CN116474160A

CN116474160A - Injury repair promoting and anti-scar material and preparation method thereof

Info

Publication number: CN116474160A
Application number: CN202310204722.8A
Authority: CN
Inventors: 高秀伟; 郑红霞; 高秀岩; 姜红; 任孝敏; 董平格
Original assignee: Shandong Junxiu Biotechnology Co ltd
Current assignee: Shandong Junxiu Biotechnology Co ltd
Priority date: 2023-02-28
Filing date: 2023-02-28
Publication date: 2023-07-25

Abstract

The invention discloses a material for promoting injury repair and resisting scar and a preparation method thereof, comprising the following steps: mixing bioactive conjugated linoleic acid with mixed solution of alkali metal solution and soluble salt (calcium or zinc) to obtain conjugated linoleate solution, and regulating pH value of the conjugated linoleate solution to 5-8 with acid; the soluble salt is soluble calcium salt or soluble zinc salt; and adding the conjugated linoleate solution into the biogenic gel material, and uniformly mixing and reacting the conjugated linoleate and the biogenic gel material to obtain the conjugated linoleate gel, wherein the mass ratio of the conjugated linoleate to the biogenic gel material is 1:10-50.

Description

Injury repair promoting and anti-scar material and preparation method thereof

Technical Field

The invention belongs to the technical field of skin repair materials, and particularly relates to a material for promoting injury repair and resisting scar and a preparation method thereof.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

The large-area injury of the skin, such as knife cuts and scar hyperplasia after burn, seriously affects the recovery of the morphology and the function of the healed patients, and is one of the difficult problems of clinical medicine. Factors such as imbalance of collagen metabolism, proliferation and contraction of fibroblasts, and change of proportion of proteoglycan components in dermis matrix are the basis of hypertrophic scar formation after burn. Currently, related scholars are actively looking for artificial or natural antagonists, such as anti-TGF-interferon, etc. But the results are not satisfactory or have not been used clinically.

The wound healing dressing and the repairing material in the prior art can promote the growth of new epidermal tissues to a certain extent, but the new tissues tend to gradually grow into scars, the tensile strength and the apparent appearance of the scars are greatly different from those of the original skin tissues, the wound healing time is still longer, and especially when the wound healing dressing and the repairing material are used for repairing large-area and deeper wounds, the healed obvious scars can cause adverse effects on the life of patients.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a material for promoting injury repair and resisting scar and a preparation method thereof, and the material can promote wound healing and inhibit formation of hypertrophic scar.

In order to achieve the above object, the present invention is realized by the following technical scheme:

in a first aspect, the invention provides a method for preparing a material for promoting injury repair and resisting scar, which comprises the following steps:

mixing bioactive conjugated linoleic acid with mixed solution of alkali solution and soluble salt (calcium or zinc) to obtain conjugated linoleate solution, and regulating pH value of the conjugated linoleate solution to 5-8 with acid; the soluble salt is soluble calcium salt and/or soluble zinc salt;

and adding the conjugated linoleate solution into the biogenic gel material, and uniformly mixing and reacting the conjugated linoleate and the biogenic gel material to obtain the conjugated linoleate gel, wherein the mass ratio of the conjugated linoleate to the biogenic gel material is 1:10-50.

The biological source gel material provides a support for proliferation of fibroblast, is favorable for reconstruction of collagen components, promotes arrangement and distribution of the fibroblast to form collagen fiber with normal structural morphology, and has the functions of inducing and regulating host cells and new-born blood vessel ingrowth, promoting epithelialization and the like. In addition, extracellular matrix proteins can also promote regeneration of epidermal cells.

Conjugated linoleates have remarkable antioxidant and anti-inflammatory properties. In addition, experimental models have demonstrated that conjugated linoleates are capable of reducing the production of inflammatory mediators, such as arachidonic acid, prostaglandins and histamine, by keratinocytes, resulting in a reduction of itching in the case of atopic dermatitis, and have the effect of maintaining the biological activity of the biogenic gel-like material.

Calcium is used for bone tissue repair; zinc is used for soft tissue repair; the calcium zinc is used for simultaneously repairing soft and hard tissues, such as tooth socket filling after tooth extraction, not only needs to meet the requirement of alveolar bone regeneration, but also needs to realize the rapid repair of gum soft tissues and cover the wound surface.

