CN110917389B - Tissue sealing composition, tissue plugging gel and preparation method thereof - Google Patents
Tissue sealing composition, tissue plugging gel and preparation method thereof Download PDFInfo
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- CN110917389B CN110917389B CN201811101432.6A CN201811101432A CN110917389B CN 110917389 B CN110917389 B CN 110917389B CN 201811101432 A CN201811101432 A CN 201811101432A CN 110917389 B CN110917389 B CN 110917389B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
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- A61L24/104—Gelatin
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- A—HUMAN NECESSITIES
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- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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Abstract
The invention discloses a tissue sealing composition, a tissue plugging gel and a preparation method thereof. The tissue sealing composition comprises a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 5000-30000 cps, and the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 10-500: 1. The tissue plugging adhesive is plugged at a wound position through secondary crosslinking of the gelatin prepolymer with certain mechanical strength and a crosslinking structure and carbodiimide, so that the use of the carbodiimide is reduced, no crosslinking agent is left, the tissue plugging adhesive has good biocompatibility, the secondary crosslinking is rapid in plugging use, the sealing strength is not lower than 45Kpa, the volume elongation is not lower than 500%, the plugging effect is excellent, and the tissue plugging adhesive can be used for rapidly plugging and stopping bleeding of arterioles, venous bleeding and visceral bleeding.
Description
Technical Field
The invention relates to the technical field of biological hemostatic materials, and in particular relates to a tissue sealing composition, tissue sealing glue and a preparation method thereof.
Background
Major wounds in surgical operations usually need a medical suture to perform anastomotic closure on the major wounds, and then in order to prevent blood leakage or infection of the wounds, the gaps can be filled and closed by adopting tissue plugging glue, and small gaps among tissues can be directly closed by adopting the tissue plugging glue. At present, products such as Focalseal, Progel, Bioglue and the like in clinic are approved by European and American countries, and can be used as an auxiliary or substitute for the traditional suturing means to achieve better effects. At the present time, however, the effect of using the tissue sealing glue is far less than expected. To prevent the wound from being ruptured by excessive tension, the tissue sealant is generally required to adhere effectively to the tissue and provide sufficient traction. Meanwhile, because the tissue plugging gel directly acts on a human body, in order to avoid causing side effects such as infection, rejection reaction and the like, the tissue plugging gel also needs sufficient biocompatibility, so that the tissue plugging gel is more beneficial to organism repair and can further get rid of the constraints of ways such as suture, nailing and the like.
Gelatin is a thin sheet or powder which is white or yellowish, semitransparent and slightly lustrous and is degraded by collagen in connective tissues such as animal skin, bones, muscle membranes, muscles and the like. The water and inorganic salt in the composition account for about 16 percent, and the protein content accounts for more than 82 percent. Similar to the parent collagen, gelatin is composed of 18 amino acids, with higher levels of the imino acids Pro and Hyp. The quasi-triple helical structure in the gelatin gel mainly depends on an intramolecular hydrogen bond and a hydrogen bond hydration system, amino of Pro and hydroxyl of Hyp can form hydrogen bonds with other amino acid side chain groups and water molecules, so that the stability of the quasi-triple helical structure is facilitated, and meanwhile, the quasi-triple helical structure is easily activated and crosslinked to form a stable three-dimensional network structure, so that the quasi-triple helical structure can be used for plugging a tissue structure. But the gelatin material needs to be crosslinked to obtain enough strength performance and provide certain tissue traction adhesive force, at present, Bio-Glue can be widely applied as a sealing auxiliary means after vascular surgery suturing, but glutaraldehyde is used for crosslinking the matrix material, obvious carcinogenic risk exists in degradation, and biocompatibility is poor.
Therefore, the tissue plugging gel which can simultaneously meet the cross-linking mechanical property and has good biocompatibility has very important significance.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the existing tissue sealing material in the aspects of biocompatibility and mechanical property, and provides a tissue sealing composition which comprises a first agent of a gelatin prepolymer with certain polymerization strength and viscosity and a second agent of a polymerization crosslinking agent carbodiimide, wherein the mechanical property requirement of the tissue sealing material is met through the combined action of the first agent and the second agent.
