CN106729957B - Preparation method of in-vivo hemostatic dressing with transglutaminase as cross-linking agent - Google Patents

Abstract

The invention discloses a preparation method of an in-vivo hemostatic dressing. In the method, glutamine transaminase is added to a human-like collagen solution for cross-linking, precise cooling and pre-freezing are carried out at a set cooling speed, vacuum freeze drying is performed, and then Co60 irradiation sterilization finally obtained an in vivo hemostatic dressing. Preparation method of the present invention The in vivo hemostatic dressing prepared by the present invention has high safety, good biocompatibility, and remarkable hemostatic effect, can be used for hemostasis of various wound surfaces and postoperative hemostasis, and has a good reputation in the related fields of medical biological materials and tissue engineering. application prospects.

Description

A kind of preparation method of in vivo hemostatic dressing using transglutaminase as cross-linking agent

technical field

The invention relates to a preparation method of an in vivo hemostatic dressing, in particular to a preparation method of an in vivo hemostatic dressing using glutaminyltransferase as a cross-linking agent, and belongs to the field of biomedical materials.

Background technique

Collagen is a high-molecular-weight protein that is generally white, opaque, and fibrous. It is one of the main components of the structure of living organisms and is widely distributed in the skin, bone and muscle tissue of animals. Collagen has hemostatic properties. On the one hand, it can strengthen the adhesion and aggregation of platelets to form thrombus and prevent blood from flowing out. On the other hand, the intrinsic coagulation pathway can also be initiated by activating and inducing various coagulation factors. However, collagen is mostly derived from animal skin, tendons, etc., and its processability is poor. During the extraction process, part of the biological activity of the protein will be lost, and it is a kind of heterologous substance to the human body, which can cause adverse reactions such as allergies. It is very easy to produce a rejection reaction in the human body. Therefore, it is necessary to find a collagen with simple preparation and high safety as a raw material.

Transglutaminase is an enzyme originally contained in human tissues, which can cross-link with collagen. Transglutaminase uses the γ-carboxamide group of the glutamine residue in the peptide bond as the donor of the acyl group. , the lysyl residue in the polypeptide chain is based on the amino group as the acyl acceptor to form intramolecular and intermolecular ε-γ (-glutamyl) lysine isopeptide bonds. Thermal cross-linking is heating under vacuum conditions, causing severe dehydration of collagen molecules, esterification of free carboxyl groups and hydroxyl groups, and amide reactions between carboxyl groups and amino groups, thereby improving the mechanical properties of the hemostatic dressing, and also maintaining the internal hemostatic dressing. The pore structure remains unchanged, making the pores uniform and complete.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a preparation method of an in vivo hemostatic dressing which is safe to use, absorbable and has good hemostatic effect.

The realization process of the present invention is as follows:

A preparation method of an in vivo hemostatic dressing: using human-like collagen as a raw material, adding water for injection, stirring and dissolving, adding transglutaminase to the human-like collagen solution for cross-linking, and controlling the cooling rate to be 4-8 ℃. /min for cooling and pre-freezing, vacuum freeze-drying, and Co60 irradiation sterilization to obtain an in vivo hemostatic dressing.

The above-mentioned human-like collagen is a human-derived collagen produced by high-density fermentation of genetically recombined Escherichia coli.

The mass concentration of the above-mentioned human-like collagen after adding water for injection is 1%-5%; the ratio of transglutaminase to human-like collagen is 4-8U/g.

Specifically, the preparation method of the above-mentioned hemostatic dressing in vivo comprises the following steps:

(1) Dissolve human-like collagen in water for injection, so that the mass concentration of human-like collagen in the solution is 1%-5%;

(2) Transfer the solution into a disposable sterile petri dish or mold, 7-15 mL per dish or mold;

(3) Add transglutaminase, the ratio of transglutaminase to human-like collagen is 4-8U/g, the reaction temperature is -4-0°C, and the cross-linking time is 24-48 hours;

(4) During the pre-freezing process, the cooling rate is controlled to 4-8 ℃/min;

(5) The samples were lyophilized for 24-72 h, and the lyophilized samples were sterilized by Co60 irradiation for 10-30 h.