The mechanism of scar formation is persistent local inflammation and excessive collagen deposition. Mast cells surrounding the wound tissue can stimulate fibroblast proliferation by releasing IL-4, vascular Endothelial Growth Factor (VEGF) and basic fibroblast growth factor (bFGF), resulting in increased type I collagen synthesis. During the proliferation, re-epithelialization and remodeling stages, wound tissue is surrounded by a large number of M2 macrophages that promote angiogenesis and collagen deposition, and M2 macrophages can promote the conversion of fibroblasts to myofibroblasts by secretion of transforming growth factor-beta (TGF-beta) and platelet derived growth factor-CC (PDGF-CC), both of which promote collagen deposition and scarring. TGF-beta 1 mediates expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts. The material prepared by the invention can rapidly promote wound repair, shorten wound healing time, avoid scar formation, and be biodegradable and absorbable by up-regulating the expression of angiogenesis genes (bFGF and VEGF) and down-regulating the expression of fibrosis genes (TGF-beta 1 and alpha-SMA).

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

In some embodiments, the concentration of the soluble salt in the mixed solution of the alkali solution and the soluble salt is 1% -10%, and the% is mass percent;

the concentration of the alkali solution is 1% -20%;

the alkali solution is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide or francium hydroxide.

Preferably, the alkaline solution is selected from sodium hydroxide or potassium hydroxide. Experiments show that when the alkali solution is other alkali solutions such as calcium hydroxide, the alkali solution does not react and delaminates, and the expected product cannot be obtained.

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

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

In some embodiments, the method further comprises the step of preparing a lesion repair, anti-scarring sponge: filling the conjugated linoleate gel into a mould for gradient freeze-drying to obtain conjugated linoleate sponge;

and (3) thermally crosslinking the conjugated linoleate sponge at 100-120 ℃ for 10-100 hours to obtain the injury repair and anti-scar sponge. Not only can the mechanical property of the crosslinked material be satisfied, but also the biological activity of the material can be ensured.

Preferably, the method further comprises the step of packaging the prepared conjugated linoleate gel or the injury repair and anti-scar sponge into a medical packaging bag and sterilizing the medical packaging bag by gamma rays of 6-20 KGy.

Preferably, the material of the mold is teflon material.

In some embodiments, the biogenic gel material is a decellularized matrix gel prepared by: scraping grease on the surface of animal peritoneal tissue, cleaning the animal peritoneal tissue until no grease exists, dehydrating the animal peritoneal tissue by adopting acetone, and degreasing the animal peritoneal tissue by adopting n-hexane;

mixing and carrying out enzymolysis on the defatted membrane tissue and pepsin or nuclease, dialyzing the enzymolysis liquid to remove immunogenicity, and freeze-drying to obtain extracellular matrix;

dissolving the extracellular matrix in phosphate buffer solution to obtain the acellular matrix gel.

Preferably, the gradient freeze-drying is: the mixture is rapidly frozen at the temperature of minus 30 ℃ to minus 80 ℃ and then is gradually heated and dried.

Preferably, the mass ratio of the defatted membrane tissue to pepsin is 10-100:1;

the mass ratio of the defatted membrane tissue to the nuclease is 50-150:1.

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

In a second aspect, the invention provides a damage repair promoting and anti-scarring material prepared by the preparation method.

The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

the scar repairing material product which takes the polydimethylsiloxane as the main component in the prior art only has the anti-scar effect, cannot be used for unhealed wounds, and has no effect of promoting the wound healing. The material prepared by the invention can quickly promote wound repair, shorten wound healing time, avoid scar formation, and be biodegradable and absorbable;

the material prepared by the invention can rapidly guide the natural regeneration of tissues, but not the healing of scars;

the material prepared by the invention has no chemical cross-linking agent, and the conjugate linoleate sponge is prepared by adopting a progressive vacuum freeze-drying and thermal cross-linking technology, so that the sponge is protected to have uniform structure and avoid forming a crystal structure. Meanwhile, chemical cross-linking agent residues possibly cause toxicity risk of organism tissues, and the material prepared by the method has no chemical cross-linking agent, so that the production process chemical residue verification link is reduced; compared with the cross-linking process of ultraviolet rays or gamma rays, the method avoids the destruction of active ingredients in the material. The active ingredients of the conjugated linoleate sponge are protected, and the biocompatibility and the bioactivity of the whole material are improved.

The preparation method comprises the steps of uniformly mixing self-made conjugated linoleate (calcium, zinc and the like) with biological source materials (extracellular matrix, sodium hyaluronate, collagen and the like) for reaction, and the materials are prepared by up-regulating the expression of angiogenesis genes (bFGF and VEGF), down-regulating the expression of fibrosis genes (TGF-beta 1 and alpha-SMA) for promoting injury repair and anti-scarring.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a diagram of a product prepared in an example of the present invention, wherein a is a conjugated zinc linoleate gel and b is a conjugated zinc linoleate sponge;

FIG. 2 is a photograph of a failed zinc conjugated linoleic acid sponge prepared in comparative example 1 of the present invention;

FIG. 3 is a graph showing the anatomical contrast of wound surfaces of 1 week (a), 4 weeks (b) and 13 weeks (c) of a conjugated linoleic acid zinc sponge in an in-vivo degradation experiment according to the embodiment of the invention;