The invention also aims to provide the tissue plugging glue, which is prepared by synchronously extruding and crosslinking the gelatin prepolymer with certain mechanical strength and viscosity and the crosslinking agent carbodiimide, and cleaning and removing free carbodiimide, and has the advantages of low residual quantity of the crosslinking agent of the tissue plugging glue, good biocompatibility, excellent mechanical property and good plugging effect.
The invention aims to provide a preparation method of the tissue plugging gel.
The above purpose of the invention is realized by the following technical scheme:
a tissue sealing composition comprises a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 5000-30000 cps, and the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 10-500: 1.
Wherein the pre-polymerization cross-linking agent for preparing the gelatin prepolymer is a biocompatible cross-linking agent.
The tissue sealing composition comprises a gelatin prepolymer and a carbodiimide cross-linking agent, on one hand, the formed prepolymer gelatin has certain cross-linking strength, which is beneficial to reducing the consumption of the cross-linking agent in the secondary cross-linking process, and after the biocompatible cross-linking agent is used for prepolymerization, the obtained prepolymer is connected to form a blocky microstructure, so that the subsequent part of the carbodiimide cross-linking agent is prevented from entering a reaction system, the residual space of the cross-linking agent is reduced, and the residual amount of carbodiimide in the final product tissue sealing adhesive is reduced. On the other hand, the gelatin prepolymer has a certain cross-linking structure relative to gelatin, the viscosity is obviously increased, and the gelatin material after secondary cross-linking is easier to form a network cross-linking structure with adhesion behavior, and is beneficial to shortening the time required by the secondary cross-linking process.
The gelatin prepolymer has a certain cross-linking structure and mechanical properties, can be quickly cross-linked into a gel structure and a membrane structure after being mixed with a small amount of water-soluble carbodiimide with high reaction activity, has the sealing strength of not less than 45Kpa and the volume elongation of not less than 500 percent, and can be used for plugging a large amount of arterial bleeding and plugging trachea and other parts.
Preferably, the viscosity of the gelatin prepolymer is 10000-30000 cps. The method for measuring the viscosity of the gelatin prepolymer comprises the following steps: the sample to be tested was placed in a beaker, measured using a KJ-6050 portable viscometer, and the test results were recorded. Note that 1cps is 1mPa s.
More preferably, the viscosity of the gelatin prepolymer is 10000 cps.
Preferably, the mass ratio of the gelatin in the first gelatin prepolymer to the carbodiimide in the second gelatin prepolymer is 100: 1.
Preferably, in the first dose of gelatin prepolymer, the mass ratio of gelatin to the prepolymerization crosslinking agent is 5-25: 1.
Preferably, the carbodiimide of the second agent is an aqueous solution with the mass fraction of 0.1-5%.
Preferably, the carbodiimide is a 1% mass fraction aqueous solution.
Preferably, the gelatin prepolymer is prepared by pre-polymerizing and crosslinking gelatin by using glutamine transaminase and/or genipin.
TG enzyme or genipin are acceptable and metabolizable protein or polysaccharide substances of human tissues, the participation of the substances does not increase the stimulation of end products to organisms, and the possible severe inflammatory reaction is avoided. But only TG enzyme or genipin is used as a biological cross-linking agent to obtain the tissue plugging gel through one-step cross-linking, the cross-linking time is long, the clinical use is not facilitated, and the mechanical strength of a final product is weak.
The tissue plugging gel is prepared by synchronously extruding and crosslinking a first agent and a second agent, and removing free carbodiimide after crosslinking.
According to the invention, a gelatin prepolymer with the viscosity of 5000-30000 cps is obtained by pre-crosslinking a gelatin solution, the pre-crosslinked gelatin solution is further crosslinked by water-soluble carbodiimide with high reaction activity, and free carbodiimide is removed after crosslinking to obtain the gelatin-based tissue plugging gel which can be tightly adhered to the surface of a tissue. The cross-linking agent carbodiimide can be removed by adopting a large amount of buffer solution for washing, and most of free water-soluble carbodiimide in a final product system can be washed away from a wound in the washing process, so that the stimulation of the material to the tissue can be avoided.
