CN111643712A

CN111643712A - Wound dressing and preparation method thereof

Info

Publication number: CN111643712A
Application number: CN202010524899.2A
Authority: CN
Inventors: 不公告发明人
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2020-09-11

Abstract

The invention discloses a wound dressing and a preparation method thereof, wherein the wound dressing comprises the following raw materials, by weight, 10-15 parts of pearl oyster mantle zymolyte, 0.5-2 parts of sodium carboxymethylcellulose, 2-5 parts of alginate, 3-8 parts of glycerol, 0.3-1.5 parts of isoprene glycol and 40-60 parts of water. The pearl oyster mantle zymolyte dressing has good biocompatibility, forms specific and stable biological protein bionic mucous membrane, provides wound healing nutrient substances, provides a moderate humid environment, promotes the release of growth factors, stimulates the proliferation of cells, improves the regeneration capacity and cell movement of epidermal cells, promotes the healing of wounds and the repair of wound tissues of cavitary tracts, and is suitable for the repair of the wounds of natural cavitary tracts and skin of a human body.

Description

Wound dressing and preparation method thereof

Technical Field

The invention relates to the technical field of medical biomaterials, in particular to a wound dressing and a preparation method thereof.

Background

Wound repair refers to a series of pathophysiological processes in which local tissues are repaired by regeneration, repair and reconstruction after the tissues are lost due to the action of injury-causing factors. The body's inherent defensive adaptive response to tissue cell damage caused by the action of various deleterious factors.

When the surface of the wound surface is shallow and the histiocyte is slightly lost, the loss can be supplemented by the division and proliferation of the histiocyte of the same kind, so that the tissue cell has the same structure and function and forms complete pathological regeneration. However, when there is a great amount of tissue cells missing, the body is often repaired by using foreign materials to replace the tissue, so that the original tissue structure and function are lost, and incomplete pathological regeneration is formed. In the current wound repair process, the majority of clinical cases are regeneration of this type.

When regenerating replacement tissue, it is desirable to provide a good environment for wound repair. The wound dressing has the potential hydrophilic property, and the water absorption and moisture retention properties are used for absorbing wound exudate or discharging water to the wound, so that a moist healing environment is provided for the wound, and the granulation tissue growth and epithelial cell crawling are protected and promoted. The dressing has film forming property, forms a protective layer on the surface of a wound surface and plays a role of a physical barrier. Therefore, the wound repair wound dressing becomes a preferred material in clinical use at present. The existing wound dressing product is mainly prepared from materials such as collagen, chitosan, hyaluronic acid, alginate and the like, has the effects of stopping bleeding and promoting coagulation, partially induces various cells to proliferate and differentiate, and has the defects of poor stability, weak capability of bioactive substances and the like.

By inquiring a national medical appliance product database, the active wound dressing product without marine shellfish source extract can promote the healing performance of the wound, has better compatibility with injured tissues, can be dissolved in a cell culture medium of skin fibroblasts, and is beneficial to promoting the healing of the wound.

The main marine shell species of pearls cultivated in coastal areas are adopted, pearl sacs are formed by cell proliferation of mantle through nucleus insertion of the pearls, and the mantle of the pearl shells plays an important role in pearl formation by secreting nacres. At present, most of pearl shellfish meat is discarded after pearl picking, and the economic added value is not high. The main nutrient component of the pearl oyster mantle is multi-protein (mostly collagen), so the development and utilization of the pearl oyster mantle function are very necessary.

Disclosure of Invention

Based on the technical problems in the background art, the invention provides a wound dressing and a preparation method thereof.

The invention provides a wound dressing which comprises the following raw materials in parts by weight: 10-15 parts of pearl shell mantle zymolyte, 0.5-2 parts of sodium carboxymethylcellulose, 2-5 parts of alginate, 3-8 parts of glycerol, 0.3-1.5 parts of isoprene glycol and 40-60 parts of water.

Preferably, the pearl oyster skin enzymolysis product comprises 15 parts by weight of pearl oyster mantle zymolyte, 2 parts by weight of sodium carboxymethylcellulose, 5 parts by weight of alginate, 8 parts by weight of glycerol and 1.5 parts by weight of isoprene glycol.

