A material for promoting injury repair and anti-scarring and its preparation method

Abstract

The present invention discloses a material for promoting injury repair and anti-scarring and a preparation method thereof, comprising the following steps: mixing a biologically active conjugated linoleic acid with a mixed solution of an alkali metal solution and a soluble salt (calcium or zinc) for reaction to obtain a conjugated linoleate solution, and adjusting the pH value of the conjugated linoleate solution to 5-8 with an acid; the soluble salt is a soluble calcium salt or a soluble zinc salt; adding the conjugated linoleate solution to a biogenic gel material, and the mass ratio of the conjugated linoleate to the biogenic gel material is 1:10 to 50, and after mixing evenly and reacting, a conjugated linoleate gel is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin repair materials, and particularly relates to a material for promoting damage repair and anti-scarring, and a preparation method thereof. Background Art

[0002] The statements herein only provide background art related to the present invention and do not necessarily constitute prior art.

[0003] Severe scar hyperplasia after large-area skin injuries such as knife cuts and burns seriously affects the morphological and functional recovery of patients after healing, which is one of the difficult problems in clinical medicine. Factors such as collagen metabolism imbalance, fibroblast proliferation and contraction, and changes in the proportion of proteoglycan components in the dermal matrix are the basis for the formation of hypertrophic scars after burns. At present, relevant scholars are actively searching for artificial or natural antagonist factors, such as anti-TGF factor - interferon, etc. However, the results are not satisfactory or have not been used clinically.

[0004] The wound healing dressings and repair materials in the prior art can promote the growth of new epidermal tissues to a certain extent, but the new tissues often gradually grow into scars, with a large difference in tensile strength and apparent appearance from the original skin tissue, and the wound healing time is still relatively long. Especially when used for the repair of large-area and deeper wounds, the obvious scars after healing may have an adverse impact on the patient's life. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a material for promoting damage repair and anti-scarring, and a preparation method thereof. This material can not only promote wound healing but also inhibit the formation of hypertrophic scars.

[0006] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0007] In the first aspect, the present invention provides a preparation method of a material for promoting damage repair and anti-scarring, including the following steps:

[0008] Mix a biologically active conjugated linoleic acid with a mixed solution of an alkali solution and a soluble salt (calcium or zinc) to obtain a conjugated linoleate solution, and adjust the pH value of the conjugated linoleate solution to 5 - 8 with an acid; the soluble salt is a soluble calcium salt and / or a soluble zinc salt;

[0009] Add the conjugated linoleate solution to a biogenic gel material, and the mass ratio of the conjugated linoleate to the biogenic gel material is 1:10 - 50. After mixing evenly and reacting, a conjugated linoleate gel is obtained.

[0010] Biogenic gel materials provide a scaffold for the proliferation of fibroblasts, contribute to the remodeling of collagen components, prompt fibroblasts to arrange and distribute regularly, and form collagen fibers with normal structural morphology. In addition, they also have the functions of inducing and regulating the ingrowth of host cells and new blood vessels, and promoting epithelial formation. In addition, extracellular matrix proteins can also promote the regeneration of epidermal cells.

[0011] Conjugated linoleate has significant antioxidant and anti-inflammatory properties. In addition, experimental models have demonstrated that conjugated linoleate can reduce the production of inflammatory mediators such as arachidonic acid, prostaglandins, and histamine by keratinocytes, thereby resulting in a reduction in itching in the case of atopic dermatitis and having the effect of maintaining the biological activity of biogenic gel materials.

[0012] Calcium-based materials are used for bone tissue repair; zinc-based materials are used for soft tissue repair; calcium-zinc-based materials are used for the simultaneous repair of hard and soft tissues, such as filling the alveolar socket after tooth extraction, which requires both meeting the regeneration of alveolar bone and achieving the rapid repair and coverage of the wound surface by gingival soft tissue.

[0013] The formation mechanism of scars is persistent local inflammation and excessive collagen deposition. Mast cells around the wound tissue can stimulate fibroblast proliferation by releasing IL-4, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF), resulting in an increase in type I collagen synthesis. During the proliferation, re-epithelialization, and remodeling stages, there are a large number of M2 macrophages around the wound tissue that promote angiogenesis and collagen deposition. M2 macrophages can promote the transformation of fibroblasts into myofibroblasts by secreting transforming growth factor-β (TGF-β) and platelet-derived growth factor-CC (PDGF-CC), both of which promote collagen deposition and scar formation. TGFβ1 mediates the expression of α-smooth muscle actin (α-SMA), which is a marker of myofibroblasts. The material prepared by the present invention can rapidly promote wound repair, shorten the wound healing time, avoid scar formation, and is biodegradable and absorbable by upregulating the expression of angiogenesis genes (bFGF and VEGF) and downregulating the expression of fibrosis genes (TGF-β1 and α-SMA).

[0014] In some embodiments, the soluble salt is a mixture of calcium chloride and zinc chloride, and the mass ratio of calcium chloride to zinc chloride is 1-10:1.

[0015] In some embodiments, in the mixed solution of the alkali solution and the soluble salt, the concentration of the soluble salt is 1%-10%, where % is the mass percentage;

[0016] The concentration of the alkali solution is 1%-20%;

[0017] The alkali solution is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, or francium hydroxide.

