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, which comprises the steps of adding glutamine transaminase into a human-like collagen solution for crosslinking, accurately cooling and pre-freezing at a set cooling speed, carrying out vacuum freeze drying, and then irradiating and sterilizing by Co60 to finally obtain the in vivo hemostatic dressing. The in-vivo hemostatic dressing prepared by the preparation method has high safety, good biocompatibility and obvious hemostatic effect, can be used for hemostasis of various wound surfaces and postoperative hemostasis, and has good application prospect in related fields of medical biomaterials, tissue engineering and the like.

Description

Preparation method of in-vivo hemostatic dressing with 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 taking transglutaminase as a cross-linking agent, belonging to the field of biomedical materials.

Background

Collagen is a high molecular protein, generally in the form of white, opaque fibers. It is one of the major components of the constitutive structure of an organism and is widely distributed in the skin, bones and muscle tissues of animals. The collagen has hemostatic effect, and can promote platelet adhesion and aggregation to form thrombus and prevent blood outflow. 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, tendon and the like, has poor processability, can lose partial biological activity of protein in the extraction process, belongs to a heterologous substance for human bodies, can cause adverse reactions such as allergy and the like, and is very easy to generate rejection reaction when being applied to the human bodies. Therefore, it is necessary to find a collagen which is easy to prepare and has high safety as a raw material.

Transglutaminase is an enzyme originally contained in human tissues and can undergo a crosslinking reaction with collagen, and forms an epsilon-gamma (-glutamyl) lysine isopeptide bond within and between protein molecules by using a gamma-carboxamide group of a glutamine residue in a peptide bond as an acyl group donor and a lysyl residue in a polypeptide chain as an acyl group acceptor. The thermal crosslinking is heating under vacuum condition, so that collagen molecules are seriously dehydrated, free carboxyl and hydroxyl generate esterification reaction and amide reaction between carboxyl and amino occurs, thereby improving the mechanical property of the hemostatic dressing, and keeping the internal porous structure of the hemostatic dressing unchanged, so that pores are uniform and complete.

Disclosure of Invention

The invention aims to provide a preparation method of an in-vivo hemostatic dressing which is safe to use, absorbable and good in hemostatic effect.

The realization process of the invention is as follows:

a preparation method of an in vivo hemostatic dressing comprises the following steps: the in vivo hemostatic dressing is prepared by taking human-like collagen as a raw material, adding water for injection, stirring and dissolving, adding glutamine transaminase into a human-like collagen solution for crosslinking, cooling and pre-freezing by controlling the cooling rate to be 4-8 ℃/min, performing vacuum freeze drying, and irradiating and sterilizing Co 60.

The human-like collagen is a human-derived collagen produced by high-density fermentation of genetically recombinant escherichia coli.

The mass concentration of the human-like collagen added into the water for injection is 1-5 percent; the ratio of glutamine transaminase to human-like collagen is 4-8U/g.

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

(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.

The invention has the following advantages:

(1) the collagen related to the invention is human collagen fermented by genetically engineered bacteria at high density, which fundamentally solves the problems of water insolubility, virus hidden danger and the like of collagen extracted from animals, has the advantages of good biocompatibility, promotion of cell growth, no virus hidden danger, low immunological rejection reaction and the like, and improves the safety of the in-vivo hemostatic dressing;

(2) the in vivo hemostatic dressing is prepared by adopting enzymatic crosslinking, specifically, the glutamine transaminase crosslinking collagen is used for preparing the in vivo hemostatic dressing, the mechanical strength of the hemostatic dressing can be improved, the hemostatic effect is improved, the glutamine transaminase is an enzyme contained in human tissues, the in vivo hemostatic dressing is safe and non-toxic, and the prepared hemostatic dressing is applied to clinic, so that the good biological safety of the in vivo hemostatic dressing is ensured;

(3) the in-vivo hemostatic dressing prepared by the invention has the advantages of simple and easy realization of synthesis process, stable product quality, high safety, easy process amplification and good reproducibility, and has good application prospect in the related fields of medical biomaterials, tissue engineering and the like.

