CN114917396A - Degradable hemostatic and healing-promoting sponge and preparation method and application thereof - Google Patents
Degradable hemostatic and healing-promoting sponge and preparation method and application thereof Download PDFInfo
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- CN114917396A CN114917396A CN202210766391.2A CN202210766391A CN114917396A CN 114917396 A CN114917396 A CN 114917396A CN 202210766391 A CN202210766391 A CN 202210766391A CN 114917396 A CN114917396 A CN 114917396A
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- dermal matrix
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/0005—Ingredients of undetermined constitution or reaction products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0036—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0042—Materials resorbable by the body
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Surgery (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Materials Engineering (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a degradable hemostatic and healing promoting sponge, and a preparation method and application thereof. The degradable hemostatic and heal-promoting sponge is prepared by foaming, molding and freeze-drying a foaming agent and acellular dermal matrix glue; the mass volume ratio of the foaming agent to the acellular dermal matrix adhesive is 0.25-2 g:100 mL. The degradable hemostatic healing-promoting sponge has extremely high porosity, excellent imbibition performance, controllable degradation speed and excellent hemostatic performance, overcomes the defects of high degradation speed, poor imbibition performance and easy collapse of collagen sponge, and overcomes the defect of over-compact acellular dermal matrix. The degradable hemostatic and healing-promoting sponge can be used for preparing hemostatic materials or wound healing materials.
Description
Technical Field
The invention belongs to the field of medical polymer materials, and relates to a degradable hemostatic and heal-promoting sponge, and a preparation method and application thereof.
Background
With the cross fusion of biomedicine and the subjects of material science, chemistry, physics and the like, particularly the continuous development of nanotechnology, the development of novel medical functional materials has made a rapid progress. The application of the functional biological material can not only rebuild the barrier function of the skin, but also actively regulate and control the microenvironment of the wound surface, clear pathogenic microorganisms, promote vascularization and the like, and further accelerate the healing process of the wound surface. In recent years, medical absorbable hemostatic materials have attracted great attention from various medical and industrial fields, and many large-scale pharmaceutical companies are dedicated to research and develop novel hemostatic materials. The hemostatic materials commonly used at present comprise fibrin glue, albumin glue, chitosan, absorbable gelatin sponge, alpha-cyanoacrylate tissue glue and cellulose. The absorbable gelatin sponge is the most common hemostatic material in clinic at present, has long use history, more definite use effect and low price, and is suitable for bleeding, bleeding and deep trauma of trauma and operation. However, the clinical use of the absorbable gelatin sponge has the defects of high secondary infection rate, large tissue foreign body reaction, poor adhesiveness, easy falling and the like.
The porcine acellular dermal matrix is mainly used as a temporary effective covering in the field of wound repair treatment, and is too compact, so that after the porcine acellular dermal matrix is implanted, cells and blood supply cannot permeate the acellular dermal matrix easily, the early nutritional requirement of a transplanted skin can not be effectively maintained, and finally the skin is necrotic and shed. The collagen sponge has excellent hemostatic property and biocompatibility, and can cause platelet aggregation when contacting with a bleeding wound surface, stimulate the platelets to release subcellular particles and blood coagulation factors, and the blood coagulation factors can be actively attached and fill damaged blood vessels, so that the blood coagulation process is promoted. In addition, the collagen can form stable gel on a contact surface, guide wound revascularization, repair damaged wounds and greatly shorten the wound healing time. However, in the application process, the collagen sponge is difficult to maintain the inherent shape under a humid environment, has low porosity and is easy to collapse, and the application is greatly limited.
In view of the defects and limitations of the existing acellular dermal matrix and collagen material, a new method for preparing a new product with high porosity and water absorption rate, which can rapidly and effectively stop bleeding and promote wound coagulation is necessary to be researched.