[0018] Preferably, the alkaline solution is selected from sodium hydroxide or potassium hydroxide. Through experiments, it was found that when the alkaline solution is other alkaline solutions such as calcium hydroxide, no reaction occurs and layering occurs, and the expected product cannot be obtained.

[0019] In some embodiments, the acid is hydrochloric acid, glacial acetic acid, citric acid or lactic acid.

[0020] In some embodiments, the biogenic gel material is selected from one, two or more of natural collagen, recombinant collagen, sodium hyaluronate, and extracellular matrix.

[0021] In some embodiments, it further includes the steps of preparing a damage repair and anti-scar sponge: loading the conjugated linoleate gel into a mold and freeze-drying it gradient to obtain a conjugated linoleate sponge;

[0022] Thermally crosslink the conjugated linoleate sponge at 100-120 °C for 10-100 h to obtain the damage repair and anti-scar sponge. It can not only meet the mechanical properties of the material after crosslinking but also ensure the biological activity of the material.

[0023] Preferably, it further includes the step of encapsulating the prepared conjugated linoleate gel or damage repair and anti-scar sponge in a medical packaging bag and sterilizing it with 6-20 KGy of gamma rays.

[0024] Preferably, the material of the mold is Teflon material.

[0025] In some embodiments, the biogenic gel material is acellular extracellular matrix gel, and its preparation method is: scraping the grease on the surface of the animal peritoneal tissue, washing until there is no grease, dehydrating with acetone, and degreasing with n-hexane;

[0026] Mix the degreased membrane tissue with pepsin or nuclease for enzymatic hydrolysis, dialyze the enzymatic hydrolysate to remove immunogenicity, and obtain the extracellular matrix after freeze-drying;

[0027] Dissolve the extracellular matrix in phosphate buffer to obtain acellular matrix gel.

[0028] Preferably, the gradient freeze-drying is: rapidly freezing the mixture at -30 to -80 °C first, and then drying by gradually increasing the temperature.

[0029] Preferably, the mass ratio of the degreased membrane tissue to pepsin is 10-100:1;

[0030] The mass ratio of the degreased membrane tissue to nuclease is 50-150:1.

[0031] Preferably, the dialysis membrane used for dialysis is a 10-50 kDa dialysis membrane.

[0032] Second aspect, the present invention provides a material for promoting injury repair and anti-scarring, which is prepared by the preparation method.

[0033] The beneficial effects obtained by one or more embodiments of the present invention are as follows:

[0034] In the prior art, the scar repair material products mainly composed of polydimethylsiloxane only have the function of anti-scarring, cannot be used for unhealed wounds, and have no effect of promoting wound healing. The material prepared by the present invention can rapidly promote wound repair, shorten the wound healing time, avoid scar formation, and is biodegradable and absorbable;

[0035] The material prepared by the present invention can rapidly guide natural tissue regeneration rather than scar healing;

[0036] The material prepared by the present invention has no chemical cross-linking agent. The conjugated linoleate sponge is prepared by the step-by-step vacuum freeze-drying and thermal cross-linking technology, which protects the uniform structure of the sponge and avoids the formation of crystal structure. At the same time, the residual chemical cross-linking agent may cause the risk of tissue toxicity in the body. The material prepared in the present invention has no chemical cross-linking agent, which reduces the verification link of chemical residues in the production process; compared with cross-linking processes such as ultraviolet or γ-ray, it avoids the damage of active ingredients in the material. It protects the active ingredients of the conjugated linoleate sponge and improves the biocompatibility and biological activity of the overall material.

[0037] It is prepared by uniformly mixing and reacting self-made conjugated linoleate (calcium zinc, etc.) with biogenic materials (extracellular matrix, sodium hyaluronate, collagen, etc.). The material promotes injury repair and anti-scar formation by up-regulating the expression of angiogenesis genes (bFGF and VEGF) and down-regulating the expression of fibrosis genes (TGF-β1 and α-SMA). Description of the Drawings

[0038] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0039] Figure 1 It is a product diagram prepared in the embodiment of the present invention. Among them, a is zinc conjugated linoleate gel, and b is zinc conjugated linoleate sponge;

[0040] Figure 2 It is a picture of the failed zinc conjugated linoleate sponge prepared in Comparative Example 1 of the present invention;

[0041] Figure 3 It is a comparison diagram of wound anatomy of the zinc conjugated linoleate sponge in vivo degradation experiment at 1 week (a), 4 weeks (b), and 13 weeks (c) in the embodiment of the present invention;

[0042] Figure 4It is a comparison diagram of treating wounds with zinc conjugate linoleate gel and untreated wounds in the embodiments of the present invention;

[0043] Figure 5 It is a comparison diagram of the wound healing test of rats using zinc conjugate linoleate sponge (Group 1), collagen material (Group 2), and sodium hyaluronate material (Group 3) in the embodiments of the present invention;

[0044] Figure 6 It is a comparison diagram of the recovery of wounds after 36 days of the wound healing test of rats using zinc conjugate linoleate sponge (Group 1), collagen material (Group 2), and sodium hyaluronate material (Group 3) in the embodiments of the present invention;

[0045] Figure 7 It is the RNA expression level of scar-related genes after treating wounds with zinc conjugate linoleate sponge (Group 1) for 15 days in the embodiments of the present invention;

[0046] Figure 8 It is the expression result of bFGF protein level detected by immunohistochemistry after treating wounds with each group for 15 days in the embodiments of the present invention. Among them, a is Group 1, under the epidermis, bFGF, 400×; b is Group 2, under the epidermis, bFGF, 400×; c is Group 3, under the epidermis, bFGF, 400×; d is the wound group, under the epidermis, bFGF, 400×.

