CN107349462B

CN107349462B - Absorbable semi-flowable crosslinked polypeptide biological surgical hemostat

Info

Publication number: CN107349462B
Application number: CN201611186959.4A
Authority: CN
Inventors: 江涛; 何迎辉
Original assignee: Beijing Nash International Biotechnology Co ltd
Current assignee: Beijing Nash International Biotechnology Co ltd
Priority date: 2016-05-09
Filing date: 2016-12-20
Publication date: 2020-07-14
Anticipated expiration: 2036-12-20
Also published as: CN107349462A

Abstract

The invention provides an absorbable semi-fluid cross-linked polypeptide biological surgical hemostat, which comprises gelatin and a cross-linking agent, wherein the cross-linking agent is added into a gelatin solution, so that amino acid side chains with high reactivity can react with each other to form cross-linking. The crosslinking degree can be increased by adding iron and calcium ions, and finally the three-dimensional network crosslinked macromolecular polypeptide is formed. The hemostatic material can be safely absorbed by human tissues, has semi-fluidity, viscosity and adhesiveness, can promote the blood coagulation process of human bodies by physical and chemical principles, and has quick hemostatic effect. The hemostatic material can treat surgical operation bleeding under the condition of not damaging tissues, increases the convenience and flexibility of surgical operation hemostasis, improves the safety and effectiveness of surgical operation hemostasis, and achieves satisfactory hemostasis effect under the conditions of multi-point bleeding, large-area bleeding, bleeding in narrow lacuna, hidden bleeding points or difficult discrimination, and difficult operation and difficult bleeding control of the existing hemostasis method.

Description

Absorbable semi-flowable crosslinked polypeptide biological surgical hemostat

Technical Field

The invention belongs to a hemostatic material for surgical operation in the medical apparatus and instruments, and particularly relates to an absorbable semi-fluid cross-linked polypeptide biological surgical hemostat and a preparation method thereof.

Background

Bleeding of tissues and organs during surgery is almost inevitable, bleeding during surgery can lead to prolonged operation time, poor surgical field and operation errors, ischemia during surgery can lead to tissue hypoxia, unstable blood pressure and blood coagulation disorders, and even patient blood loss shock or death. Complications can arise after surgery, slow recovery after surgery and prolonged hospital stay. In addition, rapid and effective hemostasis during surgery is increasingly difficult due to the decline of natural hemostasis function in surgical patients and the continuous increase of elderly people who take anticoagulants for a long time to treat cardiovascular and cerebrovascular diseases in recent years. If blood transfusion is used to supplement the blood loss of the patient during the operation, a series of problems caused by the blood transfusion such as transfusion reaction, blood incompatibility and transfusion infection can be caused. Therefore, sufficient and effective hemostasis in the surgical operation is an essential procedure, and is a key factor related to the treatment effect of the surgical operation and the prognosis health of a patient.

The natural hemostatic process of the human body after tissue or blood vessel damage is a complex and ordered physiological response involving vasoconstriction, platelet activation, soft platelet plug formation and fibrin clot formation. Recent studies based on cellular models have demonstrated that when the vessel wall is damaged, the vessel first reduces the damage gap by contraction, the collagen exposed from the vessel wall activates the platelets, deforming them, increasing their adhesiveness, and the surface factors of the platelets interact to aggregate them and form a soft platelet plug. At the same time, the tissue factor outside the blood vessel enters the blood vessel through the damaged blood vessel wall, contacts with the blood coagulation factors in the blood plasma, and initiates a series of reactions among various blood coagulation factors, and the activated blood coagulation factors convert prothrombin into active thrombin. Thrombin then binds to the damaged platelets, further activating the platelets and fibrinogen so that the molecules can crosslink with each other to form macromolecular fibrin. Then, as more and more thrombin is activated, a large amount of fibrin is formed, and fibrin binds to a large amount of platelets and red blood cells accumulated at the damaged blood vessel to form a firm fibrin clot, so that the damaged blood vessel wall is blocked.