Disclosure of Invention
The first purpose of the invention is to provide a degradable hemostatic and healing promoting sponge, which has extremely high porosity, excellent imbibition performance, controllable degradation speed and excellent hemostatic performance, overcomes the defects of high degradation speed, poor imbibition performance and easy collapse of collagen sponge, and overcomes the defect of over-compact acellular dermal matrix.
The degradable hemostatic and healing-promoting sponge is prepared by foaming, shaping and freeze-drying a foaming agent and acellular dermal matrix glue; the mass-volume ratio of the foaming agent to the acellular dermal matrix adhesive is 0.25-2 g:100 mL.
According to a further feature of the degradable sponge for hemostasis and healing promotion of the present invention, the foaming agent is one or more selected from egg white powder and rice protein hydrolysate.
According to a further feature of the degradable sponge for hemostasis and healing promotion of the present invention, the acellular dermal matrix glue is prepared by adding acetic acid solutions with different volumes into the acellular dermal matrix and dissolving.
The second purpose of the invention is to provide a preparation method of the degradable hemostatic and heal-promoting sponge.
The preparation method of the degradable hemostatic and healing-promoting sponge comprises the following steps:
A. dispersing the pig acellular dermal matrix into an acetic acid solution, stirring and dissolving to prepare uniform acellular dermal matrix glue;
B. adding a foaming agent into the acellular dermal matrix adhesive obtained in the step A, uniformly mixing, beating with an egg beater until the mixed gel is milky white and has stable volume without expansion, and pouring into a mould for standing and shaping;
C. freezing and shaping at-80 ℃, and then carrying out freeze drying to obtain the degradable hemostatic and healing promoting sponge.
According to a further feature of the preparation method of the present invention, in the step a, the concentration of the acetic acid solution is 0.5% to 2%, the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 10 to 20g:100mL, the stirring time is 1500rpm, and the time is 48 hours.
According to a further feature of the preparation method of the present invention, in the step B, the rotation speed of the eggbeater is 500-1000 rpm for 30-60 s.
According to the further characteristics of the preparation method, in the step C, the temperature of freeze drying is not lower than minus 50 ℃, and the freeze drying time is 48-72 hours; the vacuum degree was 0.1 MPa.
A third object of the present invention is to provide a hemostatic material.
The hemostatic material comprises the degradable hemostatic healing-promoting sponge.
A fourth object of the present invention is to provide a wound healing material.
The wound healing material comprises the degradable hemostatic and healing promoting sponge.
Compared with the prior art, the degradable hemostatic and healing promoting sponge has the following beneficial effects:
(1) according to the preparation method, cheap pigskin is subjected to a previous hypertonic-saline alkali method to prepare an acellular dermal matrix, an acetic acid solution is directly used for dissolving to obtain an acellular dermal matrix adhesive, rice hydrolyzed protein is added, mixed gel is stirred by an egg beater to be beaten and foamed, and finally milky white gel with a homogeneous loose porous structure is formed, and then molding and low-temperature freeze drying are carried out to obtain the sponge. Therefore, compared with the prior art, the preparation method has the advantages of simple preparation process, low cost, simple operation, safety and environmental protection.
(2) The inventor finds out through experiments that the concentration of the acellular dermal matrix adhesive has important influence on the density and the porosity of the hemostatic healing-promoting sponge, and the fiber arrangement of the hemostatic healing-promoting sponge is more and more regular along with the increase of the concentration of the acellular dermal matrix adhesive, the density is gradually increased, the porosity is gradually reduced, the increase of the density is beneficial to improving the structural stability of the hemostatic sponge, and the hemostatic sponge is not easy to break; the decreasing porosity results in a decrease in water absorption capacity. Therefore, through earlier exploration, 10-20% of acellular dermal matrix adhesive (10-20) g is selected in the preparation method of the invention: 100mL, obtaining the hemostatic and heal-promoting sponge with stable structure and high porosity, and the extracellular matrix component is favorable for re-epithelization and remodeling of the wound surface.
