CN108187129B - Absorbable gelatin styptic powder and preparation method thereof - Google Patents
Absorbable gelatin styptic powder and preparation method thereof Download PDFInfo
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- CN108187129B CN108187129B CN201810124860.4A CN201810124860A CN108187129B CN 108187129 B CN108187129 B CN 108187129B CN 201810124860 A CN201810124860 A CN 201810124860A CN 108187129 B CN108187129 B CN 108187129B
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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
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/104—Gelatin
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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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/07—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media from polymer solutions
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/12—Powdering or granulating
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
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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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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2201/00—Foams characterised by the foaming process
- C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
- C08J2201/048—Elimination of a frozen liquid phase
- C08J2201/0484—Elimination of a frozen liquid phase the liquid phase being aqueous
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2207/00—Foams characterised by their intended use
- C08J2207/10—Medical applications, e.g. biocompatible scaffolds
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- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
Abstract
The invention provides absorbable gelatin hemostatic powder and a preparation method thereof, and the BET specific surface area of the absorbable gelatin hemostatic powder is 30m2/g~70m2The preparation process comprises the steps of cross-linking treatment, water absorption swelling, mechanical crushing, freeze drying and secondary crushing of gelatin. The hemostatic powder can be well attached to a wound surface, so that the moisture in blood can be quickly absorbed, and a good hemostatic effect is realized. The preparation method is simple and convenient in process, and further, the cross-linking agent which does not participate in the reaction is adopted, and the operations of mechanical crushing, ultrasonic-centrifugal circulation cleaning or ethanol water solution cleaning are carried out after the gelatin is cross-linked and fully swelled, so that the cross-linking agent is effectively removed, the cross-linking agent is free from residue in the hemostatic powder, the irritant influence on organisms caused by the existence of the cross-linking agent is avoided, the safety of the product is improved, and the preparation method has good popularization and application values.
Description
Technical Field
The invention relates to the technical field of hemostatic materials, and particularly relates to absorbable gelatin hemostatic powder and a preparation method thereof.
Background
Surgical hemostasis is one of the important steps in clinical surgery, and good hemostatic technology and hemostatic materials are the key to ensure the success of the surgery. The existing commonly used hemostatic materials comprise various forms such as hemostatic sponge, hemostatic gauze, hemostatic gel, hemostatic powder and the like, wherein the hemostatic powder is not influenced by the size and the position of a wound surface, can be used for not only surgical wound surfaces, but also minimally invasive surgeries such as endoscopes and the like, and has unique advantages.
In the prior art, a powdery hemostatic material is a microporous polysaccharide product mainly prepared from starch, and the microporous polysaccharide product is prepared by performing ultrasonic treatment, wet-heat treatment, microwave treatment, mechanical treatment or enzyme perforation and the like on the surface of starch granules to improve the specific surface area and the hydrophilic property of the material, wherein micropores on the surface of microspheres play a role of a molecular sieve to adsorb water in blood to improve the concentration of a blood coagulation factor and accelerate the generation of a blood coagulation mechanism, so that the hemostatic effect is realized. The product is made of natural polysaccharide, is non-toxic and has good biocompatibility, but has the problems of complex preparation process, easy environmental pollution, poor water absorption performance, poor adhesion with wound surfaces and the like.
Gelatin is used as a natural water-soluble biodegradable high molecular material, has a structure similar to that of organism tissues, has good biocompatibility, can realize biodegradation and absorption without generating inflammatory reaction, and is widely applied to the field of medicines. In the aspect of hemostatic materials, gelatin hemostatic sponges and injectable gelatin hemostatic products are common, but gelatin hemostatic powder meeting clinical use requirements is not reported.
In addition, in the process of preparing gelatin hemostatic products, in order to meet the requirements of mechanical strength and/or degradability, crosslinking treatment is generally required. Finally, the cross-linking agent exists in the final product and is gradually released in the subsequent degradation process of the gelatin material, so that the tissue irritation is brought, even carcinogenicity may exist, and the safety of the product is influenced.
Therefore, the research and development of the gelatin absorbable hemostatic powder with good hemostatic effect further improves the safety performance of the hemostatic powder, and has important significance.
