CN110141677B - Local acute hemostasis absorbable material and preparation method thereof - Google Patents
Local acute hemostasis absorbable material and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a local acute hemostasis absorbable material and a preparation method thereof, which is prepared by preparing oxidized regenerated cellulose by taking bacterial cellulose as a substrate and then carrying out electrostatic adsorption self-assembly with chitosan or chitosan and collagen. The invention has the characteristics of acute hemostasis, broad-spectrum antibiosis, healing promotion and in-vivo absorption; the preparation method is environment-friendly, the compounding is uniform and efficient, and the obtained composite material is high in safety and has good market application prospect.
Description
Technical Field
The invention belongs to the technical field of biomedical composite materials, and particularly relates to an absorbable material for local acute hemostasis and a preparation method thereof.
Background
The absorbable hemostatic material is a biomedical material which is used for wound bleeding parts, can rapidly stop bleeding and can be absorbed by a human body within a certain time. The method is characterized in that: the hemostatic bag is applied to hemostasis of local wounds which are difficult to suture in human bodies or animal bodies, can be degraded in vivo and does not need to be taken out when rapid hemostasis is realized, and therefore secondary injury of dressing removal after hemostasis is avoided. Currently, the commonly used absorbable hemostatic materials include fibrin glue hemostatic materials, gelatin sponge hemostatic materials, oxidized cellulose hemostatic materials, chitosan hemostatic materials and the like. The hemostatic materials have different hemostatic mechanisms and application methods, and have different hemostatic effects.
The oxidized regenerated cellulose is successfully prepared for the first time in 1960, is a tissue-shaped or cotton-shaped absorbable hemostatic material of cellulose acid after the cellulose is oxidized, has the appearance and the texture of cotton yarn, is soft and thin, and is easy to pack, apply, fill and the like. The hemostasis mechanism is that blood platelets in the blood of the wound surface of the operation are gathered on mesh gauze, the hemostatic gauze is used as a hemostasis substrate, and a gelatinous black substance is quickly formed after the blood meets the hemostatic gauze, so that blood clots are coagulated, and the aim of hemostasis is fulfilled. It is independent of the normal blood coagulation mechanism in human body, but can quickly promote blood coagulation by means of physical action, and can effectively control small blood vessel hemorrhage. Currently, only the united states and the united kingdom are available to produce such products, and the clinical absorbable hemostatic gauze Surgicel (fast yarn) is the product of the american obsession company, and the main component of fast yarn is oxidized regenerated cellulose. The oxidized regenerated cellulose is a derivative of cellulose obtained by oxidizing C6 primary hydroxyl in cellulose into carboxyl with high selectivity, and is applied to various industries at present, and products sold in the market mainly depend on import, and only a few companies produce similar hemostatic gauze in China. Although oxidized regenerated cellulose has many excellent properties, it also has some disadvantages, such as low hemostatic efficiency, unsuitability for massive bleeding, weak antibacterial properties, low tissue compatibility, etc.
At present, in some of the patent publications or granted, natural/synthetic polymer materials or inorganic materials have been used to modify oxidized regenerated cellulose to improve its antibacterial and hemostatic properties. Such as collagen (CN105079886A), alginate (CN104013991A) and carbon nanotubes (CN 105056284A). Although the composite hemostatic material has a certain improvement on the performance of the oxidized regenerated cellulose, the improvement effect is poor, only part of the performance can be improved, and the biological absorbability of the material is adversely affected.
Disclosure of Invention
The invention aims to solve the technical problems of providing a local acute hemostasis absorbable material and a preparation method thereof, and solving the problems of weak antibacterial property, low histocompatibility and low hemostasis speed of oxidized regenerated cellulose.
The invention provides a local acute hemostasis absorbable material, which is prepared by preparing oxidized regenerated cellulose (OBC) by taking bacterial cellulose as a substrate and then carrying out electrostatic adsorption self-assembly with chitosan or chitosan and collagen.
The bacterial cellulose is obtained by taking acetobacter xylinum as a strain, performing static culture for 7-20 days at constant temperature by using a liquid culture medium, placing the solution into a sodium hydroxide solution, treating the solution at 70-90 ℃ for 2-4 hours, taking out the solution, and rinsing the solution to be neutral by using deionized water.