(3) The inventor finds out through experiments that the content of the added rice protein hydrolysate has important influence on the porosity and the mechanical property of the sponge for promoting healing and hemostasis, and with the increase of the added rice protein hydrolysate, the fiber arrangement of the sponge for promoting healing and hemostasis is more and more regular, the porosity is gradually increased, but the sponge is soft in texture and extremely easy to collapse, and the water absorption and hemostasis effects of the sponge are influenced. Therefore, the rice protein hydrolysate contained in the sponge for hemostasis and healing promotion prepared by the invention is 0.25% -2%, namely the mass-volume ratio of the rice protein hydrolysate to the acellular dermal matrix gel is (0.25-2) g:100 mL. Most preferably 0.25 percent, the porosity reaches 96.85 to 99.45 percent, the rice protein hydrolysate can rapidly absorb water in blood, adsorb red blood cells and white blood cells, and aggregate platelets, thereby achieving the purpose of rapid and effective hemostasis. The cosmetic composition can be applied to cosmetics, and can exert high-efficiency moisturizing effect, strengthen the defense function of skin, keep normal keratinization of keratinocytes and inhibit bacterial reproduction.
(4) According to different wound parts and wound shapes, the hemostatic and healing promoting sponge prepared by the method can be put into molds of different shapes for pressing treatment, and then irradiation sterilization and vacuum packaging are carried out after pressing, so that sponge supports of different shapes and different thicknesses can be obtained. The specific application method of the invention comprises the following steps: the hemostatic and healing promoting sponge prepared by the method is covered on the surface of the bleeding of the wound, the pressure is applied for 5-15 s by lightly pressing with hands, and then the wound can be stopped by taking off the sponge. The hemostatic sponge stent also can be applied to wounds after debridement, combined with micro negative pressure drainage, can accelerate revascularization of the wound surface, and can directly shift a skin grafting sheet on the prevascularized hemostatic sponge stent in the second stage.
(5) The sponge material for stopping bleeding and promoting healing has light and soft texture and high elasticity, and does not stimulate and rub wounds.
(6) The preparation method provided by the invention is easy to operate, mild in condition, free of any cross-linking agent, free of harmful substance residue, high in safety, simple in process, low in raw material price, easy to operate and easy for large-scale production.
Drawings
FIG. 1 is a diagram illustrating a process for preparing (A) a rice hydrolyzed protein-acellular dermal matrix sponge according to the present invention; (B) is a rough physical diagram of rice protein hydrolysate-acellular dermal matrix gel in different proportions before and after freeze-drying;
FIG. 2 is a schematic diagram of the optimized 0.25% -1% rice hydrolyzed protein-acellular dermal matrix gel before and after freeze-drying;
FIG. 3 is a scanning electron micrograph of a sponge with blood;
FIG. 4 is a graph showing the result of measuring the hydrophilic property of a contact angle of Tschemata;
fig. 5 is a diagram of the test results of the water absorption performance and the moisture retention performance of different hemostatic sponges.
FIG. 6 is a graph of the staining test results of live and dead cells of different hemostatic sponges.
FIG. 7 is a graph showing the results of relative cell viability assays for different hemostatic sponges.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
The first embodiment is as follows: preparation of degradable hemostatic and healing promoting sponge
The degradable hemostatic and healing promoting sponge is prepared by the following steps:
step 1: dispersing the pig acellular dermal matrix into an acetic acid solution, stirring for 48 hours, filtering to remove undissolved acellular dermal matrix, and preparing the acellular dermal matrix glue, wherein the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 10 g:100mL, stirring time 1000rpm, 48 hours. The concentration of the acetic acid solution is 0.5 to 2 percent.