Disclosure of Invention
The invention aims to provide absorbable gelatin hemostatic powder aiming at the defects of the prior art, and the absorbable gelatin hemostatic powder has a high specific surface area, can be well attached to a wound surface, quickly absorbs moisture in blood and realizes a good hemostatic effect.
The invention also aims to provide a preparation method of the absorbable gelatin hemostatic powder, the preparation method is simple and convenient in process, and the prepared hemostatic powder has good performance and hemostatic effect, can obviously reduce the content of a cross-linking agent in the hemostatic powder, improves the safety of the product, and has good popularization and application values.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an absorbable gelatin hemostatic powder, wherein the BET specific surface area of the hemostatic powder is 30m2/g ~70m2The preparation process comprises the steps of cross-linking treatment, water absorption swelling, mechanical crushing, freeze drying and secondary crushing of gelatin.
The gelatin hemostatic powder has a saturated water absorption rate of 50-200% and a volume swelling rate of 250-550%.
A preparation method of absorbable gelatin styptic powder comprises the following steps:
s1, preparing a gelatin water solution;
s2, adding a cross-linking agent into the gelatin aqueous solution for cross-linking treatment;
s3, placing the cross-linked gelatin in water for full swelling;
s4, cutting the swelled gelatin in the step S3 into small blocks, and then crushing the small blocks into micron-sized gelatin particles;
s5, freezing the gelatin particles at low temperature, and freeze-drying after complete freezing to obtain dried gelatin;
and S6, carrying out secondary crushing on the gelatin after freeze drying to obtain absorbable gelatin hemostatic powder.
The absorbable hemostatic powder is obtained by swelling crosslinked gelatin with water fully, and then carrying out mechanical crushing, freeze drying and secondary crushing. The hemostatic powder obtained in this way can be well attached to the wound surface, has good water absorption performance and hemostatic effect, and can be completely degraded and absorbed in vivo.
Preferably, in step S1, the mass concentration of the gelatin aqueous solution is 5% to 50%.
Preferably, in step S2, the amount of the cross-linking agent is 4-20% of the mass of the gelatin. Further preferably, the cross-linking agent is added during stirring of the gelatin solution.
Preferably, in step S4, the pulverization is performed by an aqueous phase material pulverizer.
Preferably, in step S6, the pulverizing speed of the secondary pulverizing is 10000r/min to 40000r/min, and the pulverizing time is 10S to 5 min.
Preferably, the preparation method further comprises packaging and irradiation sterilization of the obtained gelatin hemostatic powder, so as to obtain the hemostatic product applicable to the wound surface.
Preferably, in order to reduce the residual amount of the cross-linking agent in the gelatine hemostatic powder and improve the safety of the gelatine hemostatic powder, the invention adopts the cross-linking agent water-soluble carbodiimide (EDC) or glutamine transaminase (TG enzyme) which does not participate in the reaction, and the cross-linking agent is removed through a specific step in the preparation method, so that the finally obtained hemostatic powder product has the residual amount of the cross-linking agent of 100ppm or even below 10ppm, good safety and no stimulation to tissues. The preparation method is combined with the following steps to obtain the product:
(1) in the step S2, the cross-linking agent is water-soluble carbodiimide (EDC) or TG enzyme, preferably EDC, the mass of the cross-linking agent is 4% -20% of the mass of gelatin, the cross-linking agent is added in the process of stirring the gelatin solution, the stirring speed of the gelatin solution is 100 r/min-5000 r/min, preferably 1000r/min, and the adding speed of the cross-linking agent is 0.1 mL/S-10 mL/S, preferably 2 mL/S;
(2) between step S4 and step S5, the gelatin particles are placed in pure water for ultrasonic-centrifugal cycle treatment, or repeatedly washed with an aqueous ethanol solution, and then ethanol is removed and placed again in pure water for sufficient swelling to remove the crosslinking agent.
Preferably, the rotating speed of the centrifugation is 10000 r/min-20000 r/min, the time of single centrifugation is 5 min-10 min, the ultrasonic frequency is 25 KHz-130 KHz, and the time of single ultrasonic treatment is 5 min-30 min.