Preferably, the pearl oyster skin care solution comprises, by weight, 12 parts of pearl oyster mantle zymolyte, 2 parts of sodium carboxymethylcellulose, 2 parts of alginate, 3 parts of glycerol and 1.5 parts of isoprene glycol.

Preferably, the pearl oyster skin care solution comprises, by weight, 10 parts of pearl oyster mantle zymolyte, 0.5 part of sodium carboxymethylcellulose, 5 parts of alginate, 8 parts of glycerol and 0.3 part of isoprene glycol.

Preferably, a method for preparing a wound dressing is carried out according to the following steps:

s1: firstly, preparing pearl shell mantle zymolyte, adding water into the pearl shell mantle as a raw material according to a ratio of 1:4, adjusting the pH value to be the most suitable for enzyme, adding the enzyme according to 3500-plus 5800U/g protein, carrying out enzymolysis reaction at 42-50 ℃, stirring at a rotating speed of 200r/min, and carrying out enzymolysis for 2-4 h;

s2: taking supernatant of the stirring liquid in the S1, adding a certain amount of NaCl, standing, filtering, placing in a dialysis bag MD34(1000), dialyzing, desalting, and purifying for 12 h;

s3: carrying out vacuum freeze drying on the pearl oyster mantle zymolyte purified in the step S2 for later use;

s4: sequentially adding weighed sodium carboxymethylcellulose, alginate, glycerol and isoprene glycol into water, stirring and dispersing uniformly, and then adding triethanolamine serving as a regulator to regulate the pH value to 5.0-7.0 to form a substrate solution;

s5: heating the S4 substrate solution to 45 deg.C, adding the Concha Margaritifera mantle zymolyte prepared from S1-S3, and dissolving to obtain wound dressing.

The hydrolysis degree of the pearl oyster mantle zymolyte prepared from the S1-S3 is 15.2-19.6%, the protein content of the pearl oyster mantle zymolyte prepared from the S1-S3 is 0.576-0.683g/g, and the NaCl solution purified from the S2 is 0.1-0.3 mol/l.

The beneficial effects of the invention are as follows:

1. according to the pearl oyster mantle zymolyte dressing, an enzyme method is adopted to prepare a pearl oyster mantle zymolyte product, and the pearl oyster mantle zymolyte dressing has bioactive peptides with high activity, specificity and stability, is mainly composed of protein, has the highest proportion of collagen, is V-shaped which is different from fish skin and terrestrial collagen, has different functional activities, is prepared as a molecular bionic material of a marine shellfish source, and provides nutrient substances for wound healing;

2. in the invention, because of the hydrophilic property of the alginate, the alginate has better compatibility with injured tissues, can be dissolved in a cell culture medium of skin fibroblasts, is beneficial to promoting the healing of wounds, and the glycerol has the water absorption and moisture retention property and is used for absorbing wound exudate or discharging water to the wounds, thereby providing a moist healing environment for the wounds, protecting and promoting the growth of granulation tissues and promoting the creeping of epithelial cells, forming the film forming property of the sodium carboxymethyl cellulose, forming a protective layer on the surfaces of the wounds and playing a role of a physical barrier;

3. according to the invention, the mantle enzymatic hydrolysate of the pearl oyster can inhibit the secretion of I-type collagen of keloid fibroblasts, so that the proportion of I-type and III-type collagen in the keloid fibroblasts is reduced, the secretion of I-type and III-type collagen in normal skin fibroblasts is not influenced, and the pearl oyster mantle enzymatic hydrolysate has excellent performances of promoting wound healing and reducing scars.

Drawings

FIG. 1 is a table of cytotoxic samples for a wound dressing and method of making the same according to the present invention;

FIG. 2 is a table of cytotoxicity test results of a wound dressing and a method of making the same according to the present invention;

FIG. 3 is a table of the results of genital tract wound stimulation tests using the wound dressing and the method of preparing the same according to the present invention;

fig. 4 is a comparative graph of the use effect of the wound dressing and the preparation method thereof.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Example 1

The invention provides a wound dressing which comprises the following components, by weight, 15 parts of pearl oyster mantle zymolyte, 2 parts of sodium carboxymethylcellulose, 5 parts of alginate, 8 parts of glycerol and 1.5 parts of isoprene glycol.