A preparation method of the tissue plugging gel comprises the following steps:
s1, preparing a first agent containing a gelatin prepolymer: respectively preparing a gelatin solution and a pre-polymerization cross-linking agent solution, dripping the pre-polymerization cross-linking agent solution into the gelatin solution, stirring and polymerizing to obtain a gelatin prepolymer with the viscosity of 5000-30000 cps;
s2, preparing a second agent containing a carbodiimide water solution;
and S3, synchronously extruding and mixing the gelatin prepolymer and the carbodiimide water solution to obtain a jelly, and then removing free carbodiimide to obtain the tissue plugging gel.
Preferably, the mass fraction of the gelatin solution in S1 is 20-50%, and the mass ratio of gelatin to the pre-polymerization cross-linking agent is 5-25: 1. The preparation method of the gelatin solution comprises the following steps: mixing and stirring gelatin and water, and completely dissolving the gelatin and water at 40-80 ℃ to obtain gelatin solution with corresponding mass fraction, wherein the dissolving temperature is preferably 50 ℃.
It is worth noting that the dosage of the TG enzyme and/or genipin which are pre-polymerization cross-linking agents is a key parameter of gelatin pre-cross-linking, the dosage is too small, the pre-cross-linking degree is not enough, the dosage of EDC required by secondary cross-linking is large, and the biocompatibility of the tissue sealing glue product is influenced; excessive use amount can lead to excessive pre-crosslinking of the gelatin, so that the viscosity of the gelatin prepolymer is too high, the flowability is poor, the plugging glue cannot reach small gaps among tissues, and even cannot be smoothly extruded from an injector.
Preferably, the mass fraction of the gelatin solution in S1 is 40%, and the mass ratio of the gelatin to the pre-polymerization crosslinking agent is 10: 1.
Preferably, the dropping speed of the prepolymerization crosslinking agent in S1 is 0.1-10 mL/S. The dropping rate is more preferably 2 mL/s.
Preferably, the stirring and polymerizing speed in S1 is 100-2000 r/min, and the temperature is 35-38 ℃. The cross-linking reaction initiated by the cross-linking agent is very rapid, the reaction cross-linking degree in the system is easily uneven after one-time addition, the system can be uniformly cross-linked as much as possible by dropwise adding while stirring, and the stirring speed is most preferably 500 r/min. The gelatin can reduce or even lose fluidity at low temperature, preferably is stirred and polymerized at the temperature of 35-38 ℃, and the temperature of 35-38 ℃ is close to the temperature range of a human body, so that the gelatin is beneficial to directly applying a final product to the human body.
Preferably, the polymerization time in S1 is 1-30 min. And (3) the gelatin crosslinking speed is slow under the initiation of the prepolymerization crosslinking agent, the reaction time at 37 ℃ is about 1-30 min, preferably 3min, the stirring is stopped after the viscosity of the system is obviously increased, and the gelatin prepolymer is canned in an injector for irradiation sterilization for later use.
Preferably, the first and second doses are filled and subjected to radiation sterilization before being extruded synchronously in S3. The gelatin prepolymer and the secondary cross-linking agent carbodiimide are both subjected to strict irradiation sterilization treatment, so that the safety of the tissue plugging adhesive material can be ensured to the greatest extent.