[0047] Figure 9 It is the expression result of VEGF protein level detected by immunohistochemistry after treating wounds with each group for 15 days in the embodiments of the present invention. Among them, a is Group 1, under the epidermis, VEGF, 400×; b is Group 2, under the epidermis, VEGF, 400×; c is Group 3, under the epidermis, VEGF, 400×; d is the wound group, under the epidermis, VEGF, 400×;

[0048] Figure 10 It is the expression result of TGF-β1 protein level detected by immunohistochemistry after treating wounds with each group for 15 days in the embodiments of the present invention. Among them, a is Group 1, under the epidermis, TGF-β1, 400×; b is Group 2, under the epidermis, TGF-β1, 400×; c is Group 3, under the epidermis, TGF-β1, 400×; d is the wound group, under the epidermis, TGF-β1, 400×;

[0049] Figure 11 It is the expression result of α-SMA protein level detected by immunohistochemistry after treating wounds with each group for 15 days in the embodiments of the present invention. Among them, a is Group 1, under the epidermis, α-SMA, 400×; b is Group 2, under the epidermis, α-SMA, 400×; c is Group 3, under the epidermis, α-SMA, 400×; d is the wound group, under the epidermis, α-SMA, 400×;

[0050] Figure 12 This is the HE staining image of the wound tissue pathological section at 40× on the 36th day after treating the wound with the zinc conjugate linoleate sponge (Group 1) prepared in Example 1 in the embodiments of the present invention;

[0051] Figure 13 This is the Masson staining image of the wound tissue pathological section at 40× on the 36th day after treating the wound with the zinc conjugate linoleate sponge (Group 1) prepared in Example 1 in the embodiments of the present invention;

[0052] Figure 14 This is the HE staining image of the wound tissue pathological section at 40× on the 36th day after treating the wound with the collagen material (Group 2) in the embodiments of the present invention;

[0053] Figure 15 This is the Masson staining image of the wound tissue pathological section at 40× on the 36th day after treating the wound with the collagen material (Group 2) in the embodiments of the present invention;

[0054] Figure 16 This is the HE staining image of the wound tissue pathological section at 40× on the 36th day after treating the wound with the sodium hyaluronate material (Group 3) in the embodiments of the present invention;

[0055] Figure 17 This is the Masson staining image of the wound tissue pathological section at 40× on the 36th day after treating the wound with the sodium hyaluronate material (Group 3) in the embodiments of the present invention;

[0056] Figure 18 This is the HE staining image of the wound tissue pathological section at 40× on the 36th day after treating the wound with the zinc conjugate linoleate sponge (Group 4) prepared in Comparative Example 2 in the embodiments of the present invention;

[0057] Figure 19 This is the Masson staining image of the wound tissue pathological section at 40× on the 36th day after treating the wound with the zinc conjugate linoleate sponge (Group 4) prepared in Comparative Example 2 in the embodiments of the present invention. Detailed implementation manners

[0058] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0059] The present invention will be further described below with reference to the embodiments.

[0060] Example 1

[0061] 1. Preparation of Calcium Conjugated Linoleate, Zinc Conjugated Linoleate, and Calcium-Zinc Conjugated Linoleate

[0062] (1) Preparation of calcium conjugated linoleate: Weigh 20 g of a mixture of sodium hydroxide and calcium chloride (the mass ratio of sodium hydroxide to calcium chloride is 10:1) and dissolve it in 20 mL of water, stir well, and set aside. Separately, measure 100 mL of conjugated linoleic acid into a 250 mL beaker, stir slowly, and add the sodium hydroxide and calcium chloride mixture in small portions. Stir at a constant speed of 1000 rpm for an emulsification reaction. Observe the sample state and exothermic phenomenon during stirring, and the temperature is 36 - 40 °C.

[0063] After 14 days of reaction, the temperature drops to room temperature, reaching the reaction end point, and a paste-like calcium conjugated linoleate is formed. Its pH is alkaline, and it is adjusted to neutral with glacial acetic acid, then sealed and stored in a glass bottle and kept in a 4 °C refrigerator for later use.

[0064] (2) Preparation of zinc conjugated linoleate:

[0065] Weigh 20 g of a mixture of sodium hydroxide and zinc chloride (the mass ratio of sodium hydroxide to zinc chloride is 10:1) and dissolve it in 20 mL of water, stir well, and set aside. Separately, measure 100 mL of conjugated linoleic acid into a 250 mL beaker, stir slowly, and add the sodium hydroxide and zinc chloride mixture in small portions. Stir at a constant speed of 1000 rpm for an emulsification reaction. Observe the sample state and exothermic phenomenon during stirring, and the reaction temperature is 36 - 40 °C. After 14 days of reaction, the temperature drops to room temperature, reaching the reaction end point, and a paste-like zinc conjugated linoleate is formed. Its pH is alkaline, and it is adjusted to neutral with glacial acetic acid, then sealed and stored in a glass bottle and kept in a 4 °C refrigerator for later use.