Step 2: uniformly mixing the acellular dermal matrix adhesive treated in the step (1) with rice hydrolyzed protein powder, beating with an egg beater until the mixed gel is milky, injecting the milky mixed gel into a mold, freezing and shaping at-80 ℃, and then performing freeze drying treatment on the milky mixed gel to obtain the hemostatic sponge; wherein the freeze drying time of the freeze dryer is 48-72h, the vacuum degree is 0.1MPa, and the freeze drying temperature is not lower than minus 50 ℃. The rotating speed of the egg beater is 500rpm for 30 s; the mass and volume ratio of the rice protein hydrolysate to the acellular dermal matrix gel is 2g:100 mL.
And 3, step 3: the white porous sponge was soaked in a 70% ethanol solution for 2 hours to remove the residual acetic acid solution.
And 4, step 4: vacuum drying the obtained sponge again, cutting, packaging and Co 60 Irradiating and sterilizing to obtain the degradable hemostatic healing-promoting sponge.
The second embodiment: preparation of degradable hemostatic and healing promoting sponge
The degradable hemostatic and healing-promoting sponge is prepared by the following steps:
step 1: dispersing the porcine acellular dermal matrix into an acetic acid solution, stirring for 48 hours to prepare acellular dermal matrix glue, and filtering to remove undissolved acellular dermal matrix. Wherein the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 20g:100mL, stirring time is 1500rpm, 24 hours; the concentration of the acetic acid solution is 0.5-2%.
Step 2: uniformly mixing the acellular dermal matrix adhesive treated in the step (1) with rice hydrolyzed protein powder, beating with an egg beater until the mixed gel is milky, injecting the milky mixed gel into a mold, freezing and shaping at-80 ℃, and then performing freeze drying treatment on the milky mixed gel to obtain the sponge for stopping bleeding and promoting healing; wherein, the one-time freeze thawing process comprises the following steps: the freeze drying time of the freeze dryer is 48-72h, the vacuum degree is 0.1MPa, and the freeze drying temperature is not lower than minus 50 ℃. The rotating speed of the egg beater is 1000rpm for 60 s; the mass and volume ratio of the rice protein hydrolysate to the acellular dermal matrix gel is 1 g:100 mL.
And step 3: the white porous sponge was soaked in a 70% ethanol solution for 2 hours to remove the residual acetic acid solution.
And 4, step 4: vacuum drying the obtained sponge again, cutting, packaging and Co 60 Irradiating and sterilizing to obtain the degradable hemostatic healing-promoting sponge.
Example three: preparation of degradable hemostatic and healing promoting sponge
The degradable hemostatic and healing-promoting sponge is prepared by the following steps:
step 1: dispersing the pig acellular dermal matrix into an acetic acid solution, stirring for 48 hours to prepare acellular dermal matrix glue, and filtering to remove undissolved acellular dermal matrix. Wherein the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 15 g:100mL, stirring time is 1500rpm, and 24 hours; the concentration of the acetic acid solution is 0.5 to 2 percent.
Step 2: uniformly mixing the acellular dermal matrix glue treated in the step (1) with rice hydrolyzed protein powder, beating with an egg beater until the mixed gel is milky, injecting the milky mixed gel into a mold, freezing and shaping at-80 ℃, and then carrying out freeze drying treatment on the milky mixed gel to obtain the sponge for stopping bleeding and promoting healing; wherein, the one-time freeze thawing process comprises the following steps: the freeze drying time of the freeze dryer is 48-72h, the vacuum degree is 0.1MPa, and the freeze drying temperature is not lower than minus 50 ℃. The rotating speed of the egg beater is 800rpm for 45 s; the mass and volume ratio of the rice protein hydrolysate to the acellular dermal matrix gel is 0.5 g:100 mL.
And 3, step 3: the white porous sponge was soaked in a 70% ethanol solution for 2 hours to remove the residual acetic acid solution.
And 4, step 4: vacuum drying the obtained sponge again, cutting, packaging and Co 60 And (5) irradiating and sterilizing to obtain the degradable hemostatic and healing-promoting sponge.
