CN110141677A - Absorbable material for local acute hemostasis and preparation method thereof - Google Patents

Abstract

The invention relates to an absorbable material for local acute hemostasis and a preparation method thereof. The oxidized regenerated cellulose is prepared by using bacterial cellulose as a substrate, and then electrostatically adsorbed and self-assembled with chitosan or chitosan and collagen. The invention also has the characteristics of acute hemostasis, broad-spectrum antibacterial, healing promotion and in vivo absorption; the preparation method is green and environmentally friendly, the compounding is uniform and efficient, and the obtained composite material has high safety and good market application prospect.

Description

Absorbable material for local acute hemostasis and preparation method thereof

technical field

The invention belongs to the technical field of biomedical composite materials, in particular to an absorbable material for local acute hemostasis and a preparation method thereof.

Background technique

Absorbable hemostatic material is a biomedical material that can be used in the bleeding site of a wound, can quickly stop bleeding, and can be absorbed by the human body within a certain period of time. Its biggest feature is that it can be applied to local difficult-to-suture wounds in humans or animals to stop bleeding. While achieving rapid hemostasis, it can be degraded in the body without removing it, so as to avoid the secondary injury of removing the dressing after the hemostasis is completed. Currently commonly used absorbable hemostatic materials include fibrin glue hemostatic materials, gelatin sponge hemostatic materials, oxidized cellulose hemostatic materials and chitosan hemostatic materials. The hemostatic mechanisms and usage methods of these hemostatic materials are different, and their hemostatic effects are also very different.

Oxidized regenerated cellulose was successfully prepared for the first time in 1960. It is a tissue-like or cotton-like absorbable hemostatic material in which cellulose is oxidized to become cellulose acid. It has the appearance and texture of cotton yarn, is soft and thin, and is easy to wrap, apply and pack. and so on. The hemostatic mechanism is to gather platelets in the blood of the surgical wound on the mesh gauze, and use the hemostatic gauze as the hemostatic matrix. After encountering blood, a gel-like black substance is formed quickly, and the blood clots are formed to achieve the purpose of hemostasis. It does not rely on the normal coagulation mechanism in the human body, but rapidly promotes blood coagulation through physical action, effectively controlling small blood vessel bleeding. At present, only the United States and the United Kingdom can produce such products. Surgicel, an absorbable hemostatic gauze used clinically, is a product of Johnson & Johnson in the United States. The main component of Surgicel is oxidized regenerated cellulose. Oxidized regenerated cellulose is a derivative of cellulose obtained by highly selective oxidation of C6 primary hydroxyl groups in cellulose into carboxyl groups. At present, it has been used in all walks of life, and the products sold in the market mainly rely on imports. Several companies have produced similar hemostatic gauze. Although oxidized regenerated cellulose has many excellent properties, it also has some disadvantages, such as low hemostatic efficiency, inability to apply to massive bleeding, weak antibacterial properties and low histocompatibility.

At present, in some published or authorized invention patents, 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 (CN105056284A). Although the above composite hemostatic materials have improved the properties of oxidized regenerated cellulose to a certain extent, the improvement effect is poor, only part of its properties can be improved, and the bioabsorbable properties of the materials will be negatively affected.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide an absorbable material for local acute hemostasis and a preparation method thereof, which solve the problems of weak antibacterial properties of oxidized regenerated cellulose, low histocompatibility and slow hemostasis.

The invention provides an absorbable material for local acute hemostasis, which is obtained by using bacterial cellulose as a substrate to prepare oxidized regenerated cellulose (OBC), and then performing electrostatic adsorption and self-assembly with chitosan or chitosan and collagen.

The bacterial cellulose is obtained by using Acetobacter xylinum as a strain, and after culturing it at a constant temperature in a liquid medium for 7-20 days, placing it in a sodium hydroxide solution, treating it at 70-90° C. for 2-4 hours, taking out, and using Obtained after rinsing with deionized water until neutral.