[0066] (3) Preparation of calcium-zinc conjugated linoleate:

[0067] Weigh 20 g of a mixture of sodium hydroxide, zinc chloride, and calcium chloride (the mass ratio of sodium hydroxide to the zinc chloride and calcium chloride mixture is 10:1, and the mass ratio of zinc chloride to calcium chloride is 1:9) and dissolve it in 20 mL of water, stir well, and set aside. Separately, measure 100 mL of conjugated linoleic acid into a 250 mL beaker, and under slow stirring, add the sodium hydroxide, zinc chloride, and calcium chloride mixture in small portions. Stir at a constant speed of 1000 rpm for an emulsification reaction. Observe the sample state and exothermic phenomenon during stirring, and the reaction temperature is 36 - 40 °C.

[0068] After 14 days of reaction, the temperature drops to room temperature, reaching the reaction end point, and a paste-like calcium-zinc conjugated linoleate is formed. Its pH is alkaline, and it is adjusted to neutral with glacial acetic acid, then sealed and stored in a glass bottle and kept in a 4 °C refrigerator for later use.

[0069] 2. Preparation of Decellularized Extracellular Matrix Gel

[0070] (1) Pretreatment: Scrape the grease on the surface of the animal peritoneal tissue, and wash it with pure water until there is no fat. Dehydrate with acetone and degrease with n-hexane.

[0071] (2) Removal of immunogenicity: The degreased membrane tissue is enzymolyzed with pepsin at a ratio of 50:1; the degreased membrane tissue reacts with nuclease at a ratio of 100:1, and dialysis is carried out with a 10 kDa dialysis membrane, and freeze-drying is performed to obtain acellular extracellular matrix.

[0072] (3) Preparation of 7% acellular matrix gel: Dissolve 7 g of immunogenicity-removed and freeze-dried acellular matrix in 0.01 M phosphate buffer, and stir evenly to obtain 7% acellular matrix gel.

[0073] 3. Preparation of zinc conjugate linoleate gel

[0074] (1) Dissolve 1 g of the prepared zinc conjugate linoleate in 350 g of 7% acellular matrix gel to obtain a mixture of zinc conjugate linoleate-acellular extracellular matrix gel (CG-POSTN), and stir evenly at a rotation speed of 800 rpm.

[0075] (2) Sub-packaging: 10 mL / bottle;

[0076] (3) Sterilization: Sterilize with γ-ray;

[0077] 4. Preparation of zinc conjugate linoleate sponge

[0078] (1) Dissolve 1 g of the prepared zinc conjugate linoleate in 350 g of 7% acellular matrix gel to obtain a mixture of zinc conjugate linoleate-acellular extracellular matrix gel (CG-POSTN), and stir evenly at a rotation speed of 800 rpm.

[0079] (2) Filling: 10 mL / mold; The mold uses a special mold (preferably Teflon material) with an inner diameter of 4.5 cm × 6.5 cm; an outer diameter of 7.5 cm × 5.5 cm.

[0080] (3) Drying: Rapidly freeze the mixture at -80 °C; then gradually increase the temperature for drying and cross-linking. The drying procedure is: -30 °C, 4 h; -10 °C, 10 h; 1 °C, 4 h; 10 °C, 4 h.

[0081] (4) Thermal cross-linking: Under the condition of a vacuum degree of -1.0 pa, 50 °C, 3 h; 80 °C, 30 min; 100 °C, 1 h; 110 °C, 1 h; 120 °C, 24 h.

[0082] (5) Sterilization: Sterilize with γ-ray.

[0083] Preparation of zinc conjugate linoleate sponge in Comparative Example 1

[0084] Dissolve 1 g of zinc conjugate linoleate prepared in Example 1 in 70 g of 7% decellularized matrix gel to obtain a mixture of zinc conjugate linoleate - decellularized extracellular matrix gel (CG - POSTN), and stir evenly at a rotation speed of 800 rpm.

[0085] (2) Filling: 10 mL / mold; The mold uses a special mold (preferably made of Teflon), with an inner diameter of 4.5 cm × 6.5 cm; an outer diameter of 7.5 cm × 5.5 cm.

[0086] (3) Drying: Rapidly freeze the mixture at -80 °C; then gradually increase the temperature for drying and cross - linking. The drying procedure is: -30 °C for 4 h, -10 °C for 10 h, 1 °C for 4 h; 10 °C for 4 h.

[0087] (4) Thermal cross - linking: Under the condition of a vacuum degree of -1.0 pa, 50 °C for 3 h, 80 °C for 30 min, 100 °C for 1 h, 110 °C for 1 h, 120 °C for 24 h;

[0088] (5) Sterilization: Sterilize using γ - rays.

[0089] The obtained product is shown in Figure 2 , when the proportion of zinc conjugate linoleate is too high, the film - forming effect is not good.