Example four: preparation of degradable hemostatic and healing promoting sponge
The degradable hemostatic and healing-promoting sponge is prepared by the following steps:
step 1: dispersing the porcine acellular dermal matrix into an acetic acid solution, stirring for 48 hours to prepare acellular dermal matrix glue, and filtering to remove undissolved acellular dermal matrix. Wherein the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 20g:100mL, stirring time is 1000rpm, 36 hours; the concentration of the acetic acid solution is 0.5-2%.
Step 2: uniformly mixing the acellular dermal matrix adhesive treated in the step (1) with rice hydrolyzed protein powder, beating with an egg beater until the mixed gel is milky, injecting the milky mixed gel into a mold, freezing and shaping at-80 ℃, and then performing freeze drying treatment on the milky mixed gel to obtain the sponge for stopping bleeding and promoting healing; wherein, the one-time freeze thawing process comprises the following steps: the freeze drying time of the freeze dryer is 48-72h, the vacuum degree is 0.1MPa, and the freeze drying temperature is not lower than minus 50 ℃. The rotating speed of the egg beater is 750rpm for 60 s; the mass to volume ratio of the rice protein hydrolysate to the acellular dermal matrix gel is 1 g:100 mL.
And step 3: the white porous sponge was soaked in a 70% ethanol solution for 2 hours to remove the residual acetic acid solution.
And 4, step 4: vacuum drying the obtained sponge again, cutting, packaging and Co 60 And (5) irradiating and sterilizing to obtain the degradable hemostatic and healing-promoting sponge.
Example five: preparation of degradable hemostatic and healing promoting sponge
The degradable hemostatic and healing-promoting sponge is prepared by the following steps:
step 1: dispersing the porcine acellular dermal matrix into an acetic acid solution, stirring for 48 hours to prepare acellular dermal matrix glue, and filtering to remove undissolved acellular dermal matrix. Wherein the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 20g:100mL, stirring time is 1000rpm, 36 hours; the concentration of the acetic acid solution is 0.5 to 2 percent.
Step 2: uniformly mixing the acellular dermal matrix adhesive treated in the step (1) with rice hydrolyzed protein powder, beating with an egg beater until the mixed gel is milky, injecting the milky mixed gel into a mold, freezing and shaping at-80 ℃, and then performing freeze drying treatment on the milky mixed gel to obtain the sponge for stopping bleeding and promoting healing; wherein, the one-time freeze thawing process comprises the following steps: the freeze drying time of the freeze dryer is 48-72h, the vacuum degree is 0.1MPa, and the freeze drying temperature is not lower than minus 50 ℃. The rotating speed of the egg beater is 750rpm for 60 s; the mass and volume ratio of the rice protein hydrolysate to the acellular dermal matrix gel is 0.5 g:100 mL.
And 3, step 3: the white porous sponge was soaked in a 70% ethanol solution for 2 hours to remove the residual acetic acid solution.
And 4, step 4: the obtained sponge is again subjected toVacuum drying, cutting, packaging, Co 60 And (5) irradiating and sterilizing to obtain the degradable hemostatic and healing-promoting sponge.
Example six: preparation of degradable hemostatic and healing promoting sponge
The degradable hemostatic and healing-promoting sponge is prepared by the following steps:
step 1: dispersing the porcine acellular dermal matrix into an acetic acid solution, stirring for 48 hours to prepare acellular dermal matrix glue, and filtering to remove undissolved acellular dermal matrix. Wherein the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 20g:100mL, stirring time is 1000rpm, 36 hours; the concentration of the acetic acid solution is 0.5 to 2 percent.