Most of the traditional hemostasis methods in the existing surgical operations stop the blood flow by closing the blood vessel through compression of a mechanical or physical principle, help and promote the natural hemostasis process of a human body, and comprise gauze pressing, hemostasis forceps clamps, suture ligation, electrotome, laser, argon gas and the like, wherein the blood vessel broken end is burnt through the thermal action, so that blood in the blood vessel is coagulated and surrounding tissue protein is denatured. The purpose of the above mechanical or physical methods is to close the blood vessel so that bleeding stops.

The conventional hemostatic methods work on the premise that (1) the site of bleeding during surgery must be clearly identified, and (2) the site of bleeding is accessible by conventional hemostatic instruments and has sufficient operating space. However, in actual surgical operations, many bleeding conditions cannot satisfy the above two requirements, and thus satisfactory hemostasis effect cannot be achieved by using the conventional techniques. For example, it is difficult to achieve complete hemostasis by sealing each bleeding point with hemostats and sutures during open surgery for multiple bleeding points or bleeding, and although burning methods such as electrotome, laser and argon can treat multiple bleeding points, excessive temperature can damage surrounding tissues and organs, especially weak tissues such as brain and spinal cord, which can easily cause postoperative unrecoverable sequelae; if the gauze is used for hemostasis, continuous pressing is needed, the time is long, and the operation process is affected. For another example, laparoscopic surgery and ear, nose and throat surgery, surgeons operate in a small space with no open space to stop bleeding with instruments or gauze. For another example, in the case of a hidden bleeding site, the bleeding site cannot be determined so that the conventional hemostatic method is not applicable.

The biological hemostatic products in the market at present comprise gelatin sponge, fibrin glue, absorbable hemostatic gauze, chitosan hemostatic powder, starch polysaccharide hemostatic materials and the like. Gelatin sponge is formed by animal collagen denaturation, has good biocompatibility and biodegradability, and is widely applied to clinical hemostasis at present. However, most of the gelatin sponges are made of toxic chemical glutaraldehyde, and residues of the glutaraldehyde can cause unnecessary damage to human tissues. In addition, the gelatin sponge has some defects which cannot be overcome in the aspect of practicability, such as poor water absorption of the gelatin sponge, and slow absorption speed on surgical bleeding; the gelatin sponge has longer in-vivo degradation time, can stay in a human body for a plurality of months, and is easy to cause wound healing difficulty and chronic inflammatory reaction; gelatin sponges are solid and lack fluidity, and in many cases stop bleeding due to the inability to reach bleeding sites.

Absorbable hemostatic gauze, chitosan hemostatic powder and starch polysaccharide hemostatic materials in a biogenic hemostatic product are applied clinically in a solid form, and although the absorbable hemostatic gauze, the chitosan hemostatic powder and the starch polysaccharide hemostatic material have small damage to surrounding tissues, can treat multi-point bleeding and can be absorbed by a human body, the absorbable hemostatic gauze meets clinical requirements to a certain extent, but generally cannot perform hemostatic operation in a narrow space, and is inconvenient to use. Moreover, the powder of the chitosan hemostatic powder and the starch polysaccharide hemostatic material is easily diluted and even dispersed by excessive bleeding, so that the hemostatic effect is not ideal. In addition, the powder is often scattered in the operation field, which may make the tissues, organs, blood vessels and nerves in the operation difficult to identify, resulting in the difficulty of the operation of the surgeon.

Fibrin glue in biological hemostatic products in domestic markets at present is also called fibrin sealant, medical bioprotein glue and fibrin adhesive are animal plasma products, may be polluted by animal therapy sources, and intraoperative infection caused by animal therapy sources increases a great risk for operations, and is a taboo of surgeons. At present, the domestic fibrin glue belongs to medicines but not medical appliances and faces higher supervision risk.