[0090] Preparation of zinc conjugate linoleate sponge in Comparative Example 2

[0091] Dissolve 1 g of zinc conjugate linoleate prepared in Example 1 in 850 g of 7% decellularized matrix gel to obtain a mixture of zinc conjugate linoleate - decellularized extracellular matrix gel (CG - POSTN), and stir evenly at a rotation speed of 800 rpm.

[0092] (2) Filling: 10 mL / mold; The mold uses a special mold (preferably made of Teflon), with an inner diameter of 4.5 cm × 6.5 cm; an outer diameter of 7.5 cm × 5.5 cm.

[0093] (3) Drying: Rapidly freeze the mixture at -80 °C; then gradually increase the temperature for drying and cross - linking. The drying procedure is: -30 °C for 4 h, -10 °C for 10 h, 1 °C for 4 h, 10 °C for 4 h.

[0094] (4) Thermal cross - linking: Under the condition of a vacuum degree of -1.0 pa, 50 °C for 3 h, 80 °C for 30 min, 100 °C for 1 h, 110 °C for 1 h, 120 °C for 24 h;

[0095] (5) Sterilization: Sterilize using γ - rays.

[0096] Physical and chemical property detection of materials in Example 2

[0097] 1. Material porosity

[0098] 1.1 Measurement method

[0099] Compare the porosity of the zinc conjugate linoleate sponge prepared in Example 1 with that of the collagen sponge (a commercially available product). The drainage method is used, that is, by means of cyclic vacuum pumping, the gas in the internal pores of the material is discharged and filled with liquid, and the percentage of the weight of this part of the liquid in the total weight of the material is calculated. Weigh about 0.1 g (W1) of the dried sample, cut it into strips and put it into a volumetric flask filled with water. Cyclically pump vacuum until no more bubbles overflow from the surface of the sample and the sample sinks to the bottom. Weigh the volumetric flask containing the sample and water (W2), then take out the sample containing water inside, weigh the remaining volumetric flask and water weight (W3), and calculate the porosity (P). The formula is as follows:

[0100] P = (W2 - W3 - W1) / (W2 - W3) × 100%.

[0101] 1.2 The test results are shown in Table 1.

[0102] Table 1

[0103] Sample Name Porosity (%) Zinc Conjugated Linoleate Film 94.4 Commercially Available Collagen Sponge 97.8

[0104] 1.3 Summary: There is no difference in porosity between the two.

[0105] 2. Ignition residue

[0106] Precisely weigh 1 g of the zinc conjugate linoleate sponge material prepared in Example 1, place it in a crucible that has been ignited to constant weight, precisely weigh it, slowly ignite it until completely carbonized, cool it; add 1 mL of sulfuric acid to moisten it, heat it at low temperature until the sulfuric acid vapor is completely removed, then ignite it at 600 °C until completely ashed, transfer it to a desiccator, cool it, precisely weigh it, and then ignite it at 600 °C until constant weight to obtain the result.

[0107] 2.2 The test results are shown in Table 2 as follows:

[0108] Table 2

[0109] Sample Name Zinc Conjugated Linoleate Sponge Comparative Example 1 Comparative Example 2 Sulfate Content (%) 0.97 4.92 0.31

[0110] 2.3 Summary: According to "YY / T 1511-2017 Collagen Sponge", the sulfate ash should not be more than 2.0% (mass fraction). The materials prepared in Example 1 and Comparative Example 2 meet the requirements, while the material obtained in Comparative Example 1 does not meet the requirements, and the content of zinc conjugate linoleate is too high.

[0111] 3. Total protein content

[0112] 3.1 Weigh accurately about 10 mg of zinc conjugate linoleate prepared in Example 1 (equivalent to a nitrogen content of 1.0 mg - 2.0 mg), denoted as m1, and place it in a digestion tube. Add 0.3 g of digestion agent and 2.0 mL of concentrated sulfuric acid. Place it on an electric digestion stove and digest it in a fume hood until it becomes clear and bluish-green. Continue digestion for 60 min. At the same time, conduct a blank digestion control.

[0113] 3.2 Take 10 mL of 2% boric acid absorption solution and place it in a 250 mL conical flask. Immerse the end of the condenser tube of the nitrogen determination instrument into the boric acid absorption solution. Transfer the digested sample (m1) into the nitrogen determination tube, wash the digestion tube 3 - 4 times with a small amount of water, transfer the washing solution into the nitrogen determination tube, then add 10 mL of 50% sodium hydroxide, and then carry out distillation. When the total volume of the receiving solution is about 35 mL - 50 mL, remove the end of the condenser tube from the liquid surface, let the steam continue to flush for about 1 min, rinse the end of the condenser tube with a small amount of distilled water, and then stop distillation. Titrate the receiving solution with 0.005 mol / L sulfuric acid titrant until the solution changes from bluish-green to gray-violet, and record the volume of the sulfuric acid titrant consumed, V1. Transfer the digested sample (m2) into the nitrogen determination tube, repeat the above distillation and titration steps, and record the volume of the sulfuric acid titrant consumed, V2. Transfer the blank digestion control into the nitrogen determination tube, repeat the above distillation and titration steps, and record the volume of the sulfuric acid titrant consumed, V0, and correct it with the blank test.