The invention also provides a preparation method of the local acute hemostasis absorbable material, which comprises the following steps:
(1) preparing oxidized regenerated cellulose suspension by taking bacterial cellulose as a substrate;
(2) dropwise adding chitosan/dilute acid solution into the oxidized regenerated cellulose suspension for electrostatic adsorption self-assembly reaction, taking out, rinsing and freeze-drying to obtain a local acute hemostatic absorbable material (oxidized regenerated cellulose/chitosan absorbable antibacterial hemostatic material OC);
or, adding the collagen aqueous solution into the oxidized regenerated cellulose suspension for soaking and adsorption, then dropwise adding the chitosan/dilute acid solution into the oxidized regenerated cellulose suspension for electrostatic adsorption self-assembly reaction, then taking out, rinsing and freeze-drying to obtain the local acute hemostatic absorbable material (the oxidized regenerated cellulose/collagen/chitosan absorbable antibacterial hemostatic material OCC).
The preparation method of the oxidized regenerated cellulose suspension in the step (1) comprises the following steps: breaking up a bacterial cellulose membrane, oxidizing the bacterial cellulose membrane by a TEMPO/NaBr/NaClO mixed oxidation system at room temperature, centrifugally cleaning and dialyzing to obtain the bacterial cellulose membrane.
The chitosan/dilute acid solution in the step (2) is prepared by dissolving chitosan in 0.1-5% of dilute acid solution to form 1-5% of chitosan/dilute acid solution.
The collagen in the step (2) is fish-derived collagen; the concentration of the collagen aqueous solution is 0.1-10 mg/mL.
The mass ratio of the collagen to the oxidized regenerated cellulose in the step (2) is 1:1-10: 1.
The dipping and adsorbing time in the step (2) is 0.5-5 hours.
The electrostatic adsorption self-assembly reaction time in the step (2) is 1-60 minutes.
The bacterial nano-cellulose is used as a substrate to prepare the oxidized regenerated cellulose (OBC), and the coagulation process is accelerated by utilizing the characteristics of high purity and large specific surface area. Meanwhile, the opposite charge of OBC and chitosan is utilized to carry out electrostatic adsorption self-assembly reaction for compounding, and collagen can be introduced into the OBC and the chitosan to prepare the absorbable composite material with the functions of acute hemostasis, antibiosis and healing promotion.
Advantageous effects
(1) The local acute hemostasis absorbable material provided by the invention is prepared from oxidized regenerated cellulose by taking bacterial cellulose as a substrate, and has the advantages of higher carboxyl content, larger specific surface area, lower polymerization degree, better hemostasis effect, better biodegradability and easier absorption by a human body.
(2) The invention utilizes the charge property of the oxidized regenerated cellulose opposite to that of chitosan to carry out electrostatic adsorption self-assembly preparation without using an additional cross-linking agent; meanwhile, the addition of chitosan improves the procoagulant and antibacterial properties of the material.
(3) According to the invention, the large specific surface area of oxidized regenerated cellulose is utilized, and after collagen molecules are fully adsorbed, the oxidized regenerated cellulose and chitosan are subjected to electrostatic adsorption self-assembly reaction, so that the finally obtained material has the characteristics of acute hemostasis, antibiosis, healing promotion and absorbability.
(4) The raw materials of the invention are all green and environment-friendly biological materials, do not contain chemical substances harmful to human bodies, can be prepared into various shapes according to actual conditions and characteristics, are safe and environment-friendly, can be rapidly degraded in the environment after being discarded, and can be used as a functional material with good performance and environmental protection.
(5) The preparation method is simple and easy to implement, uniform and efficient in compounding, free of cross-linking agent and damage to the original structure of the synthetic substance, green and environment-friendly, capable of realizing industrial production and good in market application prospect.