The invention also provides a preparation method of the wound dressing, which is carried out according to the following steps: firstly, preparing a pearl shell mantle zymolyte, adding water into the pearl shell mantle serving as a raw material according to a ratio of 1:4, adjusting the pH to be the most suitable for enzyme, adding the enzyme according to 3500U/g protein, carrying out enzymolysis reaction at 42 ℃, and setting the rotating speed of a stirrer to be 200r/min for enzymolysis for 2 hours; taking supernatant, adding 0.3mol/l NaCl, standing, filtering, placing in a dialysis bag MD34(1000) for dialysis and desalting, purifying for 12h, and carrying out vacuum freeze drying on the zymolyte with the hydrolysis degree of 15.2% and the protein content of 0.576g/g for later use.

Adding 2 parts of sodium carboxymethylcellulose, 5 parts of alginate, 8 parts of glycerol and 1.5 parts of isoprene glycol into water in sequence, stirring and dispersing uniformly, adding a triethanolamine regulator to adjust the pH to 5.0, heating to 45 ℃, adding 15 parts of prepared pearl oyster mantle zymolyte, and dissolving to obtain the wound dressing A1.

Example 2

The invention provides a wound dressing which comprises, by weight, 12 parts of pearl oyster mantle zymolyte, 2 parts of sodium carboxymethylcellulose, 2 parts of alginate, 3 parts of glycerol and 1.5 parts of isoprene glycol.

The invention also provides a preparation method of the wound dressing, which is carried out according to the following steps: firstly, preparing a pearl shell mantle zymolyte, adding water into a pearl shell mantle which is taken as a raw material according to a ratio of 1:4, adjusting the pH value to be the most suitable for enzyme, adding the enzyme according to 4000U/g protein, carrying out enzymolysis reaction at 48 ℃, and setting the rotating speed of a stirrer to be 200r/min for enzymolysis for 3 hours; taking supernatant, adding 0.2mol/l NaCl, standing, filtering, placing in a dialysis bag MD34(1000) for dialysis and desalting, purifying for 12h, wherein the hydrolysis degree after purification is 16.4%, and the protein content is 0.623g/g, and performing vacuum freeze drying on the zymolyte for later use.

2 portions of sodium carboxymethylcellulose, 2 portions of alginate, 3 portions of glycerol and 1.5 portions of isoprene glycol are weighed and then added into water, stirred and dispersed evenly, and then triethanolamine regulator is added to adjust the pH value to 5.0. Heating to 45 deg.C, adding 12 parts of prepared mantle zymolyte of pearl shell, and dissolving to obtain wound dressing A2.

Example 3

The invention provides a wound dressing which comprises, by weight, 10 parts of pearl oyster mantle zymolyte, 0.5 part of sodium carboxymethylcellulose, 5 parts of alginate, 8 parts of glycerol and 0.3 part of isoprene glycol.

The invention also provides a preparation method of the wound dressing, which is carried out according to the following steps: firstly, preparing a pearl shell mantle zymolyte, adding water into a pearl shell mantle which is taken as a raw material according to a ratio of 1:4, adjusting the pH value to be the most suitable for enzyme, adding the enzyme according to 5800U/g protein, carrying out enzymolysis reaction at 50 ℃, and setting the rotating speed of a stirrer to be 200r/min for enzymolysis for 4 hours; taking supernatant, adding 0.1mol/l NaCl, standing, filtering, placing in a dialysis bag MD34(1000) for dialysis and desalting, purifying for 12h, wherein the hydrolysis degree after purification is 19.6%, and the zymolyte with the protein content of 0.683g/g is subjected to vacuum freeze drying for later use.

0.5 part of sodium carboxymethylcellulose, 5 parts of alginate, 8 parts of glycerol and 0.3 part of isoprene glycol are sequentially added into water, stirred and dispersed uniformly, then triethanolamine regulator is added to regulate the pH value to 5.0, the temperature is raised to 45 ℃, 10 parts of prepared pearl oyster mantle zymolyte are added, and the wound dressing A3 is obtained after dissolution.

Correlation test

The pearl oyster mantle zymolyte wound dressings A1, A2 and A3 prepared in examples 1, 2 and 3 were selected for the following tests.

(ii) cytotoxicity assay

Dressing samples A1, A2 and A3 are mixed according to the requirement of GB/T16886.12 according to the volume of 1: 1 adding physiological saline for leaching. Placing the material into a leaching container, wherein the leaching temperature is 37 +/-1 ℃, and the leaching time is 48 h. The leaching solution was sterilized by filtration through a filter having a mesh size of 0.22 μm, and then subjected to MTT method for cytotoxicity test.