At present, a non-biogenic artificial synthetic adhesive is also used for surgical hemostasis in the domestic market, is a fully imported product, avoids infection risks brought by blood products, but is expensive and causes heavy burden to most patients.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an absorbable semi-fluid cross-linked polypeptide biological surgical hemostat. The hemostatic material can be safely absorbed by human tissues, is between liquid and solid, has semiliquidity, viscosity and adhesiveness, can promote the human coagulation process by physical and chemical principles, and has quick hemostatic effect. The hemostatic material can treat surgical operation bleeding under the condition of not damaging tissues, increases the convenience and flexibility of surgical operation hemostasis, improves the safety and effectiveness of surgical operation hemostasis, and achieves satisfactory hemostasis effect under the conditions of multi-point bleeding, large-area blood seepage, bleeding in narrow lacuna, hidden bleeding points or difficult discrimination, and difficult operation and difficult bleeding control of the existing hemostasis method, thereby obviously shortening the surgical operation hemostasis time and reducing blood loss in the surgical operation.

In order to achieve the purpose, the invention adopts the technical scheme that:

an absorbable semi-fluid cross-linked polypeptide as hemostatic for biological surgery is composed of gelatin (80-99 wt.%) and cross-linking agent (1-20 wt.%). Preferably 90-99% by weight of gelatin and 1-10% by weight of cross-linking agent, more preferably 95-99% by weight of gelatin and 1-5% by weight of cross-linking agent.

Further, the cross-linking density Cd of the hemostat satisfies: cd is not less than 0.3200 and not more than 0.4000, the cross-linking effect is good, and the prepared hemostatic can form a jelly with semi-fluidity, viscosity and adhesiveness in a short time and can keep the semi-fluidity for a longer time.

Specifically, the gelling time of the hemostatic is 5-10 minutes, and the gelling stability is maintained for 8-12 hours.

Specifically, the cross-linking agent comprises any one or more of acrylamide, trimethylpropane, polyethylene glycol and bis-succinimidyl glutarate.

The main component of the hemostat is animal collagen (or called gelatin), and the hemostat has the biological characteristics of no toxicity, low allergenicity and good compatibility with human bodies, can be safely absorbed and metabolized by human bodies after being degraded by protease in the human bodies, and is very suitable for application in human body surgical operations. Animal gelatin is protein polypeptide, and during the preparation process, crosslinking agent is added, and the amino acid side chains with high reactivity, such as lysine, cystine and arginine side chains, can react with each other to form crosslinked polypeptide.

The cross-linked macromolecular polypeptide structure of the jelly simulates collagen exposed when the vascular wall is damaged, the collagen can directly activate platelets, and the reticular molecular structure of the jelly provides a good attachment support for the platelets, prothrombin and fibrinogen in a human body, so that the opportunity of mutual contact and interaction among all blood coagulation components is increased, the hemostasis time is shortened, and the hemostasis effect is improved. The reticular molecular structure can bind the blood platelet and the red blood cell without waiting for the formation of macromolecular fibrin, thereby greatly shortening the clot generation time. Our earlier experiments demonstrated that the greater the density of the hemostatic material per unit volume, the more platelets, prothrombin and fibrinogen can be attached, and the faster the intermolecular interactions, so that the shorter the hemostatic time, the better the hemostatic effect. However, increasing the density of the hemostatic material will lose some of its fluidity, thereby affecting the convenience of use and the directional function of the hemostatic material, and therefore, the optimal hemostatic effect can only be achieved by carefully selecting the balance between density and fluidity.

In order to optimize the product, iron and calcium ions can be added into the hemostat to increase the crosslinking degree, so that the hemostat finally becomes the three-dimensional network crosslinked macromolecular polypeptide.