The present invention has the following advantages:

(1) The collagen involved in the present invention is human-derived collagen fermented at high density by genetically engineered bacteria, which fundamentally solves the problems of water insolubility and hidden danger of viruses in animal-extracted collagen, and has good biocompatibility, It has the advantages of promoting cell growth, no hidden virus and low immune rejection, which improves the safety of hemostatic dressings in vivo;

(2) The present invention adopts enzymatic cross-linking, and specifically uses transglutaminase to cross-link collagen to prepare hemostatic dressings in vivo, which can improve the mechanical strength of the hemostatic dressing and improve the hemostatic effect. The enzyme contained is safe and non-toxic, and the prepared hemostatic dressing ensures its good biological safety when it is used in clinical practice;

(3) The synthesis process of the in vivo hemostatic dressing prepared by the present invention is simple and easy to realize, the product quality is stable, the safety is high, the process is easy to scale up and the reproducibility is good, and it has a good application prospect in related fields such as medical biomaterials and tissue engineering.

Description of drawings

Fig. 1 is the appearance diagram of in vivo hemostatic dressing;

Fig. 2 is the SEM image of in vivo hemostatic dressing;

Fig. 3 is the hemostatic effect diagram of rabbit ear of hemostatic dressing in vivo;

Fig. 4 is the hemostatic effect diagram of the rabbit liver of hemostatic dressing in vivo;

Figure 5 is a graph of the cytotoxicity experiment of hemostatic dressings in vivo;

Figure 6 shows the subcutaneous implantation of in vivo hemostatic dressings.

Detailed ways

The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments. Any equivalent transformations taken by those of ordinary skill in the art to the technical solutions of the present invention by reading the description of the present invention are The present invention is covered by the claims.

A preparation method of a hemostatic dressing in vivo, comprising the following steps:

(1) Dissolve human-like collagen in water for injection, stir to dissolve it completely, so that the mass concentration of human-like collagen in the solution is 1%-5%. A human-derived collagen produced by high-density fermentation;

(2) Transfer the solution into a disposable sterile petri dish or mold, 7-15 mL per dish or mold;

(3) Add transglutaminase, the ratio of adding transglutaminase to human-like collagen is 4-8U/g, the reaction temperature is -4-0°C, and the cross-linking time is 24-48 hours;

(4) During the pre-freezing process of the programmed freezer, the cooling rate is controlled to be 4-8°C/min for precise cooling, and it has been lowered to -40--80°C;

(5) Freeze the samples in a vacuum freeze dryer for 24-72 h;

(6) The lyophilized samples are sterilized by irradiation for 10-30 h.

The present invention uses human-like collagen as the main raw material to prepare the hemostatic dressing in vivo, and the existing methods mostly use an ultra-low temperature refrigerator to directly prefreeze, and the protein solution is directly placed at a low temperature. During freeze-drying, the ice crystals sublime to form numerous uneven holes, and the obtained in vivo hemostatic dressing has uneven surface, uneven edges, poor mechanical strength and low hemostatic effect. The pre-freezing method in the present invention is to perform programmed freezing according to the set cooling law. The formation of ice crystals is relatively regular. When the temperature drops to a certain level, many crystal nuclei are first formed. As the temperature continues to drop, the surrounding small droplets adsorb After crystallizing on the surface of these nuclei, due to the constant and consistent rate of temperature drop, the growth rates of all nuclei are very similar, and finally ice crystals with similar shapes and sizes are formed. , The pore size is uniform, the mechanical strength is large, and it is not easy to be broken by blood flow during the hemostasis process and cause secondary bleeding, which greatly improves the hemostasis effect of the sponge.

In addition, an in-depth study of the cooling rate of the programmed freezer is carried out. If the cooling rate is greater than 10ºC/min, the temperature jump will be relatively large, and the formation of ice crystals will be affected to a certain extent. If the cooling rate is less than 3ºC/min, the concentration in The droplets on the nuclei cannot be frozen quickly and have the potential to flow, so all the crystals are irregular in size and shape. The research cooling rate of the present invention is controlled at 4-8° C./min, and the obtained material has the best physical and chemical properties and hemostatic effect.

Example 1

Dissolve human-like collagen in water for injection, after dissolving, add transglutaminase, stir to dissolve it completely, so that the mass concentration of human-like collagen in the mixed solution is 2.0%, and the mass of transglutaminase is 2.0%. 4U per gram of protein, placed in a -4°C refrigerator for cross-linking reaction for 24 hours, frozen to -80°C at a speed of -4°C/min in a programmed freezer, kept for 2 hours, and quickly transferred to a vacuum freeze dryer for freezing After drying for 24 h, the package was taken out and sterilized by Co60 irradiation for 24 h to obtain an in vivo hemostatic dressing. The prepared sponge has a porosity of 92% and a water absorption rate of 1500%.