Drawings
FIG. 1 is a schematic flow chart of the production process of the present invention;
FIG. 2 is a product diagram of a topical acute hemostatic absorbable material; wherein, 1 is oxidized regenerated cellulose (OBC), 2 is oxidized regenerated cellulose/chitosan (OC), and 3 is oxidized regenerated cellulose/collagen/chitosan (OCC);
FIG. 3 is a graph showing the bacteriostatic effects of examples 1-3 on Escherichia coli and Staphylococcus aureus at the same dilution factor, with sterile gauze as a control group;
FIG. 4 is the results of the in vitro whole blood clotting time test of examples 1-3.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
(1) Taking acetobacter xylinum as a strain, performing static culture for 10 days at constant temperature by using a liquid culture medium, taking out a bacterial cellulose membrane, placing the bacterial cellulose membrane in a sodium hydroxide solution, treating the bacterial cellulose membrane for 2 hours at 80 ℃, taking out the bacterial cellulose membrane, and rinsing the bacterial cellulose membrane to be neutral by using deionized water to obtain the bacterial cellulose membrane; soaking in hyaluronic acid solution for 6h to obtain a bacterial cellulose membrane soaked by hyaluronic acid;
(2) weighing 4g of purified bacterial cellulose membrane, shearing the bacterial cellulose membrane, and then scattering the bacterial cellulose membrane in a high-speed homogenizer to obtain bacterial cellulose suspension.
(3) And (3) oxidizing the bacterial cellulose suspension (with the solid content of 1-10g/L) obtained in the step (2) by a TEMPO/NaBr/NaClO mixed oxidation system at room temperature, wherein the use amount of TEMPO is 0.1mmol/g, and NaBr is 1 mmol/g. The reaction was started by adding a certain amount of 10% NaClO solution, the pH of the reaction system was maintained at 10 ± 0.5 by dropwise addition of 0.5M NaOH solution, and the reaction was terminated by adding ethanol until no more NaOH solution was consumed. The obtained oxidation product is stored in an environment of 4 ℃ after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized regenerated cellulose suspension was determined by conductometry to be 23%.
(4) And (4) freeze-drying the oxidized regenerated cellulose suspension obtained in the step (3) to finally obtain the oxidized regenerated cellulose hemostatic material.
Example 2
(1) Taking acetobacter xylinum as a strain, performing static culture for 10 days at constant temperature by using a liquid culture medium, taking out a bacterial cellulose membrane, placing the bacterial cellulose membrane in a sodium hydroxide solution, treating the bacterial cellulose membrane for 2 hours at 80 ℃, taking out the bacterial cellulose membrane, and rinsing the bacterial cellulose membrane to be neutral by using deionized water to obtain the bacterial cellulose membrane; soaking in hyaluronic acid solution for 6h to obtain a bacterial cellulose membrane soaked by hyaluronic acid;
(2) weighing 4g of purified bacterial cellulose membrane, shearing the bacterial cellulose membrane, and then scattering the bacterial cellulose membrane in a high-speed homogenizer to obtain bacterial cellulose suspension.
(3) And (3) oxidizing the bacterial cellulose suspension (with the solid content of 1-10g/L) obtained in the step (2) by a TEMPO/NaBr/NaClO mixed oxidation system at room temperature, wherein the use amount of TEMPO is 0.1mmol/g, and NaBr is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and 0.5M NaOH solution was added dropwise to maintain the pH of the reaction system at 10 ± 0.5, and when no more NaOH solution was consumed, ethanol was added to terminate the reaction. The obtained oxidation product is stored in an environment of 4 ℃ after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized regenerated cellulose suspension was determined by conductometry to be 23%.
(4) 2g of chitosan was weighed and dissolved in 100mL of 3% acetic acid solution to form a dilute acid chitosan solution with a final concentration of 2%.
(5) And (3) dropwise adding 5mL of the chitosan dilute acid solution prepared in the step (4) into 50mL of the oxidized regenerated cellulose suspension prepared in the step (3), reacting for 60 minutes, repeatedly centrifuging and rinsing the product, and finally performing freeze drying to obtain the oxidized regenerated cellulose/chitosan composite hemostatic material.
Example 3
(1) Taking acetobacter xylinum as a strain, performing static culture for 10 days at constant temperature by using a liquid culture medium, taking out a bacterial cellulose membrane, placing the bacterial cellulose membrane in a sodium hydroxide solution, treating the bacterial cellulose membrane for 2 hours at 80 ℃, taking out the bacterial cellulose membrane, and rinsing the bacterial cellulose membrane to be neutral by using deionized water to obtain the bacterial cellulose membrane; soaking in hyaluronic acid solution for 6h to obtain a bacterial cellulose membrane soaked by hyaluronic acid;
(2) weighing 4g of purified bacterial cellulose membrane, shearing the bacterial cellulose membrane, and then scattering the bacterial cellulose membrane in a high-speed homogenizer to obtain bacterial cellulose suspension.