Step 2: uniformly mixing the acellular dermal matrix adhesive treated in the step (1) with rice hydrolyzed protein powder, beating with an egg beater until the mixed gel is milky, injecting the milky mixed gel into a mold, freezing and shaping at-80 ℃, and then performing freeze drying treatment on the milky mixed gel to obtain the hemostatic sponge; wherein, the one-time freeze thawing process comprises the following steps: the freeze drying time of the freeze dryer is 48-72h, the vacuum degree is 0.1MPa, and the freeze drying temperature is not lower than minus 50 ℃. The rotating speed of the egg beater is 750rpm for 60 s; the mass to volume ratio of the rice protein hydrolysate to the acellular dermal matrix gel is 0.25 g:100 mL.
And 3, step 3: the white porous sponge was soaked in a 70% ethanol solution for 2 hours to remove the residual acetic acid solution.
And 4, step 4: vacuum drying the obtained sponge again, cutting, packaging and Co 60 And (5) irradiating and sterilizing to obtain the degradable hemostatic and healing-promoting sponge.
Example seven: preparation of degradable hemostatic and healing promoting sponge
The degradable hemostatic and healing promoting sponge is prepared by the following steps:
step 1: dispersing the porcine acellular dermal matrix into an acetic acid solution, stirring for 48 hours to prepare acellular dermal matrix glue, and filtering to remove undissolved acellular dermal matrix. Wherein the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 20g:100mL, stirring time is 1000rpm, and the stirring time is 36 hours; the concentration of the acetic acid solution is 0.5-2%.
And 2, step: uniformly mixing the acellular dermal matrix glue treated in the step (1) with egg white powder, beating with an egg beater until the mixed gel is milky, injecting the milky mixed gel into a mold, freezing and shaping at-80 ℃, and then performing freeze drying treatment on the milky mixed gel to obtain the hemostatic sponge; wherein, the one-time freeze thawing process comprises the following steps: the freeze drying time of the freeze dryer is 48-72h, the vacuum degree is 0.1MPa, and the freeze drying temperature is not lower than minus 50 ℃. The rotating speed of the egg beater is 750rpm for 60 s; the mass and volume ratio of the egg white powder to the acellular dermal matrix gel is 0.25 g:100 mL.
And step 3: the white porous sponge was soaked in a 70% ethanol solution for 2 hours to remove the residual acetic acid solution.
And 4, step 4: vacuum drying the obtained sponge again, cutting, packaging and Co 60 Irradiating and sterilizing to obtain the degradable hemostatic healing-promoting sponge.
Example eight: preparation of degradable hemostatic and healing promoting sponge
The degradable hemostatic and healing-promoting sponge is prepared by the following steps:
step 1: dispersing the pig acellular dermal matrix into an acetic acid solution, stirring for 48 hours to prepare acellular dermal matrix glue, and filtering to remove undissolved acellular dermal matrix. Wherein the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 20g:100mL, stirring time is 1000rpm, and the stirring time is 36 hours; the concentration of the acetic acid solution is 0.5-2%.
And 2, step: uniformly mixing the acellular dermal matrix glue treated in the step (1) with egg white powder, beating with an egg beater until the mixed gel is milky, injecting the milky mixed gel into a mold, freezing and shaping at-80 ℃, and then performing freeze drying treatment on the milky mixed gel to obtain the hemostatic sponge; wherein, the one-time freeze thawing process comprises the following steps: the freeze drying time of the freeze dryer is 48-72 h; the rotating speed of the egg beater is 1000rpm for 60 s; the mass to volume ratio of the rice protein hydrolysate to the acellular dermal matrix gel is 0.5 g:100 mL.
And 3, step 3: the white porous sponge was soaked in a 70% ethanol solution for 2 hours to remove the residual acetic acid solution.
And 4, step 4: vacuum drying the obtained sponge again, cutting, packaging and Co 60 Irradiating and sterilizing to obtain the degradable hemostatic healing-promoting sponge.
Example nine: physical and chemical property experiment of degradable hemostatic healing-promoting sponge
The implementation process comprises the following steps: the hemostatic and healing promoting sponges prepared in examples 1 to 8 were cut into small disks with a diameter of 12mm, and used in the following physicochemical performance experiments.