The present invention also provides a preparation method of a local acute hemostatic absorbable material, comprising:

(1) using bacterial cellulose as a substrate to prepare an oxidized regenerated cellulose suspension;

(2) adding the chitosan/dilute acid solution dropwise to the oxidized regenerated cellulose suspension for electrostatic adsorption self-assembly reaction, then taking out, rinsing, and freeze-drying to obtain a local acute hemostatic absorbable material (oxidized regenerated cellulose / Chitosan absorbable antibacterial hemostatic material OC);

Alternatively, the collagen aqueous solution is added to the oxidized regenerated cellulose suspension for immersion and adsorption, and then the chitosan/dilute acid solution is added dropwise to the oxidized regenerated cellulose suspension for electrostatic adsorption and self-assembly reaction, and then taken out, Rinse and freeze-dry to obtain local acute hemostatic absorbable material (oxidized regenerated cellulose/collagen/chitosan absorbable antibacterial hemostatic material OCC).

The preparation steps of the oxidized regenerated cellulose suspension in the step (1) are as follows: the bacterial cellulose membrane is broken up, oxidized in a TEMPO/NaBr/NaClO mixed oxidation system at room temperature, and obtained after centrifugal cleaning and dialysis.

The chitosan/dilute acid solution in the step (2) is to dissolve chitosan in a 0.1-5% dilute acid solution to form a chitosan/dilute acid solution with a final concentration of 1-5%.

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 collagen to oxidized regenerated cellulose in the step (2) is 1:1-10:1.

The soaking adsorption 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.

Oxidized regenerated cellulose (OBC) was prepared from bacterial nanocellulose as a substrate, and its high purity and large specific surface area were utilized to accelerate the coagulation process. At the same time, the opposite charge of OBC and chitosan is used to conduct electrostatic adsorption self-assembly reaction for composite, and collagen can also be introduced into it to prepare absorbable composite material with acute hemostasis, antibacterial and healing promotion functions.

beneficial effect

(1) The absorbable material for local acute hemostasis of the present invention uses bacterial cellulose as a substrate to prepare oxidized regenerated cellulose, which has higher carboxyl group content, larger specific surface area, lower degree of polymerization, better hemostatic effect, and biodegradable performance. Better and more easily absorbed by the body.

(2) The present invention utilizes the opposite charge properties of oxidized regenerated cellulose and chitosan for electrostatic adsorption self-assembly preparation, without using additional cross-linking agent; at the same time, the addition of chitosan improves the procoagulant and antibacterial properties of the material.

(3) The present invention utilizes the large specific surface area of oxidized regenerated cellulose to conduct electrostatic adsorption and self-assembly reaction with chitosan after fully adsorbing collagen molecules. absorption properties.

(4) The raw materials of the present invention are all green and environmentally friendly biological materials, do not contain chemical substances harmful to the human body, can be prepared into various shapes according to actual conditions and characteristics, are safe and environmentally friendly, can be rapidly degraded in the environment after being discarded, and can be used as a kind of Functional materials with good performance and green environmental protection.

(5) The preparation method of the present invention is simple and feasible, the compounding is uniform and efficient, the cross-linking agent is not used, and the original structure of the synthetic substance is not damaged, and the invention is green and environmentally friendly, can be industrially produced, and has a good market application prospect.

Description of drawings

Fig. 1 is the schematic flow sheet of the preparation method of the present invention;

Figure 2 is the product diagram of the absorbable material for topical acute hemostasis; wherein, 1 is oxidized regenerated cellulose (OBC), 2 is oxidized regenerated cellulose/chitosan (OC), and 3 is oxidized regenerated cellulose/collagen/shell glycans (OCC);

Fig. 3 is the bacteriostatic effect diagram of embodiment 1-3 to Escherichia coli and Staphylococcus aureus under the same dilution ratio, and sterile gauze is a control group;

FIG. 4 is the results of the in vitro whole blood clotting time test of Examples 1-3.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

(1) Using Acetobacter xylinum as the strain, after culturing at a constant temperature in the liquid medium for 10 days, take out the bacterial cellulose membrane and place it in sodium hydroxide solution, treat it at 80 °C for 2 hours, take it out, and rinse it with deionized water until After neutralization, bacterial cellulose membrane was obtained; soaked in hyaluronic acid solution for 6 hours to obtain bacterial cellulose membrane soaked in hyaluronic acid;

(2) Weigh 4 g of the purified bacterial cellulose membrane, cut it into pieces, and then disperse it in a high-speed homogenizer to obtain a bacterial cellulose suspension.