FIG. 4 is a graph comparing a wound surface treated with a zinc conjugated linoleate gel with an untreated wound surface in an example of the present invention;

FIG. 5 is a graph showing comparative experiments of wound healing in rats using a zinc conjugated linoleic acid sponge (group I), a collagen material (group II) and a sodium hyaluronate material (group III) in an embodiment of the present invention;

FIG. 6 is a graph comparing wound healing after 36 days in a wound healing test of rats using a zinc conjugated linoleic acid sponge (group I), a collagen material (group II) and a sodium hyaluronate material (group III) in accordance with an embodiment of the present invention;

FIG. 7 shows RNA expression levels of scar related genes after 15 days of wound treatment with zinc conjugated linoleic acid sponge (group I) in an embodiment of the invention;

FIG. 8 shows the results of detecting bFGF protein level expression of each group after 15 days of wound treatment by immunohistochemistry in the examples of the present invention, wherein a is group I, under epidermis, bFGF,400×; b is group two, under epidermis, bFGF,400×; c is group three, under epidermis, bFGF,400×; d is the trauma group, under epidermis, bFGF,400×.

FIG. 9 shows the results of measuring VEGF protein levels after 15 days of wound treatment using immunohistochemistry in each group according to the present invention, wherein a is group one, subepithelial, VEGF, 400X; b is group two, under epidermis, VEGF,400×; c is group three, under epidermis, VEGF,400×; d is the trauma group, subepidermal, VEGF,400×;

FIG. 10 shows the results of detecting TGF-beta 1 protein levels of each group after 15 days of wound treatment using immunohistochemistry in accordance with the present invention, wherein a is group I, under epidermis, TGF-beta 1, 400X; b is group two, under epidermis, TGF-. Beta.1, 400×; c is group three, under epidermis, TGF- β1, 400×; d is the trauma group, under epidermis, TGF- β1, 400×;

FIG. 11 shows the results of detecting the expression of alpha-SMA protein levels after 15 days of wound treatment for each group using immunohistochemistry in the examples of the present invention, wherein a is group one, alpha-SMA under the epidermis, 400X; b is group two, under the epidermis, α -SMA,400×; c is group three, under the epidermis, α -SMA,400×; d is the trauma group, under the epidermis, α -SMA,400×;

FIG. 12 is a chart of HE staining of pathological sections of wound tissue on day 36 after treatment of the wound with the zinc conjugated linoleic acid sponge (group I) prepared in example 1, according to the invention, at 40X;

FIG. 13 is a Masson staining chart of a pathological section of a wound tissue on day 36 after the treatment of the wound with the conjugated zinc linoleate sponge (group I) prepared in example 1, in an example of the invention, 40X;

FIG. 14 is a chart of HE staining of pathological sections of wound tissue on day 36 after treatment of the wound with collagen material (group II), 40X, in an embodiment of the invention;

FIG. 15 is a map of Masson's staining of a pathological section of wound tissue on day 36 after treatment of the wound with collagen material (group II), 40X, in an example of the invention;

FIG. 16 is a chart of HE staining of a pathological section of wound tissue on day 36 after treatment of the wound with sodium hyaluronate material (group III), 40X in an example of the present invention;

FIG. 17 is a Masson's staining pattern of a pathological section of wound tissue at day 36 after treatment of the wound with sodium hyaluronate material (group III), 40X, in an example of the present invention;

FIG. 18 is a chart of HE staining of pathological sections of wound tissue on day 36 after treatment of the wound with a zinc conjugated linoleic acid sponge (group IV) prepared in comparative example 2, 40X in the examples of the present invention;

fig. 19 is a Masson staining chart of a pathological section of wound tissue on day 36 after the treatment of the wound with the zinc conjugated linoleic acid sponge (group four) prepared in comparative example 2, in the example of the present invention, 40×.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention is further illustrated below with reference to examples.

Example 1

1. Preparation of conjugated calcium linoleate, conjugated zinc linoleate and conjugated calcium zinc linoleate

(1) Preparation of conjugated calcium linoleate: weighing 20g of a mixture of sodium hydroxide and calcium chloride (the mass ratio of the sodium hydroxide to the calcium chloride is 10:1), dissolving in 20mL of water, and uniformly stirring for later use; and (3) measuring 100mL of conjugated linoleic acid in a 250mL beaker, slowly stirring, adding a small amount of mixed solution of sodium hydroxide and calcium chloride for multiple times, stirring at a constant speed of 1000rpm, and performing an emulsification reaction, wherein the sample state and the exothermic phenomenon are observed during stirring, and the temperature is 36-40 ℃.