The experimental method comprises the following steps:
(1) and (3) microstructure observation: dried sample circular pieces with diameters of 6mm in various proportions were attached to a test bench via a conductive adhesive and were subjected to a gold spraying treatment under vacuum using a sputtering machine under conditions of 1kV and 5mA for 60 seconds. The microstructure of the material was observed using scanning electron microscopy (SEM; TM3030, HITACHI) at an accelerating voltage of 20 kV.
(2) Contact angle: the contact angle was measured on the surface of the material using a contact angle measuring instrument (Theta Flex) (n is 3). When the contact angle is less than or equal to 90 degrees, the material is hydrophilic; when the contact angle is larger than 90 degrees, the material is hydrophobic.
(3) Moisture retention: the sample mass Wdry was precisely weighed and immersed in distilled water to make it fully water-swellable. Putting the mixture into a centrifuge tube, centrifuging the mixture for 3min at a speed of 500r/min, taking out the mixture and weighing the mixture W. Each group was measured in triplicate. Calculating the moisture retention of the material: wr ═ W-Wdry)/Wdry.
(4) Water absorption: taking a proper amount of material, weighing the material as M1, immersing the sample in purified water, soaking at normal temperature for 24h, taking out and absorbing excessive water on the surface of the material after the sample fully absorbs water, weighing the wet mass as M2, and calculating the water absorption rate according to the following formula, wherein the water absorption rate is (M2-M1)/M1 multiplied by 100 percent.
(5) And (3) detecting cytotoxicity: soaking the materials in complete culture medium at 37 deg.C for 24 hr to obtain leaching solution. The L929 cells were cultured at 2X 10 4 Inoculating the cells in a 96-well plate at a density of one cell per ml, adding the leaching liquor after the cells are attached to the wall, and taking a blank control group as a complete culture medium. CCK-8 reagent solution (DOJINDO, Japan) was added at 48-72h and 72h, respectively, incubated at 37 ℃ for 1h, and then applied to a microplate reader (BioTek,china) was measured for the optical density value (D) of each well at a wavelength of 450nm and subjected to statistical analysis (n-3).
(6) Staining live and dead cells: preparing a staining solution from 10uL of 1mmol/L Calcein-AM stock solution and 15uL of 1.5mmol/L PI stock solution (Solarbio, China) to 5mL of PBS, and freezing and storing at 0 ℃ in the dark for later use. After the L929 cells were inoculated to the material for 3 days, the original culture medium was blotted, 100uL of the prepared PI/CA staining solution was replaced to completely immerse the material, the material was placed in a cell incubator in the dark for 20min, the staining solution was washed with physiological saline, and the number and state of the cells were observed by obtaining an image of cell proliferation and adhesion at 490nm under an inverted fluorescence microscope (Axio Observer D1, ZEISS, Germany). Each set was run in duplicate for 3 samples.
The experimental results are as follows: the hemostatic and healing promoting sponges obtained in examples 1 to 8 are porous structures, the porosity of each sponge is more than 90%, the pore size of each sponge is 300 mu m-2 mm, and the pores are communicated in a staggered mode. After contacting with liquid or blood, the pore structure of the hemostatic gauze rapidly sucks the liquid into pores, blood platelets are gathered in the pores, and in-vivo and in-vitro blood coagulation paths are started, so that rapid hemostasis is achieved.
FIG. 1 is a schematic diagram of a process for preparing a rice hydrolyzed protein-acellular dermal matrix sponge according to the present invention; (B) is a rough physical diagram before and after freeze-drying of rice hydrolyzed protein-acellular dermal matrix gel with different proportions. As shown in figure 1, the hemostatic and healing promoting sponge prepared by the invention has complete shape and simple preparation process.
Fig. 2 is a general real object diagram of the optimized 0.25% -1% rice protein hydrolysate-acellular dermal matrix gel before and after freeze-drying, and as shown in fig. 2, the hemostatic and heal-promoting sponge prepared in the interval has good comprehensive performance.