[0114] 3.3 Result calculation

[0115] Calculate the total nitrogen content in the sample according to the following formula:

[0116]

[0117] In the formula:

[0118] W1: Total nitrogen content in the sample, %.

[0119] V1: Volume of sulfuric acid titrant consumed for titrating sample m1, mL.

[0120] V0: Volume of sulfuric acid titrant consumed for blank titration, mL.

[0121] m1: Sample mass, mg.

[0122] c: Concentration of sulfuric acid titrant, mol / L.

[0123] m0: Sample dry weight loss, %.

[0124] 3.4 The test results are shown in Table 3:

[0125] Table 3

[0126] Item Zinc Conjugated Linoleate Sponge Protein Content, % 93.3±1.0％

[0127] 3.5 Summary: According to the protein content in "YY / T 1511-2017 Collagen Sponge" 4.7, the protein content in the collagen sponge should be not less than 90%, and this sample meets the requirements.

[0128] 4. In vitro degradation test

[0129] 4.1 Weigh about 10 mg of the conjugated linoleic acid zinc sponge prepared in Example 1 and immerse it in a 15 mL centrifuge tube filled with water. Gently press it with a glass rod until it is completely wetted. Take it out, remove the excess water with filter paper, and then put it back into the centrifuge tube. 10 mL of a hydrochloric acid solution (hydrochloric acid concentration 0.1 mol / L) of pepsin with a mass fraction of 1% preheated to 37°C ± 1°C has been previously loaded into the centrifuge tube. Shake it at about 90 revolutions per minute at 37°C ± 1°C until it is completely digested, and observe the digestion time of the sample.

[0130] 4.2 The test results are shown in Table 4 as follows:

[0131] Table 4

[0132] Sample Name Zinc Conjugated Linoleate Sponge Digestion Time (h) 17.5±1.0

[0133] 4.3 Summary: Under the conditions of this test, the digestion time of the conjugated linoleic acid zinc sponge is not more than 24 hours, supporting the degradability of the conjugated linoleic acid zinc sponge.

[0134] Material safety performance detection of Example 3

[0135] Cytotoxicity test

[0136] 1.1 Negative control test solution: That is, cell culture medium, MEM containing 10% fetal bovine serum.

[0137] 1.2 Sample test solution: Take 0.08 g of the conjugated linoleic acid zinc sponge sample prepared in Example 1 in 2 mL of cell culture medium, and oscillate and extract it at 60 r / min at 37°C ± 1°C for (24 ± 2) h.

[0138] 1.3 Positive control test solution: 10% DMSO solution, prepared and used immediately.

[0139] 1.4 MTT solution: Prepare an MTT solution with a mass concentration of 1 mg / mL from MTT powder, and sterilize it by sterile filtration through an injection filter (pore size ≤ 0.22 μm) for standby.

[0140] 1.5 Place the negative control test solution, sample test solution, and positive control test solution into the wells of a cell culture plate containing L929 mouse fibroblasts respectively, and culture them in a cell culture incubator with 5% CO2 at 37°C. After 24 h, observe the morphological changes of the cells in the test sample group, negative control group, and positive control group under a microscope. Carefully remove the culture medium, add 50 μL of MTT solution to each well, continue to culture in the cell culture incubator for 2 h, then discard the liquid in the wells. Add 100 μL of DMSO to each well, shake the plate, and measure the absorbance at a wavelength of 570 nm (reference wavelength 650 nm) using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the survival rate (%) according to the following formula.

[0141]

[0142] In the formula:

[0143] OD570e - Absorbance of each test article group (sample group, positive control group);

[0144] OD570b - Absorbance of the medium control group.

[0145] And use the MTT method to determine the cell survival rate of each test article group (sample group, positive control group) relative to the medium control group.

[0146] 1.6 The test results are as follows:

[0147] Sample Name Positive Control Zinc Conjugated Linoleate Sponge Survival Rate (%) 19.8±0.1 115.5±0.3

[0148] 1.7 Summary: Under the conditions of this test, the cell survival rate of the zinc conjugate linoleate sponge is 115.5 ± 0.3%, and there is no cytotoxicity.

[0149] Example 4 In vivo Degradation Detection of Materials

[0150] 1. Animal test method: Select SD rats, anesthetize them, shave the hair on their backs, disinfect with medical iodine, make four skin incisions on both sides of the midline of the back, insert forceps through the incisions, and bluntly separate to prepare subcutaneous sacs. The bottom of the sac is 10 mm away from the incision, and they are marked as ①, ②, ③, ④ respectively. Place a zinc conjugate linoleate sponge prepared in Example 1 into each of the sacs ①, ②, and ③, and cut the size of the zinc conjugate linoleate sponge to 1 cm × 1 cm. ④ is the blank control and no treatment is done. Use absorbable suture to suture the incisions to ensure that the implants do not contact each other.

[0151] The positions of the implantation points and the sampling times of the implants are as follows:

[0152] ④ Blank ① - 1 Week ③ - 13 Weeks ② - 4 Weeks

[0153] 2. The wound tissues were taken for anatomical observation at 1 week, 4 weeks, and 13 weeks respectively. It was found that undegraded materials were clearly visible under the skin in the samples taken after 1 week, a small part of undegraded materials could be observed in the samples taken after 4 weeks, and the materials were completely degraded at 13 weeks. See Figure 3 。

[0154] 3. Summary: The zinc conjugate linoleate sponge can be completely degraded within 13 weeks, supporting the degradation performance of the zinc conjugate linoleate sponge.