The clinical application method of the tissue plugging gel comprises the following steps:
filling the gelatin prepolymer into an injector for irradiation sterilization, and placing in an environment at 37 ℃ for later use; sucking injection water with 37 ℃ by using an injector filled with carbodiimide, and dissolving for later use; when in use, the two sets of injectors are arranged on the blending combined injector to be synchronously extruded for use, and the blending combined injector can be changed according to different use amounts; after sufficient reaction the occluded site is flushed with a large volume of physiological saline to remove most of the free carbodiimide. The tissue plugging rubber has excellent plugging effect, and can be used for rapidly plugging and stopping bleeding aiming at arteriole and venous bleeding and visceral bleeding.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a tissue sealing composition, a tissue plugging adhesive and a preparation method thereof, wherein a gelatin prepolymer with certain mechanical strength and a cross-linking structure is subjected to secondary cross-linking with carbodiimide to plug at a wound position, so that the use of the carbodiimide is reduced, and most of free carbodiimide is removed by flushing a large amount of physiological saline, so that the residue of a cross-linking agent is greatly reduced, and the tissue plugging adhesive has good biocompatibility; the secondary crosslinking is rapid in the use process of plugging, the sealing strength is not lower than 45Kpa, the volume elongation is not lower than 500%, the plugging effect is excellent, and the plugging hemostatic effect can be realized on arteriole and venous hemorrhage and visceral hemorrhage.
Drawings
FIG. 1 is a diagram showing a gel film.
Fig. 2 is a diagram of the hemostatic and plugging effect of the liver of a rabbit.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Example 1
A tissue sealing composition comprises a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 21250cps, the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 40:1, the mass ratio of gelatin to a pre-polymerization cross-linking agent TG enzyme in the gelatin prepolymer in the first agent is 10:1, and the mass fraction of carbodiimide in the second agent is 1% of aqueous solution.
A tissue plugging gel is formed by synchronously extruding and crosslinking a first dose of gelatin prepolymer and a second dose of carbodiimide of a tissue plugging composition, and is prepared by the following steps:
s1, preparing 40% aqueous solution from existing gelatin, placing the prepared gelatin in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the gelatin solution at 37 ℃ at the rotating speed of 1000r/min, slowly dropwise adding TG enzyme into the beaker at the adding speed of 2ml/s, wherein the mass ratio of the gelatin to the TG enzyme is 10:1, observing the crosslinking condition of the gelatin aqueous solution, reacting for 3 minutes at 37 ℃, determining the viscosity of 21250cps, and canning the gelatin solution pre-crosslinked by the TG enzyme in an injector for irradiation sterilization for later use;
s2, taking carbodiimide, canning in an injector for radiation sterilization for later use, preparing a 1% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 40:1, and the mass ratio of gelatin to glutamine transaminase to carbodiimide is 40:4:1, arranging two sets of injectors on a blending combined injector when in use, synchronously extruding for use, and flushing a plugging position by using a large amount of physiological saline after full reaction.
Example 2
A tissue sealing composition comprises a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 29880cps, the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 100:1, the mass ratio of gelatin to a pre-polymerization cross-linking agent TG enzyme in the gelatin prepolymer in the first agent is 5:1, and the mass fraction of carbodiimide in the second agent is 1% of aqueous solution.
A tissue plugging gel is formed by synchronously extruding and crosslinking a first dose of gelatin prepolymer and a second dose of carbodiimide of the tissue plugging composition, and is prepared by the following steps:
s1, preparing 40% aqueous solution from existing gelatin, placing the prepared gelatin in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the gelatin solution at 37 ℃ at the rotating speed of 1000r/min, slowly dropwise adding TG enzyme into the beaker at the adding speed of 5mL/s, wherein the mass ratio of the gelatin to the TG enzyme is 5:1, observing the crosslinking condition of the gelatin aqueous solution, reacting for 2 minutes at 37 ℃, determining the viscosity of 29880, and canning the gelatin solution pre-crosslinked by the TG enzyme in an injector for irradiation sterilization for later use;
s2, taking carbodiimide, canning in an injector for radiation sterilization for later use, preparing a 1% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 100:1, and the mass ratio of gelatin to glutamine transaminase to carbodiimide is 100:20:1, arranging two sets of injectors on a blending combined injector when in use, synchronously extruding for use, and flushing a plugging position by using a large amount of physiological saline after use.