Preferably, the ethanol water solution is prepared by adopting the volume ratio of ethanol to water of 4: 1.
Gelatin crosslinking is a main mode for adjusting the degradation speed and the hemostatic performance of gelatin, but glutaraldehyde is generally adopted as a gelatin crosslinking agent in the prior art, and the crosslinking agent cannot be well removed after crosslinking, so that the subsequent irritation and potential safety problems can be caused. The method can well remove the cross-linking agent of the gelatin by carrying out ultrasonic-centrifugal circulation cleaning and secondary crushing operation after swelling and crushing the cross-linked gelatin, achieve good purification effect of the hemostatic material and effectively avoid residues and toxicity of irritant substances.
The crosslinking agent selected by the invention is water-soluble carbodiimide (EDC) or glutamine transaminase (TG enzyme) which does not participate in the reaction, and the crosslinking agent can be removed from a final system after crosslinking, and the crosslinking agent does not exist in a final product, thereby avoiding the irritation influence and potential safety problems on organisms caused by the existence of impurities.
In addition, the cross-linking agent initiates the cross-linking reaction very rapidly, the reaction cross-linking degree in the system is easy to be uneven after one-time addition, and the system can be uniformly cross-linked as much as possible by dropwise adding while stirring. In the crosslinking process, amino and carboxyl in the gelatin form amide bonds under the action of a crosslinking agent, the degradation process of the material is prolonged due to the increase of the amide bonds in the system, and the degradation time is prolonged along with the increase of the crosslinking agent in the system, so that the swelling rate and the degradation period of the hemostatic powder can be well adjusted by controlling the crosslinking reaction.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides absorbable gelatin hemostatic powder and a preparation method thereof. The preparation method is simple and convenient in process, and further, the cross-linking agent which does not participate in the reaction is adopted, and the operations of mechanical crushing, ultrasonic-centrifugal circulation cleaning or ethanol water solution cleaning are carried out after the gelatin is cross-linked and fully swelled, so that the cross-linking agent is effectively removed, the cross-linking agent is free from residue in the hemostatic powder, the irritant influence on organisms caused by the existence of the cross-linking agent is avoided, the safety of the product is improved, and the preparation method has good popularization and application values.
Drawings
Fig. 1 is a scanning electron microscope image of the absorbable gelatin hemostatic powder of the invention.
FIG. 2 is the result of culturing cells for 24 hours in the leaching solution of absorbable gelatin styptic powder of example 1.
FIG. 3 shows the result of culturing the cells of the leaching solution of absorbable gelatin styptic powder of example 1 for 72 hours.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way.
Example 1
A preparation method of absorbable styptic powder comprises the following steps:
s1, adding 60g of water into 40g of existing gelatin, and placing the gelatin in an oven at 80 ℃ until the gelatin is completely dissolved to prepare a gelatin water solution with the mass concentration of 40%;
s2, dissolving 1.6g of water-soluble carbodiimide (EDC) in 10mL of purified water (the mass of EDC is 4% of the mass of gelatin), adding a cross-linking agent into the gelatin aqueous solution while stirring, and carrying out cross-linking treatment, wherein the rotation speed of mechanical stirring is 1000r/min, and the adding speed of the cross-linking agent is 2 mL/S;
s3, placing the cross-linked gelatin in water for full swelling;
s4, cutting the swelled gelatin in the step S3 into small blocks, and then crushing the small blocks into micron-sized gelatin particles by adopting a water-phase material crusher;
s5, placing gelatin particles in pure water in a centrifugal deposition mode to carry out ultrasonic-centrifugal circulation treatment, wherein the centrifugal rotation speed is 15000r/min, the single centrifugation time is 5min, the ultrasonic frequency is 100KHz, the single ultrasonic treatment time is 10min, and the circulation frequency is 3 times, then filtering, rinsing, laying flat, placing in a low-temperature refrigerator for freezing, and after complete freezing, carrying out freeze drying to obtain dry gelatin;
and S6, carrying out secondary crushing on the freeze-dried gelatin, wherein the crushing speed is 30000r/min, the crushing time is 1min, and the absorbable gelatin hemostatic powder is obtained, filled in an auxiliary device and irradiated for later use.
