. There was no significant difference in weight between mice injected with different doses of physiological saline or patented preservation solution.

[0106] Behavioral observation: No abnormal behavior (such as convulsions or difficulty breathing). All animals exhibited normal activity.

[0107] Conclusion: The preservation solution of this invention has no acute toxicity, and the median lethal dose (LD50) is >50 ml / kg.

[0108] Summarize: The compound formulation range of "glycerol 1.5%-2.5%, 1,2-hexanediol 0.8%-1.5%, dipropylene glycol 0.4%-1.0%, and p-hydroxyacetophenone 0.3%-0.6%" in this invention ensures no hemolysis, no cytotoxicity, and no acute toxicity, while also taking into account antibacterial and stability properties, making it the preferred technical solution of this invention.

[0109] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A decellularized matrix dressing preservation solution, characterized in that, It includes glycerol, 1,2-hexanediol, dipropylene glycol, p-hydroxyacetophenone, and water.

2. The preservation solution according to claim 1, characterized in that, The weight percentages of the components are as follows: glycerol 1.5-2.5%; 1,2-hexanediol 0.8-1.5%; dipropylene glycol 0.4-1.0%; p-hydroxyacetophenone 0.3-0.6%; and the balance is water.

3. The preservation solution according to claim 2, characterized in that, The weight percentages of the components are as follows: glycerol 2%; 1,2-hexanediol 1%; dipropylene glycol 0.6%; p-hydroxyacetophenone 0.4%; and the balance is water.

4. The preservation solution according to claim 1 or 2, characterized in that, The weight ratio of 1,2-hexanediol to p-hydroxyacetophenone is (2:1)-(3:1); and / or The weight ratio of glycerol to dipropylene glycol is (3:1) to (5:1).

5. The preservation solution according to claim 4, characterized in that, The weight ratio of 1,2-hexanediol to p-hydroxyacetophenone is 2.5:1; and / or The weight ratio of glycerol to dipropylene glycol is 3.3:

1.

6. The method for preparing the preservation solution according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Weigh out glycerin, 1,2-hexanediol, dipropylene glycol, p-hydroxyacetophenone and water according to the weight percentage; S2: Add glycerol, 1,2-hexanediol, dipropylene glycol and p-hydroxyacetophenone to water in sequence and stir until completely dissolved to obtain the decellularized matrix dressing preservation solution.

7. The method according to claim 6, characterized in that, In S1, weigh out 1.5-2.5% glycerol, 0.8-1.5% 1,2-hexanediol, 0.4-1.0% dipropylene glycol, 0.3-0.6% p-hydroxyacetophenone, and the remainder is water.

8. The use of the preservation solution according to any one of claims 1-5 or the preservation solution obtained by the method according to any one of claims 6-7 in the preparation of wound repair medical devices.

9. The application according to claim 8, characterized in that, The medical device is a burn dressing, an ulcer repair membrane, or a tissue engineering scaffold.

Images