Example 2

Dissolve human-like collagen in water for injection, after dissolving, add transglutaminase, stir to dissolve it completely, so that the mass concentration of human-like collagen in the mixed solution is 2.5%, and the mass of transglutaminase is 2.5%. 4U per gram of protein, placed in a -4°C refrigerator for cross-linking reaction for 30 hours, frozen to -80°C at a speed of -4°C/min in a programmed freezer, kept for 2 hours, and quickly transferred to a vacuum freeze dryer for freezing After drying for 30 h, the package was taken out and sterilized by Co60 irradiation for 24 h to obtain an in vivo hemostatic dressing.

Example 3

Dissolve human-like collagen in water for injection, after dissolving, add transglutaminase, stir to dissolve it completely, so that the mass concentration of human-like collagen in the mixed solution is 4.0%, and the mass of transglutaminase is 4.0%. 6U per gram of protein, placed in a -4°C refrigerator for cross-linking reaction for 40 hours, frozen to -80°C at a speed of -6°C/min in a programmed freezer, and kept for 2 hours, then quickly transferred to a vacuum freeze dryer for freezing After drying for 48 h, the package was taken out and sterilized by Co60 irradiation for 24 h to obtain an in vivo hemostatic dressing.

Example 4

Dissolve human-like collagen in water for injection, add transglutaminase after dissolving, stir to dissolve it completely, so that the mass concentration of human-like collagen in the mixed solution is 5.0%, and the mass of transglutaminase is 5.0%. 8U per gram of protein, placed in a -4°C refrigerator for cross-linking reaction for 48 hours, frozen to -80°C at a speed of -8°C/min in a programmed freezer, kept for 2 hours, and quickly transferred to a vacuum freeze dryer for freezing After drying for 48 h, the package was taken out and sterilized by Co60 irradiation for 24 h to obtain an in vivo hemostatic dressing.

Example 5

The following is the relevant performance test of the in vivo hemostatic dressing prepared in Example 1 of the present invention:

1. Appearance observation of in vivo hemostatic dressing

In Figure 1, a is the sample appearance of the enzymatically cross-linked human collagen-like hemostatic dressing pre-frozen with controlled cooling range, and b is the sample appearance of the enzymatic cross-linked human-like collagen hemostatic dressing that is naturally pre-frozen in an ultra-low temperature refrigerator. It can be seen from the figure that compared with the in vivo hemostatic dressing that is directly pre-frozen in the ultra-low temperature refrigerator, the surface of the in-vivo hemostatic dressing that adopts the programmed cooling and pre-freezing is more flat and uniform, and there are fewer wrinkles.

2. SEM observation of hemostatic dressings in vivo

In Figure 2, a is the SEM image of the enzymatic cross-linked human collagen-like hemostatic dressing pre-frozen with controlled cooling range, and b is the surface SEM image of the enzymatic cross-linked human collagen-like hemostatic dressing that is naturally pre-frozen in an ultra-low temperature refrigerator picture. It can be seen from the figure that compared with the in vivo hemostatic dressing that is directly pre-frozen in the ultra-low temperature refrigerator, the hole formed by the in-vivo hemostatic dressing that is pre-frozen by controlling the cooling range is relatively complete and uniform in size, which can effectively increase the contact area between the dressing and the wound surface. Improve hemostatic effect.

3. Determination of the hemostatic effect of hemostatic dressings in vivo

Rabbit ear hemostasis: The New Zealand white rabbit was used as the animal hemostasis model, and 2 mL of 2.5% sodium pentobarbital prepared in advance was slowly injected into the ear vein. After the rabbit was anesthetized, it was fixed on the operating table, and the center of the rabbit was cut off with curved scissors. The rabbit hair from the artery was then disinfected with Aner iodine disinfectant in the area where the wound was to be made in the rabbit ear, and the color of the iodine solution was removed by repeated scrubbing with 75% alcohol, and then the central ear artery was cut off with a scalpel blade. And tear off the skin of the wound. When a large amount of arterial blood flows out, quickly absorb it with sterile medical gauze, then quickly apply a sponge of a certain size prepared in advance to the cut wound, and press it with your hands to stop the bleeding. At the same time, the adhesion of the sponge on the wound surface was observed, the complete hemostasis time was recorded, and the surface was photographed.