FIG. 1 is a scanning electron microscope image of the surface structure of the gelatin hemostatic powder prepared in example 1, which can be seen to have a microscopic porous structure.
Example 2
A preparation method of absorbable styptic powder comprises the following steps:
s1, taking 20g of existing gelatin, adding 80g of water, placing the mixture in a 50 ℃ oven until the gelatin is completely dissolved, and preparing a gelatin water solution with the mass concentration of 20%;
s2, dissolving 2g of water-soluble carbodiimide (EDC) in 10mL of purified water (the mass of the EDC is 10% of that of gelatin), adding a cross-linking agent into the gelatin aqueous solution while stirring, and carrying out cross-linking treatment, wherein the rotation speed of mechanical stirring is 2000r/min, and the addition speed of the cross-linking agent is 5 mL/S;
s3, placing the cross-linked gelatin in water for full swelling;
s4, cutting the swelled gelatin in the step S3 into small blocks, and then crushing the small blocks into micron-sized gelatin particles by adopting a water-phase material crusher;
s5, placing gelatin particles in pure water in a centrifugal deposition mode to perform ultrasonic-centrifugal circulation treatment, wherein the centrifugal rotation speed is 10000r/min, the single centrifugation time is 5min, the ultrasonic frequency is 25KHz, the single ultrasonic treatment time is 5min, and the circulation frequency is 3 times, then filtering, rinsing, laying flat, placing in a low-temperature refrigerator for freezing, and performing freeze drying after complete freezing to obtain dry gelatin;
and S6, carrying out secondary crushing on the freeze-dried gelatin, wherein the crushing speed is 25000r/min, the crushing time is 2min, and the absorbable gelatin hemostatic powder is obtained, filled in an auxiliary device and irradiated for later use.
Example 3
A preparation method of absorbable styptic powder comprises the following steps:
s1, adding 95g of water into 5g of existing gelatin, and placing the gelatin in a 50 ℃ oven until the gelatin is completely dissolved to prepare a gelatin water solution with the mass concentration of 5%;
s2, dissolving 0.5g of water-soluble carbodiimide (EDC) in 10mL of purified water (the mass of EDC is 10% of that of gelatin), adding a cross-linking agent into the gelatin aqueous solution while stirring, and carrying out cross-linking treatment, wherein the rotation speed of mechanical stirring of the gelatin solution is 2000r/min, and the addition speed of the cross-linking agent is 5 mL/S;
s3, placing the cross-linked gelatin in water for full swelling;
s4, cutting the swelled gelatin in the step S3 into small blocks, and then crushing the small blocks into micron-sized gelatin particles by adopting a water-phase material crusher;
s5, repeatedly stirring the crushed gelatin particles in pure water and an ethanol solution, wherein the volume of ethanol is four times of that of water, repeatedly cleaning, completely removing an ethanol phase, re-dispersing the ethanol phase in a water phase, saturating and swelling the water phase, flatly spreading the mixture, freezing the mixture in a low-temperature refrigerator, and freeze-drying the mixture in a freeze dryer after complete freezing to obtain dry gelatin;
and S6, carrying out secondary crushing on the freeze-dried gelatin, wherein the crushing speed is 40000r/min, the crushing time is 3min, and the absorbable gelatin hemostatic powder is obtained, filled in an auxiliary device and irradiated for later use.
Performance detection
1. Detection of residual amount of crosslinking agent
The detection of the residual amount of the cross-linking agent adopts LC-MS/MS detection, and LC-MS/MS adopts tandem mass spectrometry, so that not only can a molecular ion peak be obtained, but also a fragment ion peak is obtained, and therefore, the method can be used for qualitative and quantitative analysis of materials. The detection result of the residual amount of the cross-linking agent is shown in table 1, and the content of the cross-linking agent in the prepared gelatin hemostatic powder is below 10ppm, which shows that the preparation method of the invention can obviously reduce the content of the cross-linking agent and greatly improve the safety of the product.