(3) And (3) oxidizing the bacterial cellulose suspension (with the solid content of 1-10g/L) obtained in the step (2) by a TEMPO/NaBr/NaClO mixed oxidation system at room temperature, wherein the use amount of TEMPO is 0.1mmol/g, and NaBr is 1 mmol/g. The reaction was started by adding a certain amount of 10% NaClO solution, the pH of the reaction system was maintained at 10 ± 0.5 by dropwise addition of 0.5M NaOH solution, and the reaction was terminated by adding ethanol until no more NaOH solution was consumed. The obtained oxidation product is stored in an environment of 4 ℃ after centrifugation, rinsing and autoclaving. The carboxyl content of the TEMPO oxidized regenerated cellulose suspension was determined by conductometry to be 23%.
(4) 30mg of fish-derived collagen was weighed and dissolved in 10mL of sterile water thoroughly to form a protein solution with a final concentration of 3 mg/mL.
(5) And (3) adding 10mL of the protein solution prepared in the step (4) into 40mL of the oxidized regenerated cellulose suspension prepared in the step (3), and fully stirring and adsorbing for 1 hour to obtain the oxidized regenerated cellulose suspension adsorbed with the collagen.
(6) 2g of chitosan was weighed and dissolved in 100mL of 3% acetic acid solution to form a dilute acid chitosan solution with a final concentration of 2%.
(7) And (3) dropwise adding 5mL of the chitosan dilute acid solution prepared in the step (6) into 50mL of the oxidized regenerated cellulose suspension liquid adsorbed with the collagen prepared in the step (5), reacting for 60 minutes, repeatedly centrifuging and rinsing the product, and finally performing freeze drying to obtain the oxidized regenerated cellulose/collagen/chitosan composite hemostatic material.
As can be seen from fig. 3, the oxidized regenerated cellulose has a certain bacteriostatic effect compared to the gauze, which is mainly due to the low pH environment formed by the large amount of carboxyl groups in the oxidized regenerated cellulose inhibiting the proliferation and reproduction of bacteria. However, its bacteriostatic effect is very limited and a considerable number of bacteria still survive. On the contrary, the antibacterial performance of the chitosan experimental group (OC, OCC) is obviously improved compared with that of oxidized regenerated cellulose, and the chitosan experimental group has the same bacteriostatic tendency on escherichia coli and staphylococcus aureus. The excellent broad-spectrum antibacterial property shows that the OCC has the potential of being applied to clinic.
As can be seen from FIG. 4, the whole blood clotting times of OBC, OC and OCC were 248s, 220s and 180s, respectively, indicating that OCC has excellent hemostatic properties. The mechanism may be due to the synergistic effect of oxidized regenerated cellulose, chitosan and collagen. The procoagulant performance of the three materials is proved in a plurality of documents, the three materials are skillfully combined together under the condition of not damaging the original structure, a novel composite material is formed, the excellent hemostatic performance is obtained, and the composite material has the potential of being applied to clinical use as an acute hemostatic material.
Claims (9)
1. A local acute hemostasis absorbable material, which is characterized in that: the preparation method comprises the steps of preparing oxidized regenerated cellulose by taking bacterial cellulose as a substrate, and performing electrostatic adsorption self-assembly on the oxidized regenerated cellulose, chitosan and collagen to obtain the oxidized regenerated cellulose;
the local acute hemostasis absorbable material is prepared by the following method: adding a collagen aqueous solution into the oxidized regenerated cellulose suspension for dipping and adsorption, then dropwise adding a chitosan/dilute acid solution into the oxidized regenerated cellulose suspension for electrostatic adsorption self-assembly reaction, then taking out, rinsing and freeze-drying.