Rabbit liver hemostasis: 2 mL of pre-prepared 2.5% sodium pentobarbital was slowly injected into the ear vein, fixed on the operating table after anesthesia, and the abdomen of the rabbit was opened with a surgical blade until the liver was exposed. Make a wound of the same size on the leaf. When a large amount of blood flows out, first absorb it with medical sterile gauze, then quickly use the prepared collagen sponge to compress the wound, and press it with your hands to stop the bleeding, while observing the adhesion of the sponge to the wound. , the time to complete hemostasis was recorded, and the liver surface was photographed.

In Figure 3, a is the rabbit ear hemostasis diagram of the enzymatic cross-linked human-like collagen hemostatic dressing pre-frozen with controlled cooling range, and b is the rabbit ear hemostasis of the heat-cross-linked human-like collagen hemostatic dressing that is naturally pre-frozen in the ultra-low temperature refrigerator. In Figure 4, a is the hemostasis diagram of the rabbit liver with the enzymatic cross-linked human-like collagen hemostatic dressing pre-frozen with controlled cooling range, b is the rabbit liver hemostasis with the enzymatic cross-linked human-like collagen hemostatic dressing naturally pre-frozen in the ultra-low temperature refrigerator picture. It was determined that the complete hemostasis time of rabbit ears with programmed cooling and pre-freezing in vivo hemostatic dressing was about 56s, and the complete hemostasis time of liver was 44s. It can be seen from the figure that compared with the in vivo hemostatic dressing that is directly pre-frozen in the ultra-low temperature refrigerator, the rabbit ear wound with the pre-freezing in vivo hemostatic dressing with controlled cooling range has a faster hemostasis speed and better hemostasis effect. Because the smooth and flat structure of its surface is conducive to its tight adhesion to the wound surface, and the internal pore-like structure of uniform size is conducive to its interaction with platelets and coagulation factors in the blood, which enhances the hemostatic effect of the hemostatic dressing in vivo.

4. In vivo cytotoxicity assay of hemostatic dressings

Preparation method of leaching solution: All operations in this experiment were completed in a biological safety cabinet. Immerse the sterilized sponge sample in the RPMI1640 medium at a ratio of 0.1g/mL, seal it with parafilm and place it at a constant temperature of 37ºC for leaching for 72±2 h, take out the sample and filter it with a 0.2μm sterile filter , to remove particulate matter from the leachate.

Cell culture: After the passaged BHK-21 cells were digested with trypsin, fresh culture medium was added, and the cells were evenly distributed in the culture medium by repeated pipetting and beating, and counted under an inverted microscope using a hemocytometer, and the cell suspension was diluted to 10 ⁴ to 10 ⁵ cells/mL were seeded on 96-well plates, 100 μL per well, and a control group was set up. Then the inoculated 96-well plate was placed in a CO ₂ incubator at 37°C for 24 hours. After the cells adhered to the wall, the original culture solution was aspirated and the sample extract was added. The control group and blank group were added with fresh culture liquid, and then placed in a biochemical incubator to continue cultivation.

Determination: Take out a well plate at 1, 3, 5 and 7 days respectively, add 10 μL of CCK-8 solution to each well, continue to culture at 37ºC for 4 hours, use a microplate reader to measure the absorbance at 450 nm, and calculate the relative proliferation by the following Rate (relative growthrate, RGR):

RGR=A1/A2×100%

In the formula: A1: Absorbance of experimental group A2: Absorbance of control group

Cytotoxicity assays were performed using the CCK-8 kit. The Cytotoxicity Detection Kit (Cell Counting Kit-8, CCK-8) uses the principle that water-soluble tetrazolium salt can be reduced to orange-yellow water-soluble substance Formazan by dehydrogenase in mitochondria to detect cell proliferation. , the amount of Formazan generated is positively related to the number of cells, that is, the more the number of living cells, the darker the color, and the less the color, the lighter the color, that is, the greater the toxicity to the cells, and the absorption value is indirectly analyzed by the microplate reader. Changes in the number of cells.