TABLE 1 detection results of crosslinker content
| Serial number | Crosslinking agent | Number of samples to be tested/g | Detection method | Test results/mg/kg |
| Example 1 | EDC | 0.1157 | LC-MS/MS | 5.3 |
| Example 2 | EDC | 1.9830 | LC-MS/MS | 9.8 |
| Example 3 | EDC | 2.3627 | LC-MS/MS | 6.7 |
2. Specific surface area test and saturated water absorption and volume swelling rate test
Specific surface area test method: and putting a product to be detected into a sample tube of an analytical instrument, wherein the analytical instrument is a rapid full-automatic specific surface area and aperture analyzer and is of a model of American Congta NOVA 4200 e. Under the condition of low temperature (liquid nitrogen bath), a certain amount of adsorbate gas (N) is introduced into the sample tube2) Determining adsorbate molecules (N) of the sample to be detected according to the change of gas volume before and after adsorption2) The adsorption amount of (c); the specific surface area of the solid substance is determined by referring to the national standard GB/T243358-2009-gas adsorption BET principle.
The specific surface area was calculated as: in a sample placed in a gaseous environment, the surface of its material (surface area of the external and internal voids of the particles) will be physisorbed at low temperatures. When the adsorption reaches the equilibrium, measuring the amount of adsorbed gas of the equilibrium adsorption pressure, and calculating the adsorption amount of the monomolecular layer of the sample according to the BET equation so as to calculate the specific surface area of the sample. Wherein the BET equation is:
in the formula:
p-adsorbate partial pressure in Pa;
P0-the adsorbent saturation vapour pressure in Pa;
v-actual adsorption of sample in cm3；
VmThe single layer saturated adsorption capacity in cm3；
C-constant related to the adsorption capacity of the sample.
The gelatin hemostatic powders obtained in examples 1 to 3 were tested, and the test results are shown in Table 2.
Saturated Water absorption test method: a certain mass of sample (m)1) Placing the sample in a culture dish, weighing the mass m of the sample and the culture dish2Then, 0.9% physiological saline preheated to (37. + -. 1) ° C is added, and the mass of the physiological saline is 20 times of that of the test material. The dish was transferred to a drying oven and maintained at (37. + -. 1) ° C for 60 min. Taking out the culture dish, carefully and obliquely pouring out the unabsorbed excessive water until no water drops within 30s, and accurately weighing the mass m of the sample added into the culture dish by using an electronic balance3The assay was performed 3 times in parallel. Calculating to obtain the saturated water absorption rate (X) of the hemostatic powder, wherein the saturated water absorption rateThe calculation formula of (X) is: x = (m)3-m2)/m1×100%。
Method for measuring volume swelling ratio: measurement of the volume V of the sample by solvent filling1. Then the sample (V)1) Placing in a measuring cylinder filled with a certain volume of normal saline preheated to 37 + -1 deg.C, placing the measuring cylinder in a environment of 37 + -1 deg.C for 60min, and obtaining a sample volume V of the hemostatic powder after saturated absorption of water according to the height of the liquid level rising caused by the sample addition2The assay was performed 3 times in parallel. The volume swelling ratio (Y) of the hemostatic powder is obtained through calculation, and the calculation formula of the volume swelling ratio (Y) is as follows: y = (V)2-V1)/V1×100%。。
The specific surface area and swelling ratio test results of the hemostatic powders are shown in table 2.
TABLE 2 test results of specific surface area and swelling ratio of hemostatic powder
| Serial number | BET specific surface area/m2/g | Volume swell ratio/% | Mass swelling ratio/% |
| Example 1 | 30 | 296 | 53 |
| Example 2 | 40 | 396 | 126 |
| Example 3 | 70 | 512 | 180 |
3. Hemostasis effectiveness test
(1) Design of experiments
18 healthy New Zealand rabbits are selected and divided into six groups, wherein three groups are experimental groups, namely experimental group 1, experimental group 2 and experimental group 3, and the experimental groups adopt the gelatin hemostatic powder materials prepared in the examples 1 to 3. The other three groups are control groups, namely a gauze control group, a gelatin sponge control group and other brand hemostatic powder control groups, wherein the other brand hemostatic powders are chitosan hemostatic powders, and the manufacturer is Guangxi Nanning Bo Enkang Biotech limited company.