2. The absorbable material for local acute hemostasis by compression according to claim 1, wherein: the bacterial cellulose is obtained by taking acetobacter xylinum as a strain, performing static culture for 7-20 days at constant temperature by using a liquid culture medium, placing the solution into a sodium hydroxide solution, treating the solution at 70-90 ℃ for 2-4 hours, taking out the solution, and rinsing the solution to be neutral by using deionized water.
3. A method for preparing a local acute hemostasis absorbable material comprises the following steps:
(1) preparing oxidized regenerated cellulose suspension by taking bacterial cellulose as a substrate;
(2) adding a collagen aqueous solution into the oxidized regenerated cellulose suspension for soaking and adsorption, then dropwise adding a chitosan/dilute acid solution into the oxidized regenerated cellulose suspension for electrostatic adsorption self-assembly reaction, then taking out, rinsing and freeze-drying to obtain the local acute hemostasis absorbable material.
4. The production method according to claim 3, characterized in that: the preparation method of the oxidized regenerated cellulose suspension in the step (1) comprises the following steps: the bacterial cellulose membrane is broken up, oxidized by a TEMPO/NaBr/NaClO mixed oxidation system at room temperature, centrifugally cleaned and dialyzed to obtain the bacterial cellulose membrane.
5. The production method according to claim 3, characterized in that: the chitosan/dilute acid solution in the step (2) is prepared by dissolving chitosan in 0.1-5% of dilute acid solution to form 1-5% of chitosan/dilute acid solution.
6. The production method according to claim 3, characterized in that: the collagen in the step (2) is fish-derived collagen; the concentration of the collagen aqueous solution is 0.1-10 mg/mL.
7. The production method according to claim 3 or 6, characterized in that: the mass ratio of the collagen to the oxidized regenerated cellulose in the step (2) is 1:1-10: 1.
8. The production method according to claim 3, characterized in that: the immersion adsorption time in the step (2) is 0.5 to 5 hours.
9. The production method according to claim 3, characterized in that: the electrostatic adsorption self-assembly reaction time in the step (2) is 1-60 minutes.
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| CN110507844A (en) * | 2019-09-06 | 2019-11-29 | 东华大学 | A kind of absorbable composite material and preparation method for topical acute hemostasis based on oxidizing bacteria cellulose |
| CN111939314A (en) * | 2020-07-28 | 2020-11-17 | 天津科技大学 | Preparation method of bacterial cellulose ointment for wound surface |
| CN112773929A (en) * | 2020-12-31 | 2021-05-11 | 东华大学 | Absorbable hemostatic composite material based on polyanionic cellulose and preparation method thereof |
| CN112773941B (en) * | 2020-12-31 | 2022-09-16 | 东华大学 | Chitosan microsphere-bacterial cellulose composite material and preparation and application thereof |
| CN114288480B (en) * | 2021-12-06 | 2022-10-11 | 盐城工学院 | Biologically modified oxidized regenerated cellulose hemostatic anti-adhesion material and preparation method thereof |
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| JPS6094401A (en) * | 1983-10-31 | 1985-05-27 | Asahi Chem Ind Co Ltd | Cellulose dervative having excellent liquid absorption characteristic, its production and structure containing the same |
| EP2173297B1 (en) * | 2007-07-18 | 2021-03-31 | Marine Polymer Technologies, Inc. | Application of polymeric materials to screens to facilitate hemostasis and wound healing |
| US9149511B2 (en) * | 2011-06-30 | 2015-10-06 | Ethicon, Inc. | Procoagulant peptides and their derivatives and uses therefor |
| CN102908651B (en) * | 2012-10-30 | 2014-04-02 | 威高集团有限公司 | Preparation method of oxidized regenerated cellulose hemostatic material with micro-nano composite structure |
| CN103550817B (en) * | 2013-10-29 | 2017-03-01 | 上海昌颌医药科技有限公司 | A kind of Bacterial cellulose/shitosan composite sponge dressing and preparation method thereof |
| CN104383587B (en) * | 2014-12-01 | 2016-10-19 | 哈尔滨工业大学 | A kind of fullerene/oxidized regenerated cellulose compound hemostatic material and preparation method thereof |
| CN106178066A (en) * | 2015-05-07 | 2016-12-07 | 中国科学院化学研究所 | A kind of modified cellulose/chitosan compound hemostatic material and preparation method and application |
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