After 14 days of reaction, the temperature is reduced to room temperature, namely, the reaction end point is reached, and the pasty conjugated calcium linoleate is formed. The pH of the solution is alkaline, glacial acetic acid is adjusted to be neutral, the solution is stored in a glass bottle in a sealing way, and the solution is stored in a refrigerator at 4 ℃ for standby.

(2) Preparation of conjugated zinc linoleate:

weighing 20g of a mixture of sodium hydroxide and zinc chloride (the mass ratio of the sodium hydroxide to the zinc chloride is 10:1), dissolving in 20mL of water, and uniformly stirring for later use; and (3) measuring 100mL of conjugated linoleic acid in a 250mL beaker, slowly stirring, adding a small amount of mixed solution of sodium hydroxide and zinc chloride for multiple times, stirring at a constant speed of 1000rpm, and performing an emulsification reaction, wherein the sample state and the exothermic phenomenon are observed during stirring, and the reaction temperature is 36-40 ℃. After 14 days of reaction, the temperature is reduced to room temperature, namely the reaction end point is reached, the pasty conjugated zinc linoleate is formed, the pH is alkaline, glacial acetic acid is adjusted to be neutral, and the zinc linoleate is stored in a glass bottle in a sealing manner and is stored in a refrigerator at 4 ℃ for standby.

(3) Preparing conjugated calcium zinc linoleate:

weighing 20g of mixture of sodium hydroxide, zinc chloride and calcium chloride (the mass ratio of the sodium hydroxide to the zinc chloride to the calcium chloride is 10:1, and the mass ratio of the zinc chloride to the calcium chloride is 1:9), dissolving in 20mL of water, and uniformly stirring for later use; and additionally measuring 100mL of conjugated linoleic acid in a 250mL beaker, adding a small amount of mixed solution of sodium hydroxide, zinc chloride and calcium chloride for many times under the action of slow stirring, stirring at a constant speed of 1000rpm, and performing emulsification reaction, wherein the sample state and exothermic phenomenon are observed during stirring, and the reaction temperature is 36-40 ℃.

After 14 days of reaction, the temperature is reduced to room temperature, namely the reaction end point is reached, the pasty conjugated calcium zinc linoleate is formed, the pH is alkaline, glacial acetic acid is adjusted to be neutral, the mixture is sealed and stored in a glass bottle, and the mixture is stored in a refrigerator at 4 ℃ for standby.

2. Preparation of extracellular matrix-free gel

(1) Pretreatment: animal peritoneal tissue is scraped to remove grease on the surface of the tissue, and pure water is used for washing until no grease exists. Acetone dehydration and n-hexane degreasing.

(2) Immunogenicity removal: performing enzymolysis on the defatted membrane tissue and pepsin in a ratio of 50:1; the defatted membrane tissue reacts with nuclease in a ratio of 100:1, and a 10kDa dialysis membrane is dialyzed and freeze-dried to obtain the extracellular matrix.

(3) Preparation of 7% acellular matrix gel: 7g of the deimmunized and freeze-dried acellular matrix is dissolved in 0.01M phosphate buffer solution and stirred uniformly to obtain 7% acellular matrix gel.

3. Preparation of conjugated linoleic acid zinc gel

(1) 1g of the prepared conjugated zinc linoleate was dissolved in 350g of 7% acellular matrix gel to obtain a mixture of conjugated zinc linoleate and acellular matrix gel (CG-POSTN), and the mixture was stirred uniformly at 800 rpm.

(2) And (5) subpackaging: 10 mL/bottle;

(3) And (3) sterilization: gamma ray sterilization;

4. preparation of conjugated linoleic acid zinc sponge

(2) And (3) filling: 10 mL/die; the inner diameter of the die is 4.5cm multiplied by 6.5cm by adopting a special die (preferably made of Teflon material); the outer diameter is 7.5cm by 5.5cm.

(3) And (3) drying: rapidly freezing the mixture at-80 ℃; and then heating up and drying step by step, and crosslinking, wherein the drying procedure is as follows: -30 ℃ for 4 hours; -10 ℃,10h;1 ℃ for 4 hours; 10 ℃ for 4 hours.

(4) Thermal crosslinking: the vacuum degree is 50 ℃ for 3 hours under the condition of-1.0 pa; 80 ℃ for 30min;100 ℃ for 1h;110 ℃ for 1h;120 ℃ for 24h.

(5) And (3) sterilization: and adopting gamma rays for sterilization.

Comparative example 1 preparation of Zinc conjugated linoleic acid sponge

1g of the zinc conjugated linoleic acid prepared in example 1 was dissolved in 70g of 7% acellular matrix gel to obtain a mixture of zinc conjugated linoleic acid and acellular matrix gel (CG-POSTN), and the mixture was stirred uniformly at 800 rpm.