(2) Experimental methods
The specific experimental method comprises the following steps:
1. after the rabbit is anesthetized, the back of the rabbit is fixed, the abdomen of the rabbit is upward, the abdominal cavity is opened, the liver is fully exposed, and a surgical knife is used for making a wound of about 1cm multiplied by 1cm on the surface of the liver to form a liver trauma bleeding model;
2. after the model is built, weighed gauze (w) is taken0) Quickly covering the wound surface, slightly pressing while timing, removing gauze after 10s, and weighing1），w1-w0Is a blood volume value;
3. quickly covering the hemostatic material after removing the gauze, covering the hemostatic material with the gauze, forcibly pressing the gauze, moving the gauze every 5s during the pressing period to observe the bleeding condition of the wound surface, stopping timing when no bleeding occurs, and recording the hemostatic time.
(3) Results of the experiment
And (3) judging that the hemostasis fails when the hemostasis time exceeds 5min in the experimental process, performing five experiments on each group respectively, calculating the average value of each group, and showing a comparison table of the hemostasis time and the hemostasis effect as shown in table 3.
TABLE 3 hemostasis test results
| Group of | Mean amount of bleeding | Mean time to hemostasis |
| Experimental group 1 | 2.1g | 1min55s |
| Experimental group 2 | 1.9g | 1min20s |
| Experimental group 3 | 3.0g | 1min40s |
| Control group 1 | 2.0g | ＞5min |
| Control group 2 | 2.5g | 1min40s |
| Control group 3 | 2.2g | 2min00s |
4. Cytotoxicity assay
(1) Design of experiments
Experimental groups: complete medium was used: DMEM culture medium +10% Fetal Bovine Serum (FBS) +1% double antibody (mixed solution of penicillin and streptomycin), adding leaching liquor, adding cell suspension, and performing cell culture.
Control group: complete medium was used: DMEM medium +10% Fetal Bovine Serum (FBS) +1% diabase (mixture of penicillin and streptomycin) was added to the cell suspension to perform cell culture. The control group was not added with the leaching solution, and the rest of the cell culture conditions were the same as those of the experimental group.
Blank group: complete medium was used: DMEM medium +10% Fetal Bovine Serum (FBS) +1% diabase (mixed solution of penicillin and streptomycin), and the blank group was placed in the same environment as the experimental group and the control group at the same time without adding the leaching solution and the cell suspension, and used as a reference for measuring absorbance values of the experimental group and the control group.
(2) Experimental methods
Culturing the cells by contact with the extract as L929To test cells, cell proliferation was observed and the hemostatic material prepared in example 1 was evaluated for its toxic effects on cells in vitro.
The specific procedure of the experiment is as follows:
1. the gelatin styptic powder of example 1 was extracted at a ratio of 0.1g/mL with complete medium (DMEM medium +10% Fetal Bovine Serum (FBS) +1% diabody (mixed solution of penicillin and streptomycin)) to obtain 100% leaching solution, and diluted with 100% leaching solution to obtain 75% leaching solution and 25% leaching solution.
2. The gelatin styptic powder prepared in the example 1 is adopted to prepare leaching liquor, wherein the leaching temperature is (37 +/-1) DEG C, and the leaching time is (24 +/-2) h. Directly leaching to obtain 100% leaching solution, and diluting with 100% leaching solution to obtain 75% leaching solution and 25% leaching solution. Then, the L929 cells were resuspended using a complete medium to a concentration of 4X 104Individual cells/mL of cell suspension. Using 96-well plate as culture plate, wherein the experimental group and control group are prepared by adding the above cell suspension into culture plate, adding 100 μ L blank into each wellGroups were prepared by adding 100. mu.L of complete medium per well of the plate without adding cell suspension.