Referring to fig. 1, the survival rates of the cytotoxic samples relative to the blank cells are all greater than 70%, which indicates that none of the three wound dressings has potential cytotoxicity. The survival rates of the test sample of 50 percent and 100 percent of leaching liquor are higher, and repeated tests are not needed.

The fibroblasts cultured by the dressing leach liquor have normal cell morphology, good adherent growth, basically no shrinkage, suspension and death, no obvious reduction of cell density, no inhibition of cell growth, and good growth state of each group of cells. The samples have no cytotoxicity, and meet the cytotoxicity requirement of national standard on medical materials.

(II) cytotoxicity assay

Wound dressing samples A1, A2 and A3 were taken and tested according to GB/T16886.11, part 11 of the biological evaluation of medical devices: the acute systemic toxicity test recommended in the systemic toxicity test "standard requires the development of a dressing biocompatibility evaluation. 20 mice were randomly divided into sample groups and negative control groups, each group consisting of 5 mice. The sample group and the negative control group are respectively injected with the tested sample leaching solution and the negative control solution from the abdominal cavity according to the injection dosage of 50ml/kg, and then the animals are returned to the respective cages for feeding. Immediately after the injection, the mice were observed for general status, toxicity performance and number of dead animals in the test group and the control group at 4h, 24h, 48h and 72h, and the animal body weight was recorded after the 72h observation.

Referring to fig. 2, the observation results of each animal were obtained, and the animals in the test group did not respond more than the negative control group within the observation period of 72 hours, did not show death and toxicity, had increased body weight, and did not have any difference from the negative control group, as shown in table 2, indicating that the three samples did not have systemic acute toxicity.

(III) genital tract cavity wound surface stimulation test

6 New Zealand female white rabbits (1.8-2.2 kg in weight) were injected with 2ml of control physiological saline and a sample, respectively, into the test animals. After 24h, the animals are killed by an air embolism method, the abdomen is broken, the complete reproductive tract is taken out, the animals are longitudinally cut, and whether congestion, edema and other manifestations exist is visually observed. Then fixing in 10% formalin for 24h, selecting three parts at two ends and center of genital tract, making into slices, HE staining, performing histopathology examination, and evaluating vaginal mucosa irritation score and intensity according to vaginal mucosa irritation reaction score standard table and intensity grading table of Disinfection technical Specification (2002 edition).

Referring to fig. 3, the test result is as follows, the difference between two groups of average integrals of the stimulation response of the genital tract and the cavity mucosa of the test animal is 0, and the stimulation response belongs to the stimulation effect according to the classification table of the stimulation intensity of the vaginal mucosa in the technical Specification for Disinfection (2002).

Therefore, the wound dressing has no obvious cytotoxicity, no irritation and good histocompatibility in vitro, and the residue rate of the wound dressing is less than 10 percent after one month of in vitro enzymolysis. Therefore, the pearl oyster mantle zymolyte dressing prepared by the invention has good biocompatibility, can be used as wound healing promoting dressing, and is suitable for repairing natural cavities and skin wounds of human bodies.

(IV) animal experiments

60 male mice with the body mass of about 18g are randomly divided into a negative control group, a collagen dressing positive control group and a sample A3 pearl oyster mantle zymolyte dressing administration group. Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate, depilated on their backs, and full-cortical wound models of about 0.8cm in diameter were made and housed individually in cages. The administration group is smeared with 30mg of the sample A3 with mass concentration every day, the positive control group is smeared with 30mg of collagen dressing every day, and the negative control group is not treated. The wound surface was observed every other day, the size of the wound was measured, and the results were recorded.

Measuring the diameter of the wound surface for 1 time every 2d, and calculating the wound healing rate: wound healing rate-wound measurement value on day 0-nth/day 0 × 100%; scar reduction rate ═ (day 0-day 14)/day 0 wound measurements × 100%.