(3) And (3) drying: rapidly freezing the mixture at-80 ℃; and then heating up and drying step by step, and crosslinking, wherein the drying procedure is as follows: -30 ℃ for 4 hours, -10 ℃ for 10 hours, and 1 ℃ for 4 hours; and 4 hours at 10 ℃.

(4) Thermal crosslinking: the vacuum degree is 50 ℃ for 3 hours under the condition of-1.0 pa; 80 ℃ for 30min;100 ℃ for 1h;110 ℃ for 1h;120 ℃ for 24 hours;

(5) And (3) sterilization: and adopting gamma rays for sterilization.

The obtained product is shown in figure 2, the proportion of conjugated zinc linoleate is too high, and the film forming effect is poor.

Comparative example 2 preparation of Zinc conjugated linoleic acid sponge

1g of the zinc conjugated linoleic acid prepared in example 1 was dissolved in 850g of 7% acellular matrix gel to obtain a mixture of zinc conjugated linoleic acid and acellular matrix gel (CG-POSTN), and the mixture was stirred uniformly at 800 rpm.

(5) And (3) sterilization: and adopting gamma rays for sterilization.

Example 2 physical and chemical Properties detection of materials

1. Porosity of the material

1.1 measurement method

The porosities of the conjugated zinc linoleate sponge and the collagen sponge (commercially available products) prepared in example 1 are compared, and the gas in the pores inside the material is discharged by a liquid discharge method, namely a method of circularly vacuumizing, and the material is filled with liquid, and the weight of the liquid is calculated as the percentage of the total weight of the material. Weighing about 0.1g (W1) of the dried sample, shearing the dried sample into strips, putting the strips into a volumetric flask containing water, circularly vacuumizing until no bubbles overflow on the surface of the sample and the sample is sunk, weighing the volumetric flask containing the sample and the water (W2), taking out the sample containing the water, weighing the rest volumetric flask and the water (W3), and calculating the porosity (P), wherein the formula is as follows:

P＝(W2-W3-W1)/(W2-W3)×100％。

1.2 test results are shown in Table 1.

TABLE 1

Sample name	Porosity (%)
		Conjugated zinc linoleate film	94.4
Collagen sponge on the market	97.8

1.3 summary: the porosity of the two materials is not different.

2. Residue on ignition

2.1 precisely weighing 1g of the conjugated zinc linoleate sponge material prepared in example 1, placing the sponge material in a crucible which is burnt to constant weight, precisely weighing, slowly burning to complete carbonization, and cooling; adding 1mL of sulfuric acid to moisten, heating at low temperature until sulfuric acid vapor is removed, burning at 600 ℃ to completely ash, transferring into a dryer, cooling, precisely weighing, and then burning at 600 ℃ to constant weight.

2.2 test results, as shown in Table 2:

TABLE 2

Sample name	Conjugated linoleic acid zinc sponge	Comparative example 1	Comparative example 2
				Sulfate content (%)	0.97	4.92	0.31

2.3 summary: according to YY/T1511-2017 collagen sponge 4.5 sulfated ash content not more than 2.0% (mass fraction), the materials prepared in example 1 and comparative example 2 meet the requirements, the material obtained in comparative example 1 does not meet the requirements, and the conjugated linoleic acid zinc content is too high.

3. Total protein content

3.1 accurate weighing of about 10mg of the conjugated zinc linoleate prepared in example 1, (about equivalent to 1.0mg to 2.0mg of nitrogen content), denoted as m ₁ Placing in a digestion tube, adding 0.3g of digestive agent and 2.0mL of concentrated sulfuric acid, placing on an electrothermal digestion stove, digesting in a fume hood until the solution becomes clear, and continuing digestion for 60min. Blank digestion control was also performed.

3.2 taking 10mL of 2% boric acid absorption liquid, placing the 10mL into a 250mL conical flask, and immersing the end of a condensation tube of the azotometer into the boric acid absorption liquid. The digested sample (m ₁ ) Transferring into a nitrogen fixing tube, washing the digestion tube with a small amount of water for 3-4 times, transferring the washing solution into the nitrogen fixing tube, adding 10mL of 50% sodium hydroxide, and then distilling. And (3) removing the tail end of the condensing tube from the liquid level after the total volume of the receiving liquid is about 35-50 mL, allowing the steam to continue flushing for about 1min, flushing the tail end of the condensing tube with a small amount of distilled water, and stopping distillation. For receiving liquidsTitration is carried out on 0.005mol/L sulfuric acid titration solution until the solution changes from blue-green to grey-purple, and the volume V of the consumed sulfuric acid titration solution is recorded ₁ . The digested sample (m ₂ ) Transferring into nitrogen fixing tube, repeating the above distillation and titration steps, and recording volume V of consumed sulfuric acid titration solution ₂ . Transferring blank digestive control into nitrogen fixing tube, repeating the above distillation and titration steps, and recording volume V of consumed sulfuric acid titration solution ₀ Correction was performed with a blank test.