Drawings

FIG. 1 is an external view of an in vivo hemostatic dressing;

FIG. 2 is an SEM image of an in vivo hemostatic dressing;

FIG. 3 is a chart showing the hemostatic effect of rabbit ears as a hemostatic dressing in vivo;

FIG. 4 is a chart of the hemostatic effect of rabbit liver as an in vivo hemostatic dressing;

FIG. 5 is a graph of an in vivo hemostatic dressing cytotoxicity test;

fig. 6 is a view of the implantation of the hemostatic dressing in vivo subcutaneously.

Detailed Description

The technical solution of the present invention is not limited to the specific embodiments listed below, and includes any combination of the specific embodiments, and any equivalent changes to the technical solution of the present invention by a person skilled in the art after reading the description of the present invention are covered by the claims of the present invention.

A preparation method of an in vivo hemostatic dressing comprises the following steps:

(1) dissolving human-like collagen in water for injection, stirring to completely dissolve the human-like collagen, so that the mass concentration of the human-like collagen in the solution is 1-5%, wherein the human-like collagen is human-derived collagen produced by high-density fermentation of gene recombinant escherichia coli;

(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 added glutamine transaminase to human-like collagen is 4-8U/g, the reaction temperature is-4-0 ℃, and the crosslinking time is 24-48 hours;

(4) accurately cooling by controlling the cooling rate to be 4-8 ℃/min in the pre-freezing process by using a program freezer until the temperature is reduced to-40-80 ℃;

(5) freeze-drying the sample in a vacuum freeze dryer for 24-72 h;

(6) the freeze-dried sample is sterilized by irradiation for 10-30 h.

The invention adopts human-like collagen as a main raw material to prepare the in vivo hemostatic dressing, the prior method mostly adopts an ultra-low temperature refrigerator to directly pre-freeze, the protein solution is directly placed at low temperature, the liquid drops in the protein solution can not react in time, so that the formed ice crystals are disordered and have different sizes, during freeze-drying, the ice crystals are sublimated to form a plurality of uneven holes, and the obtained in vivo hemostatic dressing has uneven surface, uneven edges, poor mechanical strength and low hemostatic efficacy. The pre-freezing method comprises the steps of carrying out program freezing according to a set cooling rule, comparing the formation rule of ice crystals, when the temperature is reduced to a certain degree, firstly forming a plurality of crystal nuclei, continuously reducing the temperature, absorbing surrounding small liquid drops on the surfaces of the crystal nuclei for crystallization, wherein the reduction speed of the temperature is constant and consistent, the growth rates of all the crystal nuclei are very close to each other, finally forming the ice crystals with the shape and the size close to each other, freeze-drying to obtain the pore size close to each other, and the obtained in-vivo hemostatic dressing has the advantages of smooth surface, uniform pore size and high mechanical strength, is not easy to be broken by blood flow in the hemostatic process to cause secondary bleeding, and greatly improves the hemostatic effect of the sponge.

In addition, the temperature reduction rate of the program freezer is deeply researched, if the temperature reduction rate is more than 10 ℃ per minute, the temperature jump is large, the formation of the ice crystals is necessarily influenced to a certain extent, if the temperature reduction rate is less than 3 ℃ per minute, the liquid drops enriched on the crystal nuclei cannot be rapidly frozen and have the possibility of flowing, and thus all the sizes and the shapes of the crystals are irregular. The research of the invention controls the cooling rate at 4-8 ℃/min, and the obtained material has the best physical and chemical properties and hemostatic effect.