FIG. 3 is a scanning electron microscope image of a sponge for promoting healing of bleeding, as shown in FIG. 3, the sponge for promoting healing of bleeding prepared by the present invention has a porous structure with obvious porous structure, and the porous structure has strong adsorption and water absorption capacity.
FIG. 4 is a graph showing the result of the test of the contact angle hydrophilicity of the sponge for promoting healing of hemostasis in the present invention, as shown in FIG. 4, the sponge for promoting healing of hemostasis prepared by the present invention all showed hydrophilicity, and the hydrophilicity was the most preferable to add 0.25% of rice protein hydrolysate.
Fig. 5 is a diagram showing the results of the water absorption and moisture retention tests of different hemostatic sponges, and as shown in fig. 5, the hemostatic sponge prepared according to the present invention has a rapid and strong water absorption capacity, so that blood can rapidly form stable blood clots on the surface of the material and at the damaged sites of blood vessels, thereby achieving the purpose of rapid hemostasis.
FIG. 6 is a result of a staining test of living and dead cells of different hemostatic and healing promoting sponges, and as shown in FIG. 6, the hemostatic and healing promoting sponges prepared according to the present invention have good biocompatibility, and can promote migration of skin-related cells in pores.
FIG. 7 is a diagram showing the results of the relative cell viability tests of different hemostatic and healing promoting sponges, and as shown in FIG. 7, the hemostatic and healing promoting sponges prepared according to the present invention have no cytotoxicity, and can maintain the survival and proliferation of skin-associated cells.
Based on the physicochemical properties, the degradable hemostatic and healing promoting sponge can be used for preparing hemostatic materials or wound healing materials.
It should be noted that the above embodiments can be freely combined as necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A degradable hemostatic healing-promoting sponge is characterized in that: the gel is prepared by foaming, shaping and freeze-drying a foaming agent and acellular dermal matrix glue; the mass-volume ratio of the foaming agent to the acellular dermal matrix adhesive is 0.25-2 g:100 mL.
2. The degradable hemostatic and heal-promoting sponge according to claim 1, wherein: the foaming agent is one or more selected from egg white powder and rice hydrolyzed protein.
3. The degradable hemostatic and heal-promoting sponge according to claim 1, wherein: the acellular dermal matrix glue is prepared by adding acetic acid solutions with different volumes into an acellular dermal matrix for dissolution.
4. The method for preparing the degradable hemostatic and healing promoting sponge according to claim 1, comprising the steps of:
A. dispersing the pig acellular dermal matrix into an acetic acid solution, stirring and dissolving to prepare uniform acellular dermal matrix glue;
B. adding a foaming agent into the acellular dermal matrix adhesive obtained in the step A, uniformly mixing, beating with an egg beater until the mixed gel is milky white and has stable volume without expansion, and pouring into a mould for standing and shaping;
C. freezing and shaping at-80 ℃, and then freezing and drying to obtain the degradable hemostatic and heal-promoting sponge.
5. The method of manufacturing according to claim 4, characterized in that: in the step A, the concentration of the acetic acid solution is 0.5% -2%, the ratio of the mass of the acellular dermal matrix to the volume of the acetic acid solution is 10-20 g:100mL, the stirring time is 1500rpm, and the stirring time is 48 hours.
6. The method of manufacturing according to claim 4, characterized in that: in the step B, the rotating speed of the eggbeater is 500-1000 rpm for 30-60 s.
7. The preparation method according to claim 4, wherein in the step C, the freeze-drying temperature is not lower than minus 50 ℃, the freeze-drying time is 48-72 hours, and the vacuum degree is 0.1 MPa.
8. A hemostatic material comprising the degradable hemostatic healing promoting sponge of claim 1.
9. A wound healing material comprising the degradable hemostatic healing promoting sponge of claim 1.
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