3.3 calculation of results

The total nitrogen content in the sample was calculated as follows:

wherein:

W ₁ : total nitrogen content in the sample,%.

V ₁ : titration sample m ₁ The volume of sulfuric acid titrant, mL, was consumed.

V ₀ : blank titration consumed sulfuric acid titration volume, mL.

m ₁ : sample mass, mg.

c: sulfuric acid titration concentration, mol/L.

m ₀ Sample loss on drying,%.

3.4 test results are shown in Table 3:

TABLE 3 Table 3

Project	Conjugated linoleic acid zinc sponge
		Protein content,%	93.3±1.0％

3.5 nodules: according to the content of 4.7 proteins in YY/T1511-2017 collagen sponge, the protein content in the collagen sponge is not less than 90%, and the sample meets the requirements.

4. In vitro degradation test

4.1 weighing about 10mg of the conjugated zinc linoleate sponge prepared in example 1, immersing the sponge in a 15mL centrifuge tube containing water, slightly pressing the sponge with a glass rod until the sponge is completely immersed, taking out the sponge, removing excessive water with filter paper, putting the sponge into the centrifuge tube, and putting 10mL of hydrochloric acid solution (hydrochloric acid concentration is 0.1 mol/L) of pepsin which is preheated to 37+/-1 ℃ and has the mass fraction of 1% into the centrifuge tube in advance. The time of sample digestion was observed at 37 ℃ ± 1 ℃, shaking at about 90 rpm until complete digestion.

4.2 test results are shown in Table 4:

TABLE 4 Table 4

Sample name	Conjugated linoleic acid zinc sponge
		Digestion time (h)	17.5±1.0

4.3 summary: under the test condition, the digestion time of the conjugated linoleic acid zinc sponge is not longer than 24 hours, and the degradability of the conjugated linoleic acid zinc sponge is supported.

Example 3 Material safety Performance detection

Cytotoxicity assay

1.1 negative control test solution: i.e.cell culture broth, MEM containing 10% fetal bovine serum.

1.2 sample test solution: 0.08g of the conjugated zinc linoleate sponge sample prepared in example 1 was extracted (24.+ -. 2) in 2mL of cell culture medium at 37.+ -. 1 ℃ under shaking at 60r/min for h.

1.3 positive control test solution: 10% DMSO solution, ready to use.

1.4MTT solution: MTT powder is prepared into MTT solution with the mass concentration of 1mg/mL, and the MTT solution is sterilized by a sterile filter (the pore diameter is less than or equal to 0.22 mu m) for standby.

1.5 placing the negative control test solution, the sample test solution and the positive control test solution in the wells of the cell culture plate in which L929 mouse fibroblasts were cultured, respectively, in 5% CO ₂ Culturing in a cell culture incubator at 37 ℃ for 24 hours, observing the morphological changes of the cells after culturing of a test sample group, a negative control group and a positive control group under a microscope, carefully removing the culture medium, adding 50 mu L of MTT solution into each hole, continuously culturing in the cell culture incubator for 2 hours, discarding liquid in the holes, adding 100 mu L of DMSO into each hole, shaking the plate, measuring absorbance at 570nm (reference wavelength 650 nm) by using an enzyme-labeled instrument, and calculating the survival rate (%) according to the following formula.

Wherein:

OD570 e-absorbance of each test sample group (sample group, positive control group);

OD570 b-vehicle control absorbance.

And the cell viability of each test group (sample group, positive control group) relative to the medium control group was determined by MTT method.

1.6 test results are shown in the following table:

sample name	Positive control	Conjugated linoleic acid zinc sponge
			Survival (%)	19.8±0.1	115.5±0.3

1.7 nodules: under the test condition, the survival rate of the conjugated linoleic acid zinc sponge cells is 115.5+/-0.3 percent, and the conjugated linoleic acid zinc sponge cells have no cytotoxicity.

Example 4 in vivo degradation detection of materials

1. Animal test method: after SD rats are selected for anesthesia, the backs are shaved, medical iodophor is used for disinfection, four skin incisions are made along the two sides of the central line of the backs, forceps at the incisions are used for probing, subcutaneous sacs are prepared through blunt separation, and the bottoms of the sacs are 10mm away from the incisions and are respectively marked as (1), (2), (3) and (4). (1) (2) and (3) placing a conjugated zinc linoleate sponge prepared in example 1 into each capsule, and cutting the conjugated zinc linoleate sponge to a size of 1cm multiplied by 1cm. (4) As a blank, no treatment was performed. The incision is closed with absorbable sutures to ensure that the implants do not contact each other.