Example 1

Dissolving human-like collagen in water for injection, adding transglutaminase after dissolution, stirring to completely dissolve the human-like collagen until the mass concentration of the human-like collagen in the mixed solution is 2.0 percent and the mass of the transglutaminase is 4U per gram of protein, placing the mixed solution in a refrigerator at the temperature of-4 ℃ for crosslinking reaction for 24 hours, freezing the mixed solution in a program freezer to the temperature of-80 ℃ at the speed of-4 ℃/min, keeping the temperature for 2 hours, quickly transferring the mixed solution to a vacuum freeze dryer for freeze-drying for 24 hours, taking out the mixed solution, packaging the dried product, and performing Co60 irradiation sterilization for 24 hours to obtain the in-vivo hemostatic dressing. The prepared sponge has the porosity of 92% and the water absorption of 1500%.

Example 2

Dissolving human-like collagen in water for injection, adding transglutaminase after dissolution, stirring to completely dissolve the human-like collagen until the mass concentration of the human-like collagen in the mixed solution is 2.5 percent and the mass of the transglutaminase is 4U per gram of protein, placing the mixed solution in a refrigerator at the temperature of-4 ℃ for crosslinking reaction for 30 h, freezing the mixed solution in a program freezer to the temperature of-80 ℃ at the speed of-4 ℃/min, keeping the temperature for 2h, quickly transferring the mixed solution to a vacuum freeze dryer for freeze-drying for 30 h, taking out the mixed solution, packaging the dried product, and performing Co60 irradiation sterilization for 24h to obtain the in-vivo hemostatic dressing.

Example 3

Dissolving human-like collagen in water for injection, adding transglutaminase after dissolution, stirring to completely dissolve the human-like collagen until the mass concentration of the human-like collagen in the mixed solution is 4.0 percent and the mass of the transglutaminase is 6U per gram of protein, placing the mixed solution in a refrigerator at the temperature of-4 ℃ for crosslinking reaction for 40 hours, freezing the mixed solution in a program freezer to the temperature of-80 ℃ at the speed of-6 ℃/min, keeping the temperature for 2 hours, quickly transferring the mixed solution to a vacuum freeze dryer for freeze-drying for 48 hours, taking out the mixed solution, packaging the dried product, and performing Co60 irradiation sterilization for 24 hours to obtain the in-vivo hemostatic dressing.

Example 4

Dissolving human-like collagen in water for injection, adding transglutaminase after dissolution, stirring to completely dissolve the human-like collagen until the mass concentration of the human-like collagen in the mixed solution is 5.0 percent and the mass of the transglutaminase is 8U per gram of protein, placing the mixed solution in a refrigerator at the temperature of-4 ℃ for crosslinking reaction for 48 hours, freezing the mixed solution in a program freezer to the temperature of-80 ℃ at the speed of-8 ℃/min, keeping the temperature for 2 hours, quickly transferring the mixed solution to a vacuum freeze dryer for freeze-drying for 48 hours, taking out the mixed solution, packaging the dried product, and performing Co60 irradiation sterilization for 24 hours to obtain the in-vivo hemostatic dressing.

Example 5

The following are the relevant performance tests for the in vivo hemostatic dressing prepared in example 1 of the present invention:

1. in vivo hemostatic dressing appearance observation

In fig. 1, a is a sample appearance diagram of the enzyme-method cross-linked human collagen hemostatic dressing pre-frozen by controlling the temperature reduction amplitude, and b is a sample appearance diagram of the enzyme-method cross-linked human collagen hemostatic dressing naturally pre-frozen by an ultra-low temperature refrigerator. Compared with the in-vivo hemostatic dressing directly pre-frozen in an ultra-low temperature refrigerator, the in-vivo hemostatic dressing pre-frozen by adopting the procedure cooling has the advantages of more smooth and uniform surface and less folds.

2. Scanning electron microscope observation of in vivo hemostatic dressing

In fig. 2, a is a surface scanning electron microscope image of the enzyme-method cross-linked human-like collagen hemostatic dressing pre-frozen by controlling the cooling range, and b is a surface scanning electron microscope image of the enzyme-method cross-linked human-like collagen hemostatic dressing naturally pre-frozen by an ultra-low temperature refrigerator. Compared with the in-vivo hemostatic dressing directly pre-frozen in an ultra-low temperature refrigerator, the in-vivo hemostatic dressing pre-frozen by controlling the cooling amplitude has complete holes and uniform size, can effectively increase the contact area of the dressing and the wound surface, and improves the hemostatic effect.