Example 3
A tissue sealing composition comprises a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 10000cps, the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 10:1, the mass ratio of gelatin to a pre-polymerization cross-linking agent TG enzyme in the gelatin prepolymer in the first agent is 25:1, and the mass fraction of carbodiimide in the second agent is 5% of aqueous solution.
A tissue plugging gel is formed by synchronously extruding and crosslinking a first dose of gelatin prepolymer and a second dose of carbodiimide of the tissue plugging composition, and is prepared by the following steps:
s1, preparing 40% aqueous solution from existing gelatin, placing the prepared gelatin in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the gelatin solution at 37 ℃ at the rotating speed of 500r/min, slowly dropwise adding TG enzyme into the beaker at the adding speed of 5mL/s, wherein the mass ratio of the gelatin to the TG enzyme is 25:1, observing the crosslinking condition of the gelatin aqueous solution, reacting for 10 minutes at 37 ℃, determining the viscosity of 10000, and canning the gelatin solution pre-crosslinked by the TG enzyme in an injector for irradiation sterilization for later use;
s2, taking carbodiimide, canning in an injector, performing radiation sterilization for later use, preparing a 5% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 10:1, and the mass ratio of gelatin to TG enzyme to carbodiimide is 50:2:5, arranging two sets of injectors on a blending combined injector when in use, synchronously extruding for use, and flushing a plugging position by using a large amount of physiological saline after use.
Example 4
A tissue sealing composition comprises a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 32010cps, the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 500:1, the mass ratio of gelatin to a pre-polymerization cross-linking agent TG enzyme in the gelatin prepolymer in the first agent is 5:1, and the mass fraction of carbodiimide in the second agent is 1% of aqueous solution.
A tissue plugging gel is formed by synchronously extruding and crosslinking a first dose of gelatin prepolymer and a second dose of carbodiimide of the tissue plugging composition, and is prepared by the following steps:
s1, preparing 40% aqueous solution from existing gelatin, placing the prepared gelatin in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the gelatin solution at 37 ℃ at the rotating speed of 300r/min, slowly dropwise adding TG enzyme into the beaker at the adding speed of 0.1mL/s, wherein the mass ratio of the gelatin to the TG enzyme is 5:1, observing the crosslinking condition of the gelatin aqueous solution, reacting for 3 minutes at 37 ℃, determining the viscosity to be 32010, and canning the gelatin solution pre-crosslinked by the TG enzyme in an injector for irradiation sterilization for later use;
s2, taking carbodiimide, canning in an injector for radiation sterilization for later use, preparing a 1% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 100:0.2, and the mass ratio of gelatin to glutamine transaminase to carbodiimide is 500:100:1, arranging two sets of injectors on a blending injector when in use, synchronously extruding for use, and flushing a plugging position by using a large amount of physiological saline after use.
Example 5
A tissue sealing composition comprises a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 20050cps, the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 100:1, the mass ratio of gelatin to a pre-polymerization cross-linking agent genipin in the gelatin prepolymer in the first agent is 10:1, and the carbodiimide in the second agent is 0.1% aqueous solution by mass fraction.
A tissue plugging gel is formed by synchronously extruding and crosslinking a first dose of gelatin prepolymer and a second dose of carbodiimide of the tissue plugging composition, and is prepared by the following steps: s1, preparing 40% aqueous solution from existing gelatin, placing the prepared gelatin in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the gelatin solution at 37 ℃ in an environment with the rotating speed of 800r/min, slowly dropwise adding genipin into the beaker at the adding speed of 1mL/s, wherein the mass ratio of the gelatin to the genipin is 10:1, observing the crosslinking condition of the gelatin aqueous solution, reacting for 5 minutes at 37 ℃, determining the viscosity to be 20050, and canning the genipin pre-crosslinked gelatin solution in an injector for irradiation sterilization for later use;
s2, taking carbodiimide, canning in an injector, performing radiation sterilization for later use, preparing a 0.1% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 100:1, and the mass ratio of gelatin to genipin to carbodiimide is 100:10:1, arranging two sets of injectors on a blending injector when in use, synchronously extruding for use, and flushing a plugging position by using a large amount of physiological saline after use.