Figure 5 shows that the enzymatic cross-linked human collagen hemostatic dressing, the thermal cross-linked human collagen hemostatic dressing, the enzymatic cross-linked animal collagen hemostatic dressing and the thermal cross-linked animal collagen hemostatic dressing extract the effect of the cell The effect of growth, it can be seen from the figure that the cytotoxicity of the enzymatically cross-linked animal collagen hemostatic dressing and the thermally cross-linked animal collagen hemostatic dressing was grade 2, while the enzymatically cross-linked human-like collagen hemostatic dressing and the thermal cross-linked hemostatic dressing were evaluated as grade 2. The cytotoxicity evaluation of the combined human collagen hemostatic dressing is grade 1, and with the increase of cell culture time and days, the cell survival rate also increases, which shows that the in vivo hemostatic dressing prepared by the present invention has no hidden virus and high safety. , good biocompatibility, has the effect of promoting cell growth, and meets the requirements of biomedical materials.

5. Subcutaneous implantation of in vivo hemostatic dressings

First, inject 2 mL of 2.5% anesthetic into the ear vein of the New Zealand rabbit, and then fix it on the operating table. Four surgical cloths cover other places and only expose the place where the back is to be implanted. The skin of the back of the rabbit is disinfected with Aner's iodine. , disinfect your hands with 75% alcohol. Gently pick up the back skin by hand, and then use a surgical blade to gently cut a small incision of about 10mm in length. During the incision, you need to carefully observe the capillaries on the back of the rabbit and avoid it, and then use forceps to pick up the prepared The sample hemostatic material is placed under the skin, keeping at least 5mm with the incision as much as possible, and then the incision is sutured. One week later, the New Zealand rabbits were sacrificed, and the inner skin of the rabbits was observed for redness, purulence and ulceration, etc., to evaluate the biocompatibility of the hemostatic material in vivo.

In Fig. 6, a is a subcutaneous implantation diagram of an enzymatically cross-linked human-like collagen hemostatic dressing after 1 week, and b is a subcutaneous implantation diagram of an enzymatically cross-linked animal collagen hemostatic dressing. It can be seen from the figure that there is obvious redness, swelling and purulence around the subcutaneous implanted tissue of the animal collagen in vivo hemostatic dressing, while there is no redness, purulence and festering around the subcutaneous implanted tissue of the humanoid collagen in vivo hemostatic dressing. , indicating that the in vivo hemostatic dressing of the present invention has no hidden virus and biological rejection, has high safety and good biocompatibility, and is a good biomedical hemostatic material.

The content of the present invention is not limited to those listed in the embodiments, and any equivalent transformations taken by those of ordinary skill in the art to the technical solutions of the present invention by reading the description of the present invention are covered by the claims of the present invention.

Claims (3)

1. A preparation method of an in vivo hemostatic dressing is characterized in that: the in-vivo hemostatic dressing is prepared by taking human-like collagen as a raw material, adding water for injection, stirring and dissolving to obtain a human-like collagen solution with the mass concentration of 1% -5%, adding glutamine transaminase into the human-like collagen solution for crosslinking, wherein the ratio of the glutamine transaminase to the human-like collagen is 4-8U/g, performing temperature reduction and pre-freezing by controlling the temperature reduction rate to be 4-8 ℃/min, then performing vacuum freeze drying, and performing Co60 irradiation sterilization.

2. The method of making an in vivo hemostatic dressing according to claim 1, wherein: the human-like collagen is human-derived collagen produced by high-density fermentation of genetic recombinant escherichia coli.

3. A method of making an in vivo hemostatic dressing according to claim 1, characterized by the steps of:

(1) dissolving human-like collagen in water for injection to ensure that the mass concentration of the human-like collagen in the solution is 1-5 percent;

(2) transferring the solution into disposable sterile culture dishes or molds, wherein 7-15 mL of solution is added in each dish or mold;

(3) adding glutamine transaminase, wherein the ratio of the glutamine transaminase to the human-like collagen is 4-8U/g, the reaction temperature is-4-0 ℃, and the crosslinking time is 24-48 hours;

(4) in the pre-freezing process, the cooling rate is controlled to be 4-8 ℃/min;

(5) and (3) carrying out sample freeze-drying treatment for 24-72 h, and carrying out irradiation sterilization on the freeze-dried sample Co60 for 10-30 h.

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