(3) the bacterial cellulose suspension (solid content is 1-10g/L) obtained in step (2) is oxidized at room temperature by TEMPO/NaBr/NaClO mixed oxidation system, the amount of TEMPO is 0.1mmol/g, NaBr is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and the pH of the reaction system was maintained at 10±0.5 by adding 0.5M NaOH solution dropwise. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl group content of the TEMPO oxidized regenerated cellulose suspension determined by conductometric titration was 23%.

(4) freeze-drying the oxidized regenerated cellulose suspension obtained in step (3), and finally obtain the oxidized regenerated cellulose hemostatic material.

Example 2

(1) Using Acetobacter xylinum as the strain, after culturing at a constant temperature in the liquid medium for 10 days, take out the bacterial cellulose membrane and place it in sodium hydroxide solution, treat it at 80 °C for 2 hours, take it out, and rinse it with deionized water until After neutralization, bacterial cellulose membrane was obtained; soaked in hyaluronic acid solution for 6 hours to obtain bacterial cellulose membrane soaked in hyaluronic acid;

(2) Weigh 4 g of the purified bacterial cellulose membrane, cut it into pieces, and then disperse it in a high-speed homogenizer to obtain a bacterial cellulose suspension.

(3) the bacterial cellulose suspension (solid content is 1-10g/L) obtained in step (2) is oxidized at room temperature by TEMPO/NaBr/NaClO mixed oxidation system, the amount of TEMPO is 0.1mmol/g, NaBr is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and the pH of the reaction system was maintained at 10±0.5 by adding 0.5M NaOH solution dropwise. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl group content of the TEMPO oxidized regenerated cellulose suspension determined by conductometric titration was 23%.

(4) Weigh 2 g of chitosan and fully dissolve it in 100 mL of 3% acetic acid solution to form a chitosan dilute acid solution with a final concentration of 2%.

(5) 5 mL of the chitosan dilute acid solution prepared in step (4) was added dropwise to 50 mL of the oxidized regenerated cellulose suspension prepared in step (3), and after 60 minutes of reaction, the product was repeatedly centrifuged and rinsed, and finally freeze-dried, The oxidized regenerated cellulose/chitosan composite hemostatic material was obtained.

Example 3

(1) Using Acetobacter xylinum as the strain, after culturing at a constant temperature in the liquid medium for 10 days, take out the bacterial cellulose membrane and place it in sodium hydroxide solution, treat it at 80 °C for 2 hours, take it out, and rinse it with deionized water until After neutralization, bacterial cellulose membrane was obtained; soaked in hyaluronic acid solution for 6 hours to obtain bacterial cellulose membrane soaked in hyaluronic acid;

(2) Weigh 4 g of the purified bacterial cellulose membrane, cut it into pieces, and then disperse it in a high-speed homogenizer to obtain a bacterial cellulose suspension.

(3) the bacterial cellulose suspension (solid content is 1-10g/L) obtained in step (2) is oxidized at room temperature by TEMPO/NaBr/NaClO mixed oxidation system, the amount of TEMPO is 0.1mmol/g, NaBr is 1 mmol/g. A certain amount of 10% NaClO solution was added to start the reaction, and the pH of the reaction system was maintained at 10±0.5 by adding 0.5M NaOH solution dropwise. When the NaOH solution was no longer consumed, ethanol was added to terminate the reaction. The obtained oxidized product was stored at 4°C after centrifugation, rinsing and autoclaving. The carboxyl group content of the TEMPO oxidized regenerated cellulose suspension determined by conductometric titration was 23%.

(4) Weigh 30 mg of fish-derived collagen and fully dissolve it in 10 mL of sterile water to form a protein solution with a final concentration of 3 mg/mL.

(5) 10 mL of the protein solution prepared in step (4) was added to 40 mL of the oxidized regenerated cellulose suspension prepared in step (3), fully stirred and adsorbed for 1 hour to obtain an oxidized regenerated cellulose suspension with collagen adsorbed.