Example 6
A tissue sealing composition comprises a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 5510cps, the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 100:1, the mass ratio of gelatin to a pre-polymerization cross-linking agent genipin in the gelatin prepolymer in the first agent is 10:1, and the carbodiimide in the second agent is 0.1% aqueous solution by mass fraction.
A tissue plugging gel is formed by synchronously extruding and crosslinking a first dose of gelatin prepolymer and a second dose of carbodiimide of the tissue plugging composition, and is prepared by the following steps:
s1, preparing a 20% aqueous solution from existing gelatin, placing the prepared aqueous solution in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the solution at 37 ℃ at a rotating speed of 800r/min, slowly and dropwise adding genipin into the beaker at a adding speed of 1mL/s, wherein the mass ratio of the gelatin to the genipin is 10:1, observing the crosslinking condition of the gelatin aqueous solution, reacting for 5 minutes at 37 ℃, determining the viscosity to be 5510, and filling the genipin pre-crosslinked gelatin solution in a syringe for radiation sterilization for later use;
s2, taking carbodiimide, canning in an injector, performing radiation sterilization for later use, preparing a 0.1% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 100:1, and the mass ratio of gelatin to genipin to carbodiimide is 100:10:1, arranging two sets of injectors on a blending injector when in use, synchronously extruding for use, and flushing a plugging position by using a large amount of physiological saline after use.
Comparative example 1
A tissue plugging gel is prepared by the following steps:
preparing 40% aqueous solution from the existing gelatin, placing the prepared gelatin in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a big beaker, mechanically stirring the gelatin solution in an environment at 37 ℃ at the rotating speed of 800r/min, slowly dripping TG enzyme into the beaker at the adding speed of 1ml/s, wherein the mass ratio of the gelatin to the TG enzyme is 10:1, observing the crosslinking condition of the gelatin aqueous solution, slowing the gelatin crosslinking speed caused by the TG enzyme, reacting for about 5 minutes at 37 ℃, measuring the viscosity to be 20020, stopping stirring, and preparing the tissue plugging gel.
The obtained tissue plugging glue is used for a rabbit liver bleeding plugging experiment, a wound cannot be plugged, and the wound continuously bleeds.
Comparative example 2
A tissue plugging gel is prepared by the following steps:
preparing 40% aqueous solution from existing gelatin, placing in a 50 deg.C oven until gelatin is completely dissolved, placing gelatin solution in a syringe, placing in 37 deg.C environment, performing irradiation sterilization, and keeping at ambient temperature;
filling carbodiimide into a syringe, sucking water for injection at 37 ℃ by the syringe filled with the carbodiimide, preparing a 1% aqueous solution, dissolving, and performing radiation sterilization for later use, wherein the mass ratio of gelatin to EDC is 10: 1;
when in use, the two sets of injectors are arranged on the blending combined injector and are synchronously extruded for use.
The obtained tissue plugging glue is used for a rabbit liver bleeding plugging experiment, a window can be plugged, a large amount of normal saline is used for flushing the plugging position after the tissue plugging glue is used, and a large amount of EDC is still remained.
Comparative example 3
A tissue plugging gel is prepared by the following steps:
s1, preparing 40% aqueous solution from existing gelatin, placing the prepared aqueous solution in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the solution at 37 ℃ at the rotating speed of 500r/min, slowly and dropwise adding TG enzyme into the beaker at the adding speed of 5mL/s, wherein the mass ratio of the gelatin to the TG enzyme is 10:3, observing the crosslinking condition of the gelatin aqueous solution, reacting for 10 minutes at 37 ℃, measuring the viscosity of 42530, and canning the gelatin solution pre-crosslinked by the TG enzyme in an injector for irradiation sterilization for later use;
s2, taking carbodiimide, canning in a syringe, performing radiation sterilization for later use, and preparing a 10% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 10:1, and the mass ratio of gelatin to glutamine transaminase to carbodiimide is 10:3: 1.