Referring to fig. 4, the healing conditions of each group were obtained, and the animals of each experimental group had scabbed without exudation of fluid, and the wound surface changed significantly. The control group wound began to scab on day 8, the wound appeared dark red, and the positive control group had some scabs removed. The wound surface of the administration group is completely scabbed and becomes flesh red and new skin. The scab part of the control group wound fell off at the 10 th day, the scar area change of the negative control group was small, and the scab shrinkage of the positive control group was obvious. The wound surface skin of the administration group gradually tends to be normal skin. On day 12, each group had completely removed the scab and further scab was reduced. The positive control group had a secondary scab removal and the healed area of the administered group began to grow hair. On day 14, most of the wounds in the control group were less than 2mm in diameter, and the wounds in the administration group were all 0mm in diameter. The wound surface of the control group appeared to be flesh red new skin, and the new flesh color tissue was rough. Compared with a control group, the healing condition of the administration group is good, the wound surface has new skin and is smooth, no scar is generated, and the administration group has no difference with normal skin. The results show that the administration group has obvious effects on eschar removal and scar contraction, has related functional active peptides to play main roles, and is beneficial to healing of skin wound and scar reduction.

Preparing mouse wound tissue homogenate, subpackaging and storing at-80 ℃, and measuring related indexes according to a kit instruction. FGF-2 has stimulatory effects on the growth and differentiation of fibroblasts, the proliferation of vascular smooth muscle cells, endothelial cells, the metabolism, growth of extracellular matrix, and the motility of cells of mesodermal origin. Increase the rate and extent of granulation tissue formation and stimulate the healing process.

The pearl oyster mantle zymolyte dressing is closely related to the formation, maturation, regeneration and repair of skin and accessories thereof, promotes angiogenesis and wound repair, and plays a positive biological role in each stage of wound repair. The dressing for smearing the mantle zymolyte of the pearl oyster has no obvious promotion effect on FGF-2 secretion, and TGF-beta helps monocytes to be converted into macrophages, and then the remodeling of granulation tissues is accelerated, the release of wound healing growth factors is promoted, and the synthesis of extracellular matrix and the wound contraction are promoted. Therefore, the wound healing process can be promoted by applying the pearl oyster mantle zymolyte dressing to promote the secretion of collagen by fibroblasts, increase the extracellular matrix deposition and neovascularization. Meanwhile, the expression of cell cycle regulatory protein is accelerated, the cell cycle of fibroblast and the like is promoted to be converted to the mitosis phase, so that the mitosis of the cell is accelerated, and the wound healing effect consistent with the healing rate result is realized.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. The wound dressing is characterized by comprising the following raw materials in parts by weight: 10-15 parts of pearl shell mantle zymolyte, 0.5-2 parts of sodium carboxymethylcellulose, 2-5 parts of alginate, 3-8 parts of glycerol, 0.3-1.5 parts of isoprene glycol and 40-60 parts of water.

2. A wound dressing according to claim 1, comprising, in parts by weight, 15 parts of mantle substrate of pearl oyster, 2 parts of sodium carboxymethylcellulose, 5 parts of alginate, 8 parts of glycerol, and 1.5 parts of isoprene glycol.

3. A wound dressing according to claim 1, comprising, in parts by weight, 12 parts of mantle substrate of pearl oyster, 2 parts of sodium carboxymethylcellulose, 2 parts of alginate, 3 parts of glycerol, and 1.5 parts of isoprene glycol.

4. A wound dressing according to claim 1, comprising, in parts by weight, 10 parts of mantle substrate of pearl oyster, 0.5 part of sodium carboxymethylcellulose, 5 parts of alginate, 8 parts of glycerol, and 0.3 part of isoprene glycol.

5. A method of manufacturing a wound dressing as claimed in any one of claims 1 to 4, in which the method of manufacturing the wound dressing is carried out by the steps of:

s2: taking supernatant of the stirring liquid in the S1, adding a certain amount of NaCl, standing, filtering, placing in a dialysis bag MD34 for dialysis and desalting, and purifying for 12 hours;

6. A wound dressing as claimed in claim 5, wherein the hydrolysate of mantle substrate of pearl shell prepared in S1-S3 has a degree of hydrolysis of 15.2% -19.6%.

7. A wound dressing as claimed in claim 5, wherein the protein content of the mantle substrate of the pearl oyster prepared in S1-S3 is 0.576-0.683 g/g.

8. A method for preparing a wound dressing according to claim 5, wherein the purified NaCl solution in S2 is 0.1-0.3 mol/l.