3. The plates of the experimental, control and blank groups were pre-incubated in an incubator for 24 hours (at 37 ℃ C., 5% CO)2Under conditions). The leaching solution was added to the experimental group in the concentration gradient of 100. mu.L/well and not added to the control group and blank group, and then each plate was cultured in an incubator for 24 hours and 72 hours (at 37 ℃ C., 5% CO)2Under conditions). The culture medium was then carefully aspirated from the wells, 100. mu.L of CCK-8 was added to each well, the mixture was prepared by mixing 90. mu.L of complete medium + 10. mu.L of CCK-8, and the plates were incubated in an incubator for 2 hours. The absorbance at 450nm was then measured with a microplate reader.
(3) Results of the experiment
FIGS. 2 and 3 show absorbance values (OD values) of the control and experimental groups, respectively, minus the blank group in the cytotoxicity test. As can be seen from the figure, the cell proliferation rate after the culture by the leaching liquor is close to that of a control group, so the hemostatic material has good safety and no toxic or side effect on cell growth.
Claims (10)
1. An absorbable gelatin hemostatic powder, which is characterized in that the BET specific surface area of the hemostatic powder is 30m2/g～70m2The preparation method comprises the following steps of cross-linking treatment of gelatin, full water absorption and swelling, mechanical crushing to micron-sized particles, ultrasonic-centrifugal cycle cleaning or repeated cleaning by adopting ethanol water solution, freeze drying and secondary crushing;
wherein, the crosslinking agent adopted in the gelatin crosslinking treatment is water-soluble carbodiimide or glutamine transaminase;
the cross-linking agent residual quantity of the hemostatic powder is below 10 ppm.
2. The absorbable gelatin hemostatic powder of claim 1, wherein the gelatin hemostatic powder has a saturated water absorption of 50% to 200% and a volume swelling ratio of 250% to 550%.
3. The preparation method of the absorbable gelatin styptic powder is characterized by comprising the following steps:
s1, preparing a gelatin water solution;
s2, adding a cross-linking agent into the gelatin aqueous solution for cross-linking treatment;
s3, placing the crosslinked gelatin in water for full swelling;
s4, cutting the swelled gelatin in the step S3 into small blocks, and then crushing the small blocks into micron-sized gelatin particles;
s5, freezing the gelatin particles at a low temperature, and freeze-drying after complete freezing to obtain dried gelatin;
and S6, carrying out secondary crushing on the gelatin after freeze drying to obtain absorbable gelatin hemostatic powder.
4. The method for preparing absorbable gelatin hemostatic powder as claimed in claim 3, wherein, in step S2, the amount of the cross-linking agent is 4-20% by mass of the gelatin.
5. The method for preparing an absorbable gelatin hemostatic powder as set forth in claim 3, wherein the pulverization is performed using an aqueous phase material pulverizer in step S4.
6. The method for preparing absorbable gelatin hemostatic powder as claimed in claim 3, wherein in step S6, the crushing speed of the secondary crushing is 10000r/min to 40000r/min, and the crushing time is 10S to 5 min.
7. The method for preparing an absorbable gelatin hemostatic powder of claim 3, further comprising: (1) in the step S2, the cross-linking agent is EDC or TG enzyme, the mass of the cross-linking agent is 4-20% of that of gelatin, the cross-linking agent is added in the process of stirring the gelatin solution, the stirring speed of the gelatin solution is 100-5000 r/min, and the adding speed of the cross-linking agent is 0.1-10 mL/S;
(2) between step S4 and step S5, the gelatin particles are placed in pure water for ultrasonic-centrifugal cycle treatment, or repeatedly washed with an aqueous ethanol solution, and then ethanol is removed and placed again in pure water for sufficient swelling to remove the crosslinking agent.
8. The method for preparing absorbable gelatin hemostatic powder as claimed in claim 7, wherein the rotation speed of centrifugation is 10000r/min to 20000r/min, and the time of single centrifugation is 5min to 10 min; the ultrasonic frequency is 25 kHz-130 kHz, and the time of single ultrasonic treatment is 5 min-30 min.
9. The method for preparing absorbable gelatin hemostatic powder as claimed in claim 7, wherein the ethanol aqueous solution is prepared from ethanol and water in a volume ratio of 4: 1.
10. The method for preparing an absorbable gelatin hemostatic powder as set forth in any one of claims 3 to 9, further comprising the steps of packaging and radiation sterilization of the obtained gelatin hemostatic powder.
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