When the device is used, the two sets of injectors are arranged on the blending injector and are synchronously extruded for use, but the materials cannot be extruded to block the blending injector.
Comparative example 4
A tissue plugging gel is prepared by the following steps:
s1, preparing 40% aqueous solution from existing gelatin, placing the prepared gelatin in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the gelatin solution at 37 ℃ at the rotating speed of 500r/min, slowly dropwise adding TG enzyme into the beaker at the adding speed of 5mL/s, wherein the mass ratio of the gelatin to the TG enzyme is 30:1, observing the crosslinking condition of the gelatin aqueous solution, reacting for 10 minutes at 37 ℃, and filling the gelatin solution pre-crosslinked by the TG enzyme in a syringe for irradiation sterilization for later use;
s2, taking carbodiimide, canning in a syringe, performing radiation sterilization for later use, and preparing a 10% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 10:1, and the mass ratio of gelatin to glutamine transaminase to carbodiimide is 30:1: 6.
When the device is used, the two sets of injectors are arranged on the blending injector and are synchronously extruded for use, but the materials cannot be extruded to block the blending injector.
Comparative example 5
A tissue plugging gel is prepared by the following steps:
s1, preparing 40% aqueous solution from existing gelatin, placing the prepared gelatin in a 50 ℃ oven until the gelatin is completely dissolved, placing the completely dissolved gelatin solution in a large beaker, mechanically stirring the gelatin solution at 37 ℃ at the rotating speed of 1000r/min, slowly and dropwise adding TG enzyme into the beaker at the adding speed of 2ml/s, wherein the mass ratio of the gelatin to the TG enzyme is 10:1, observing the crosslinking condition of the gelatin aqueous solution, reacting for 3 minutes at 37 ℃, and filling the gelatin solution pre-crosslinked by the TG enzyme in a syringe for radiation sterilization for later use;
s2, taking carbodiimide, canning in an injector, performing radiation sterilization for later use, preparing a 1% aqueous solution when in use, wherein the mass ratio of gelatin to EDC is 600:1, and the mass ratio of gelatin to glutamine transaminase to carbodiimide is 600:6:1, and when in use, arranging two injectors on a blending combined injector, and synchronously extruding for use, wherein no gel is formed due to insufficient dosage of EDC.
Result detection
The volume elongation, the sealing strength and the EDC residual amount of the tissue plugging gels prepared in examples 1-6 and comparative examples 1-5 were measured, and the measurement results are shown in the following table 1.
The preparation method of the volume elongation comprises the following steps: and (3) placing the material in the process of obtaining the final product in a dumbbell-shaped mold, taking out the sample strip after complete crosslinking, placing the sample strip on a universal mechanical machine, setting the distance between the initial clamps, stretching the clamps towards two ends, recording the displacement between the clamps after the material is broken, and obtaining the volume elongation as the measured result.
The detection method of the sealing strength comprises the following steps: taking fresh pigskin or pig casing with the area of 20 x 20cm, fixing the fresh pigskin or pig casing at the opening of a closed box with only one opening, sealing the opening of the closed box, and connecting a pressure inflating pump and a pressure sensor to the other side of the closed box. Cutting a small opening with the diameter of 0.5cm +/-0.02 cm at the central position of the pigskin or the pig casing, then placing a plugging material on the surface of the small opening, uniformly spraying the first mixed solution and the second mixed solution around the plugging material through a duplex syringe, timing for 1-5 minutes after spraying, then inflating the sealed box through a pressure pump, and recording the reading of a pressure sensor when the plugging material is pushed open, namely the sealing strength.
The detection method of the EDC residual quantity comprises the following steps: EDC standard was taken using LCMS/MS tandem mass spectrometry equipment and the final product was then analyzed by comparison. The residual amount test is to simulate real operation, and the final product is subjected to EDC residual amount test after being continuously washed by a large amount of physiological saline.
TABLE 1
The tissue-sealing gel membrane of example 3 is shown in fig. 1, and the hemostatic and plugging effect is shown in fig. 2, which shows that the hemostatic and plugging membrane is placed in the liver of a rabbit to perform hemostatic and plugging, and has an obvious effect.