Implant site location and implant sampling time as shown in the following table:

(4) blank space	(1) -1 week
		(3) -13 weeks	(2) -4 weeks

2. The wound tissue was anatomically observed for 1 week, 4 weeks and 13 weeks, and after 1 week the sample was found to be evident as subcutaneous undegraded material, after 4 weeks the sample was found to be a small fraction of undegraded material, and after 13 weeks the material was completely degraded, see fig. 3.

3. The small knot: the conjugated linoleic acid zinc sponge can be completely degraded within 13 weeks, and the degradation performance of the conjugated linoleic acid zinc sponge is supported.

Example 5 Performance test of materials on scar repair

1. Establishing rabbit ear scar model

Adult healthy rabbits are selected, the ears of the rabbits are sterilized by using 75% ethanol, the ears of the rabbits are scratched by a surgical knife, and the wound is about 2cm. On the 6 th day after operation, the wound surface forms scab. The wound surface is cleaned by using 75% ethanol, the conjugated zinc linoleate gel prepared in the example 1 is smeared at the wound surface of the left ear of a rabbit on the 21 st day after operation, and the right ear is not treated.

2. Repair test of scar by conjugated linoleic acid zinc gel

2.1 from the 21 st day after operation, the wound surface is cleaned by using 75% ethanol, the wound surface of the scar of the left ear of the rabbit is smeared with the conjugated zinc linoleate gel prepared in the example 1, and the right ear is not treated at all once a day.

2.2 after operation, the healing condition of the left ear and the right ear is observed on the 45 th day, the scar is healed after the conjugated zinc linoleate gel is smeared on the left ear of the rabbit, and the untreated wound surface of the right ear has obvious scar, as shown in figure 4.

2.3 summary: under the test condition, the conjugated linoleic acid zinc gel has repair performance on the formed scar.

EXAMPLE 6 test of repair Performance against defective rat skin

1. Two wound marks are respectively made on the left side and the right side of the back of a rat by using a circular scale mould with the diameter of 1cm, the marked back tissue is cut off by a scalpel, the left side and the right side of the same rat are filled with the same material, different rats use different materials for a wound healing test, and a blank control group and a wound control group are added at the same time.

2. The materials were grouped as follows:

grouping	Filling material
		Group one	Zinc conjugated linoleate sponge prepared in example 1
Group II	Collagen material
		Group III	Sodium hyaluronate material
Group IV	Conjugate zinc linoleate sponge prepared in comparative example 2

4. And (3) experimental observation: the wound healing conditions of 1,4,7, 15, 21 and 36 days are respectively selected from different materials, the size of the wound is measured by using a flexible ruler on the 4 th day after the operation of the rat, the size of the wound is similar to the size during the operation, and the wound is basically unchanged. On day 7 after rat operation, the wound surface is measured by a flexible rule, and the wound surface is reduced and has a diameter of about 0.5cm. On day 15 post-surgery rats, the first wound had a circular defect of approximately 0.1cm in diameter, and the second and third wounds had circular defects of approximately 0.3cm in diameter. The rat is provided with a linear defect of about 0.1cm multiplied by 0.5cm on the first wound, a round defect of about 0.2cm multiplied by 0.3cm on the second wound, a round defect of about 0.2cm multiplied by 0.3cm on the third wound, and all wound tissues are taken on the 36 th day after the operation to be pathological sections. The wound healing situation is shown in fig. 5.

On day 36 post-surgery, the first and fourth groups of skin recovered substantially, and the second and third groups had scarring, as shown in fig. 6.

And taking the wound tissue on the 15 th day, and evaluating the repairing effect from the related gene expression and protein level. The selected genes are: transforming growth factor beta 1 (TGF-beta 1), alpha smooth muscle actin (alpha-SMA), basic fibroblast growth factor (bFGF), vascular Endothelial Growth Factor (VEGF). Taking the pathological section of the wound tissue on the 36 th day, and carrying out HE staining.

As shown in fig. 7, in early and mid stages of epidermal formation, the expression level of the angiogenesis genes (bFGF and VEGF) in each treatment group (group one, group two, group three) was higher than that in the blank control group and the wound control group, and the group one was the greatest in the up-regulation amplitude, significantly higher than that in the wound group; the expression level of the fibrosis genes (TGF-beta 1 and alpha-SMA) in each treatment group (group I, group II and group III) was lower than that in the blank control group and the wound control group, and the downregulation amplitude of the groups was significantly higher than that in the wound group. Up-regulation of angiogenic genes (bFGF and VEGF) and down-regulation of fibrotic genes (TGF- β1 and α -SMA) indicate that the material has anti-scarring activity during epidermal formation. As shown in figures 8-11, the immunohistochemical results are consistent with the corresponding gene expression results, and the anti-scarring effect of the material is verified from the gene expression and protein level.