(6) Weigh 2 g of chitosan and fully dissolve it in 100 mL of 3% acetic acid solution to form a chitosan dilute acid solution with a final concentration of 2%.

(7) 5 mL of the chitosan dilute acid solution prepared in step (6) was added dropwise to 50 mL of the collagen-adsorbed oxidized regenerated cellulose suspension prepared in step (5), and the product was repeatedly centrifuged and rinsed after 60 minutes of reaction. Finally, freeze-drying is performed to obtain oxidized regenerated cellulose/collagen/chitosan composite hemostatic material.

It can be seen from Figure 3 that compared with gauze, oxidized regenerated cellulose has a certain antibacterial effect, which is mainly because the low pH environment formed by a large number of carboxyl groups in oxidized regenerated cellulose inhibits the proliferation and reproduction of bacteria. However, its bacteriostatic effect is very limited, and a considerable number of bacteria still survive. In contrast, the antibacterial properties of the experimental group compounded with chitosan (OC, OCC) were significantly improved compared with the oxidized regenerated cellulose, and had the same antibacterial trend against Escherichia coli and Staphylococcus aureus. The excellent broad-spectrum antibacterial activity indicates that OCC has the potential for clinical application.

It can be seen from Figure 4 that the whole blood clotting times of OBC, OC and OCC are 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 properties of these three materials have been confirmed in many literatures. The present invention combines these three materials ingeniously without destroying the original structure to form a new composite material and obtain excellent It has hemostatic properties and has the potential to be used clinically as an acute hemostatic material.

Claims (9)

1. The absorbable material 1. a kind of topical acute stops blooding, it is characterised in that: by being aoxidized again by substrate preparation of bacteria cellulose Raw cellulose, then carry out Electrostatic Absorption self assembly with chitosan or chitosan and collagen and obtain.
2. The absorbable material 2. a kind of topical acute according to claim 1 stops blooding, it is characterised in that: the bacteria cellulose Be by after fluid nutrient medium constant temperature stationary culture 7-20 days, being placed in sodium hydroxide solution using acetobacter xylinum as strain, It takes out after handling 2-4h at 70-90 DEG C, is obtained after being rinsed with deionized water to neutrality.
3. 3. a kind of preparation method of topical acute hemostasis absorbable material, comprising:

   (1) using bacteria cellulose as substrate, oxidized regenerated cellulose suspension is prepared；

   (2) by chitosan/dilute acid soln be added dropwise in oxidized regenerated cellulose suspension carry out Electrostatic Absorption self assembly it is anti- It answers, then takes out, rinse, freeze-drying obtains topical acute hemostasis absorbable material；

   It is adsorbed alternatively, collagen aqueous solution is added to impregnate in oxidized regenerated cellulose suspension, then by chitosan/diluted acid Solution is added dropwise to progress Electrostatic Absorption self-assembling reaction in oxidized regenerated cellulose suspension, then takes out, and rinses, and freezes It is dry, obtain topical acute hemostasis absorbable material.
4. 4. preparation method according to claim 3, it is characterised in that: the oxidized regenerated cellulose in the step (1) is outstanding The preparation step of turbid is as follows: bacteria cellulose film being broken up, at room temperature through TEMPO/NaBr/NaClO mixed oxidization system It is obtained after oxidation, eccentric cleaning and dialysis.
5. 5. preparation method according to claim 3, it is characterised in that: chitosan/dilute acid soln in the step (2) is It dissolves the chitosan in the dilute acid soln of 0.1-5%, forms chitosan/dilute acid soln of final concentration of 1-5%.
6. 6. preparation method according to claim 3, it is characterised in that: the collagen in the step (2) is source of fish glue It is former；The concentration of collagen aqueous solution is 0.1-10mg/mL.
7. 7. the preparation method according to claim 3 or 6, it is characterised in that: collagen and oxidation in the step (2) The mass ratio of regenerated cellulose is 1:1-10:1.
8. 8. preparation method according to claim 3, it is characterised in that: the dipping adsorption time in the step (2) is 0.5-5 hours.
9. 9. preparation method according to claim 3, it is characterised in that: the Electrostatic Absorption self assembly in the step (2) is anti- It is 1-60 minutes between seasonable.