From the results in the table 1, it can be seen that the sealing strength and the volume elongation of the one-step polymerization only using the prepolymer crosslinking agent can not meet the requirements of the tissue plugging adhesive at all, and although the sealing strength and the volume elongation of the one-step polymerization only using the carbodiimide can reach the standards, the residual amount of the crosslinking agent carbodiimide is too high, and the biosafety of the material is unqualified.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (9)
1. The tissue sealing composition is characterized by comprising a first agent containing gelatin prepolymer and a second agent containing carbodiimide, wherein the viscosity of the gelatin prepolymer in the first agent is 5000-30000 cps, the mass ratio of gelatin to a prepolymerization crosslinking agent in the gelatin prepolymer in the first agent is 5-25: 1, and the mass ratio of gelatin in the gelatin prepolymer in the first agent to carbodiimide in the second agent is 10-500: 1.
2. The tissue sealing composition according to claim 1, wherein the carbodiimide in the second agent is an aqueous solution with a mass fraction of 0.1-5%.
3. The tissue sealing composition of claim 1, wherein the gelatin prepolymer is prepared by pre-polymerizing and cross-linking gelatin with transglutaminase and/or genipin.
4. A tissue sealing gel, characterized in that the tissue sealing gel is prepared by synchronously extruding and crosslinking a first agent containing gelatin prepolymer and a second agent containing carbodiimide of the tissue sealing composition according to any one of claims 1 to 3, and removing free carbodiimide after crosslinking.
5. A method for preparing the tissue plugging gel of claim 4, which is characterized by comprising the following steps:
s1, preparing a first dose containing a gelatin prepolymer: respectively preparing a gelatin solution and a pre-polymerization cross-linking agent solution, dripping the pre-polymerization cross-linking agent solution into the gelatin solution, stirring and polymerizing to obtain a gelatin prepolymer with the viscosity of 5000-30000 cps;
s2, preparing a second agent containing carbodiimide aqueous solution;
s3, synchronously extruding and mixing the first agent and the second agent to obtain a jelly, and then removing free carbodiimide to obtain the tissue plugging gel.
6. The preparation method of the tissue plugging gel according to claim 5, wherein the mass fraction of the gelatin solution in S1 is 20-50%, and the mass ratio of gelatin to the pre-polymerization cross-linking agent is 5-25: 1.
7. The preparation method of the tissue plugging gel according to claim 5, wherein the dropping speed of the pre-polymerization cross-linking agent solution in S1 is 0.1-10 mL/S.
8. The preparation method of the tissue plugging gel according to claim 5, wherein the stirring and polymerizing speed in S1 is 100-2000 r/min, and the temperature is 35-38 ℃.
9. The method for preparing the tissue plugging gel of claim 5, wherein the first agent and the second agent are filled and subjected to radiation sterilization treatment before being synchronously extruded in the step S3.
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| US20050079999A1 (en) * | 1998-06-23 | 2005-04-14 | James Wilkie | Methods for controlling the viscosity of polymer-based tissue sealants and adhesives |
| US20090175946A1 (en) * | 2006-07-10 | 2009-07-09 | Gelita Ag | Use of gelatin and a cross-linking agent for producing cross-linking medical glues |
| US20150196683A1 (en) * | 2006-12-15 | 2015-07-16 | Lifebond Ltd. | Gelatin-transglutaminase hemostatic dressings and sealants |
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| US20050079999A1 (en) * | 1998-06-23 | 2005-04-14 | James Wilkie | Methods for controlling the viscosity of polymer-based tissue sealants and adhesives |
| US20090175946A1 (en) * | 2006-07-10 | 2009-07-09 | Gelita Ag | Use of gelatin and a cross-linking agent for producing cross-linking medical glues |
| US20150196683A1 (en) * | 2006-12-15 | 2015-07-16 | Lifebond Ltd. | Gelatin-transglutaminase hemostatic dressings and sealants |
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