HE staining results are shown in FIGS. 12-19. The group of sections are all seen in healing of sores and ulcers, and the epidermis is keratinized stratified flat epithelium; below it is fibrous tissue, masson stained, shown to consist mainly of collagen fibers. The two sections of the group are all healed by the sore and the epidermis is keratinized stratified flat epithelium, but the local dermis layer is thinner, and is presumed to be the skin adjacent to the sore and the sore; below it is fibrous tissue, masson stained, shown to consist mainly of collagen fibers. The three sections of the group are all healed by sores, the epidermis is keratinized stratified flat epithelium, but the local dermis layer is thinner, and is presumed to be the skin adjacent to the sores; below it is fibrous tissue, masson stained, shown to consist mainly of collagen fibers. The group four sections are all healed by sores and ulcers, and the epidermis is keratinized stratified flat epithelium; below it is fibrous tissue, masson stained, shown to consist mainly of collagen fibers. The junction of dermis and subcutaneous tissue is seen as inflammatory cell localized infiltrates (within the dashed line).

It can be seen that the recovery effect of the first wound surface is better than that of the second, third and fourth wound surfaces, the wound surface tissue heals faster, and the anti-scar effect is better. And in the fourth group, the content of the conjugated linoleic acid zinc is too low, inflammatory cell infiltration occurs, and the overall repair effect is poorer than that of the first group. The test proves that the group of conjugated zinc linoleate sponges have anti-scar effect.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of a material for promoting injury repair and resisting scar is characterized by comprising the following steps: the method comprises the following steps:

mixing bioactive conjugated linoleic acid with mixed solution of alkali solution and soluble salt to obtain conjugated linoleate solution, and regulating pH value of the conjugated linoleate solution to 5-8 with acid; the soluble salt is soluble calcium salt or soluble zinc salt;

2. The method for preparing the injury repair and scar-preventing material according to claim 1, wherein the method comprises the following steps: the soluble salt is a mixture of calcium chloride and zinc chloride, and the mass ratio of the calcium chloride to the zinc chloride is 1-10:1;

or, in the mixed solution of the alkali solution and the soluble salt, the concentration of the soluble salt is 1% -10%, and the% is mass percent;

the concentration of the alkali solution is 1% -20%.

3. The method for preparing the injury repair and scar-preventing material according to claim 1, wherein the method comprises the following steps: the acid is hydrochloric acid, glacial acetic acid, citric acid or lactic acid;

or, the alkali solution is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide or francium hydroxide;

preferably, the alkali solution is sodium hydroxide or potassium hydroxide.

4. The method for preparing the injury repair and scar-preventing material according to claim 1, wherein the method comprises the following steps: the biological source gel-like material is selected from one, two or more of natural collagen, recombinant collagen, sodium hyaluronate and extracellular matrix.

5. The method for preparing the injury repair and scar-preventing material according to claim 1, wherein the method comprises the following steps: the method also comprises the steps of preparing the injury repair promotion and anti-scar sponge: filling the conjugated linoleate gel into a mould for gradient freeze-drying to obtain conjugated linoleate sponge;

and (3) thermally crosslinking the conjugated linoleate sponge at 100-120 ℃ for 10-100 hours to obtain the injury repair promoting and anti-scar sponge.

6. The method for preparing the injury repair and anti-scarring material according to claim 5, wherein the method comprises the following steps: the method also comprises the steps of packaging the prepared conjugated linoleate gel or the injury repair promotion and anti-scar sponge into a medical packaging bag and sterilizing by gamma rays of 6-20 KGy.

7. The method for preparing the injury repair and anti-scarring material according to claim 5, wherein the method comprises the following steps: the mold is made of Teflon.

8. The method for preparing the injury repair and scar-preventing material according to claim 1, wherein the method comprises the following steps: the biological source gel material is extracellular matrix gel, and the preparation method comprises the following steps: scraping grease on the surface of animal peritoneal tissue, cleaning the animal peritoneal tissue until no grease exists, dehydrating the animal peritoneal tissue by adopting acetone, and degreasing the animal peritoneal tissue by adopting n-hexane;

9. The method for preparing the injury repair and scar-preventing material according to claim 8, wherein the method comprises the following steps: the mass ratio of the defatted membrane tissue to the pepsin is 10-100:1;

the mass ratio of the defatted membrane tissue to the nuclease is 50-150:1;

10. An injury repair promoting and anti-scar material is characterized in that: prepared by the preparation method of any one of claims 1 to 9;

or the injury repair promoting and anti-scar material is conjugated linoleate gel, which comprises conjugated linoleate and biological source gel material, wherein the mass ratio of the conjugated linoleate to the biological source gel material is 1:10-50;

the conjugated linoleate is conjugated linoleic acid calcium salt or zinc salt;

preferably, the biogenic gel material is extracellular matrix-free gel;

or the injury repair promoting and anti-scar material is conjugated linoleate sponge, and the conjugated linoleate gel is prepared by freeze-drying.