3. Determination of hemostatic effect of in vivo hemostatic dressing

Stopping bleeding of rabbit ears: the method comprises the steps of taking a New Zealand white rabbit as an animal hemostasis model, slowly injecting 2mL of 2.5% sodium pentobarbital prepared in advance into ear veins, fixing the rabbit on an operating table after the rabbit is anesthetized, shearing off rabbit hair from a central artery of the rabbit by using a pair of bending scissors, disinfecting a position of a rabbit ear where a wound is to be made by using an iodine disinfectant, repeatedly scrubbing by using 75% alcohol to remove the color of the iodine disinfectant, cutting off the central artery of the ear by using an operation blade, and tearing off the skin of the made wound. When a large amount of arterial blood flows out, the arterial blood is quickly sucked by sterile medical gauze, and then sponge with certain size prepared in advance is quickly applied to the cut wound surface and pressed by hands to stop bleeding. And meanwhile, observing the adhesion condition of the sponge on the wound surface, recording the complete hemostasis time, and taking a picture of the surface.

Stopping bleeding of rabbit liver: 2mL of 2.5% sodium pentobarbital prepared in advance is slowly injected into an auricular vein, the sodium pentobarbital is fixed on an operating table after anesthesia, the abdomen of a rabbit is opened by adopting an operating blade until the liver is exposed, a wound surface with the same size is made on the liver lobe by using the blade, when a large amount of blood flows outwards, the blood is firstly absorbed by using medical sterile gauze, then the prepared collagen sponge is quickly used for compressing the wound surface, the hemostasis is realized by pressing the hand, the adhesion condition of the sponge on the wound surface is observed at the same time, the complete hemostasis time is recorded, and the picture is taken for the surface of the liver.

In fig. 3, a is a rabbit ear hemostasis map of the enzyme-crosslinked human-like collagen hemostasis dressing pre-frozen by controlling the cooling range, and b is rabbit ear hemostasis of the heat-crosslinked human-like collagen hemostasis dressing naturally pre-frozen by the ultra-low temperature refrigerator. In fig. 4, a is a rabbit liver hemostasis map of the enzyme-crosslinked human-like collagen hemostasis dressing pre-frozen by controlling the cooling range, and b is a rabbit liver hemostasis map of the enzyme-crosslinked human-like collagen hemostasis dressing naturally pre-frozen by the ultra-low temperature refrigerator. The complete hemostasis time of rabbit ears and liver of the in-vivo hemostatic dressing which is pre-frozen by adopting programmed cooling is determined to be about 56s, and the complete hemostasis time of liver is 44 s. Compared with the direct pre-frozen in-vivo hemostatic dressing in an ultra-low temperature refrigerator, the rabbit ear wound hemostasis and liver hemostasis speed of the in-vivo hemostatic dressing adopting the pre-frozen temperature reduction range is higher, and the hemostatic effect is better because the smooth flat structure on the surface of the dressing is favorable for tightly adhering to the wound surface, the porous structure with uniform inside size is favorable for the action between the blood platelet and blood coagulation factors in the dressing and the blood, and the hemostatic effect of the in-vivo hemostatic dressing is enhanced.

4. Cytotoxicity detection of in vivo hemostatic dressings

The preparation method of the leaching liquor comprises the following steps: all operations of the experiment were completed in a biosafety cabinet. Immersing the sterilized sponge sample into RPMI1640 culture solution in a ratio of 0.1g/mL, sealing with a sealing film, leaching at a constant temperature of 37 ℃ for 72 +/-2 h, taking out the sample, and filtering with a sterile filter of 0.2 mu m to remove particulate matters in the leaching solution.