[0155] Example 5 Performance Test of Materials on Scar Repair

[0156] 1. Establish a scar model on the ears of rabbits

[0157] Adult healthy rabbits were selected. The ears of the rabbits were disinfected with 75% ethanol, and the ears of the rabbits were scratched with a scalpel, with the wound being about 2 cm. On the 6th day after the operation, the wound surface scabbed. The wound surface was cleaned with 75% ethanol. On the 21st day after the operation, the zinc conjugate linoleate gel prepared in Example 1 was applied to the wound surface of the left ear of the rabbit, and no treatment was done to the right ear.

[0158] 2. Repair test of zinc conjugate linoleate gel on scars

[0159] 2.1 Starting from the 21st day after the operation, the wound surface was cleaned with 75% ethanol, and the zinc conjugate linoleate gel prepared in Example 1 was applied to the scar wound surface of the left ear of the rabbit, and no treatment was done to the right ear, once a day.

[0160] 2.2 On the 45th day after the operation, the healing conditions of the left ear and the right ear were observed. After the zinc conjugate linoleate gel was applied to the left ear of the rabbit, the scar healed, and there were obvious scars on the untreated wound surface of the right ear, as Figure 4 shown.

[0161] 2.3 Summary: Under the conditions of this test, the zinc conjugate linoleate gel has the performance of repairing the formed scars.

[0162] Example 6 Performance Test of Repairing Defective Skin in Rats

[0163] 1. Two wound marks were made on the left and right sides of the back of the rats using a circular scale mold with a diameter of 1 cm. The marked back tissues were cut off with a scalpel. The same materials were filled on the left and right sides of the same rat, and different materials were used for different rats in the wound healing test. At the same time, a blank control group and a trauma control group were added.

[0164] 2. The material groups are as follows in the table:

[0165] Grouping Filling Material Group 1 Zinc Conjugated Linoleate Sponge Prepared in Example 1 Group 2 Collagen Material Group 3 Sodium Hyaluronate Material Group 4 Zinc Conjugated Linoleate Sponge Prepared in Comparative Example 2

[0166] 4. Experimental Observation: The wound healing conditions of different materials were observed on the 1st, 4th, 7th, 15th, 21st, and 36th days respectively. On the 4th day after surgery in rats, a flexible ruler was used to measure the wound size, which was similar to the size during the operation, and the wound showed basically no change. On the 7th day after surgery in rats, the wound was measured with a flexible ruler, and all wounds became smaller, with a diameter of approximately 0.5 cm. On the 15th day after surgery in rats, there was a circular defect with a diameter of approximately 0.1 cm in Group 1, and circular defects with a diameter of approximately 0.3 cm in Group 2 and Group 3. On the 21st day after surgery in rats, there was a linear defect of approximately 0.1 cm × 0.5 cm in Group 1, a circular defect of approximately 0.2 cm × 0.3 cm in Group 2, and a circular defect of approximately 0.2 cm × 0.3 cm in Group 3. On the 36th day after surgery, tissue samples of all wounds were taken for pathological sectioning. The wound healing conditions are as Figure 5 shown.

[0167] On the 36th day after surgery, the skin in Group 1 and Group 4 had basically recovered, while scar formation occurred in Group 2 and Group 3, as Figure 6 shown.

[0168] Tissue samples of the wounds on the 15th day were taken to evaluate the repair effect at the levels of related gene expression and protein. The selected genes were: transforming growth factor β1 (TGF-β1), α-smooth muscle actin (α-SMA), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). Pathological sections of the wound tissue on the 36th day were taken for HE staining.

[0169] As Figure 7 shown, in the early and middle stages of epidermal formation, the RNA expression levels of angiogenesis genes (bFGF and VEGF) in each treatment group (Group 1, Group 2, and Group 3) were higher than those in the blank control group and the wound control group, and the up-regulation amplitude in Group 1 was the largest, significantly higher than that in the wound group; the RNA expression levels of fibrosis genes (TGF-β1 and α-SMA) in each treatment group (Group 1, Group 2, and Group 3) were lower than those in the blank control group and the wound control group, and the down-regulation amplitude in Group 1 was significantly higher than that in the wound group. The up-regulation of angiogenesis genes (bFGF and VEGF) and the down-regulation of fibrosis genes (TGF-β1 and α-SMA) indicate that the material has anti-scar formation activity during epidermal formation. As Figures 8 - 11 shown, the immunohistochemical results were consistent with the corresponding gene expression results, verifying that the material has an anti-scar effect at the levels of gene expression and protein.