Cell culture: digesting the passaged BHK-21 cells by trypsin, adding fresh culture solution, repeatedly blowing and beating to uniformly distribute the cells in the culture solution, counting under an inverted microscope by using a blood cell counting plate, and diluting the cell suspension to 10⁴~10⁵cells/mL, were seeded separately in 96-well plates at 100. mu.L per well, and a control group was set. The inoculated 96-well plate was then placed in CO at 37 deg.C₂Culturing for 24h in incubator, sucking out original culture solution after cell adherence, adding sample leaching solution, adding fresh culture solution into control group and blank group, and further culturing in biochemical incubator.

And (3) determination: taking out a pore plate at days 1, 3, 5 and 7 respectively, adding 10 mu L of CCK-8 solution into each pore, continuously culturing for 4h at 37 ℃, measuring an absorption value at 450nm by using a microplate reader, and calculating a relative proliferation rate (RGR) by the following steps:

RGR=A1/A2×100%

in the formula: a1: experimental absorbance a 2: absorbance of control group

Cytotoxicity assays were performed using the CCK-8 kit. The Cell Counting Kit-8 (CCK-8) detects the proliferation of cells by utilizing the principle that water-soluble tetrazolium salt can be reduced into orange water-soluble substance Formazan by dehydrogenase in mitochondria in the cells, the amount of the formed Formazan is in positive relation with the number of the cells, namely, the larger the number of living cells, the darker the color is, the lighter the color is, the more the toxicity to the cells is, and the change of the number of the cells is indirectly analyzed by measuring the absorption value by a microplate reader.

Fig. 5 shows the effect of the leaching liquor of 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 on cell growth, and it can be seen from the figure that the cytotoxicity evaluation of the enzymatic cross-linked animal collagen hemostatic dressing and the thermal cross-linked animal collagen hemostatic dressing is grade 2, while the cytotoxicity evaluation of the enzymatic cross-linked human collagen hemostatic dressing and the thermal cross-linked human collagen hemostatic dressing is grade 1, and the cell survival rate is increased along with the increase of cell culture time days, which shows that the in vivo hemostatic dressing prepared by the invention has no virus hidden trouble, high safety, good biocompatibility, and the effect of promoting cell growth, and meets the requirements of biomedical materials.

5. Subcutaneous implantation of intracorporeal hemostatic dressings

The method comprises the steps of firstly injecting 2mL of anesthetic into ear veins of a New Zealand rabbit, then fixing the anesthetic on an operating table, covering other places with four pieces of operating cloth to expose a place to be implanted on the back, disinfecting the skin on the back of the rabbit with an iodine, and disinfecting hands with 75% alcohol. Gently gripping back skin with hands, gently cutting a small opening with a surgical blade to a length of about 10mm, carefully observing and avoiding capillary vessels on the back of the rabbit, gripping prepared sample hemostatic material with forceps, placing under the skin, keeping at least 5mm away from the incision, suturing the incision, and smearing the surgical wound with iodine for sterilization. One week later, the New Zealand rabbits were sacrificed, and the biocompatibility of the hemostatic material in the living body was evaluated by observing whether the inner skin of the rabbits showed swelling, suppuration, and ulceration.

In fig. 6, a is a subcutaneous implantation diagram of the hemostatic dressing of the enzyme-crosslinked human-like collagen after 1 week, and b is a subcutaneous implantation diagram of the hemostatic dressing of the enzyme-crosslinked animal collagen. As can be seen from the figure, the obvious red swelling and suppuration phenomena are generated around the subcutaneous implanted tissue of the in vivo animal collagen hemostatic dressing, and the red swelling, suppuration and ulceration phenomena are not generated around the subcutaneous implanted tissue of the in vivo human collagen hemostatic dressing, which shows that the in vivo hemostatic dressing has no virus hidden trouble and biological rejection, high safety and good biocompatibility, and is a good biomedical hemostatic material.

The invention is not limited to the examples, and any equivalent changes to the technical solution of the invention by a person skilled in the art after reading the description of the invention are covered by the claims of the 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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