[0170] The results of HE staining are shown in Figures 12 - 19In all sections of Group 1, wound healing was observed. The epidermis was keratinized stratified squamous epithelium; beneath it was fibrous tissue, which, upon Masson staining, was shown to be mainly composed of collagen fibers. In all sections of Group 2, wound healing was observed. The epidermis was keratinized stratified squamous epithelium, but a thinner area was seen in the local dermis, presumably the skin adjacent to the wound; beneath it was fibrous tissue, which, upon Masson staining, was shown to be mainly composed of collagen fibers. In all sections of Group 3, wound healing was observed. The epidermis was keratinized stratified squamous epithelium, but a thinner area was seen in the local dermis, presumably the skin adjacent to the wound; beneath it was fibrous tissue, which, upon Masson staining, was shown to be mainly composed of collagen fibers. In all sections of Group 4, wound healing was observed. The epidermis was keratinized stratified squamous epithelium; beneath it was fibrous tissue, which, upon Masson staining, was shown to be mainly composed of collagen fibers. At the junction of the dermis and subcutaneous tissue, localized infiltration of inflammatory cells was seen (within the dotted line).

[0171] It can be seen that the wound recovery effect of Group 1 was better than that of Group 2, Group 3, and Group 4. The wound tissue healed faster and the anti-scar effect was better. In Group 4, due to the too low content of zinc conjugated linoleate, inflammatory cell infiltration occurred, and the overall repair effect was worse than that of Group 1. This experiment proved that the zinc conjugated linoleate sponge of Group 1 had an anti-scar effect.

[0172] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a material for promoting injury repair and anti-scarring, characterized in that: It includes the following steps: Mix and react the bioactive conjugated linoleic acid with a mixed solution of an alkali solution and a soluble salt. Under the action of slow stirring, the conjugated linoleic acid is added to the mixed solution of the alkali solution and the soluble salt in small amounts and multiple times, and stirred uniformly to carry out an emulsification reaction. Observe the sample state and exothermic phenomenon during stirring. The temperature during the reaction is 36 - 40 °C to obtain a conjugated linoleate solution, and adjust the pH value of the conjugated linoleate solution to 5 - 8 with an acid; the soluble salt is a soluble calcium salt or a soluble zinc salt; In the mixed solution of the alkali solution and the soluble salt, the concentration of the soluble salt is 1% - 10%, and % is the mass percentage; The concentration of the alkali solution is 1% - 20%; Add the conjugated linoleate solution to the bio-based gel material. The mass ratio of the conjugated linoleate to the bio-based gel material is 1:10 - 50, and after mixing evenly and reacting, a conjugated linoleate gel is obtained; The alkali solution is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide or cesium hydroxide.

2. The preparation method of the material for promoting injury repair and anti-scar according to claim 1, wherein: The soluble salt is a mixture of calcium chloride and zinc chloride, and the mass ratio of calcium chloride to zinc chloride is 1 - 10:

1.

3. The preparation method of the material for promoting injury repair and anti-scarring according to claim 1, characterized in that: The acid is hydrochloric acid, glacial acetic acid, citric acid or lactic acid.

4. The preparation method of the material for promoting injury repair and anti-scar as claimed in claim 3, wherein: The alkali solution is sodium hydroxide or potassium hydroxide.

5. The preparation method of the material for promoting injury repair and anti-scar according to claim 1, wherein: The bio-based gel material is selected from one, two or more of natural collagen, recombinant collagen, sodium hyaluronate, and extracellular matrix.

6. The preparation method of the material for promoting injury repair and anti-scar according to claim 1, characterized in that: It also includes the step of preparing a sponge for promoting injury repair and anti-scar: load the conjugated linoleate gel into a mold and freeze-dry it gradient to obtain a conjugated linoleate sponge; Thermally crosslink the conjugated linoleate sponge at 100 - 120 °C for 10 - 100 h to obtain the sponge for promoting injury repair and anti-scar.

7. The preparation method of the material for promoting injury repair and anti-scarring according to claim 6, characterized in that: It also includes the step of encapsulating the prepared conjugated linoleate gel or the sponge for promoting injury repair and anti-scar in a medical packaging bag and sterilizing it with 6 - 20 KGy of γ-rays.

8. The preparation method of the injury-promoting repair and anti-scar material according to claim 6, characterized in that: The material of the mold is Teflon.

9. The preparation method of the material for promoting injury repair and anti-scar according to claim 1, characterized in that: The bio-based gel material is a decellularized extracellular matrix gel, and its preparation method is: scrape the grease on the surface of the animal peritoneal tissue and wash it until there is no grease, and dehydrate it with acetone and degrease it with n-hexane; Mix and enzymatically hydrolyze the degreased membrane tissue with pepsin or nuclease, dialyze the enzymatic hydrolysate to remove immunogenicity, and obtain the extracellular matrix after freeze-drying; Dissolve the extracellular matrix in phosphate buffer to obtain a decellularized matrix gel.

10. The preparation method of the material for promoting injury repair and anti-scar according to claim 9, characterized in that: The mass ratio of the degreased membrane tissue to pepsin is 10 - 100:1; The mass ratio of the degreased membrane tissue to nuclease is 50 - 150:

1.

11. The preparation method of the material for promoting injury repair and anti-scar according to claim 9, characterized in that: The dialysis membrane used for dialysis is a 10 - 50 kDa dialysis membrane.

12. A material for promoting injury repair and anti-scarring, characterized in that: Prepared by the preparation method according to any one of claims 1 - 11.

13. A material for promoting injury repair and anti-scarring according to claim 12, characterized in that: The bio-based gel material is a decellularized extracellular matrix gel.

Images