In the process of manufacturing the hemostatic, iron and calcium ions assist the reaction and crosslinking between gelatin side chains, and the crosslinking degree of the final product is increased, so that a small amount of iron and calcium ions are carried in the net structure of the macromolecular polypeptide of the final product. During the application process, the hemostat is contacted with the water solution, and the iron ions in the hemostat can increase the viscosity of the hemostat so that the hemostat has good viscosity and adhesiveness. When a hemostat contacts and interacts with a coagulation factor, calcium ions may act as a catalyst in the various steps of activating the coagulation factor, accelerating the conversion of prothrombin to thrombin, and accelerating the conversion of fibrinogen to fibrin.

The preparation method of the hemostat comprises the following steps:

dissolving animal gelatin in deionized water, increasing temperature to accelerate dissolution, adding crosslinking agent (one or two of acrylamide, trimethylpropane, polyethylene glycol and disuccinimide glutarate) after dissolution, stirring for 8-16 hours under ultraviolet rays, filtering with filter paper, blocking the transparent jelly by the filter paper, washing for many times to wash off excessive crosslinking agent, drying the filtered product in flowing air flow, after weight loss of more than 90%, harvesting the product, grinding into tiny particles, and sieving to make the particles of the product uniform. The hemostat was mixed with a small amount of 0.9% aqueous sodium chloride solution to form a uniform, semi-fluid, granular white or light yellow gum.

In the preparation process, after the gelatin is dissolved, a cross-linking agent is added into the solution, and 0.05M ferric ion and 0.05M divalent calcium ion are simultaneously added.

Through the optimization process, the intensity of the ultraviolet radiation is 6000 muW-8000 muW/cm under the wavelength of 200nm-300nm²。

Compared with the prior art, the invention has the advantages that:

firstly, the hemostat of the invention has good compatibility with human body, no toxicity and low sensitization, and can be safely absorbed and metabolized by human body after being degraded by protease in human body.

Secondly, the invention enables the hemostatic substance to have semi-fluidity, viscosity and adhesiveness by adjusting the proportion of the gelatin and the cross-linking agent, thereby being convenient for the surgical hemostatic process, obviously shortening the surgical hemostatic time, making up the defects of the existing hemostatic method and solving the problems that: (1) treating multiple spots and large area bleeding and bleeding in a short time without specific positioning; (2) effective hemostasis is achieved when bleeding points are hidden or difficult to judge; (3) rapid hemostasis can be achieved under short-term compression or lack of compression; (4) satisfactory hemostasis is achieved in situations where other conventional hemostatic devices are difficult to operate and control.

Thirdly, the hemostat of the invention can assist and promote the natural hemostasis process of human body, accelerate the hemostasis process by physical and chemical principles: (1) the macromolecular reticular cross-linked structure can directly activate platelets, increase the opportunity of mutual contact and interaction among the platelets, prothrombin and fibrinogen in a human body, directly bind the platelets and red blood cells, and greatly shorten the generation time of a blood clot; (2) the water absorption of the hemostatic material enables the hemostatic material to slightly expand after absorbing bleeding, so that the blood vessel is squeezed to achieve the effect of helping hemostasis; (3) iron ions in the hemostatic material can increase the viscosity and adhesiveness of the hemostatic material, and calcium ions can act as a catalyst in various steps of activating blood coagulation factors, accelerate the conversion of prothrombin into thrombin, and accelerate the conversion of fibrinogen into fibrin.

Fourth, the hemostat of the present invention does not damage surrounding tissues, does not risk the retention of foreign bodies during surgery, and does not disturb the surgical field.

Fifthly, the mobility, the viscosity, the attachment and the absorption time in the human body of the hemostatic can be controlled by adjusting the proportion of the gelatin and the cross-linking agent in the preparation process, thereby meeting the hemostatic requirements in different operations. For example, treatment of bleeding from the sinuses in otorhinolaryngological procedures requires a somewhat less fluid and more adherent biosurgical hemostat, which makes it easier to adhere to the sinus wall; a somewhat more fluid biologic surgical hemostat is needed in spinal surgery to facilitate access to the bottom of the bleeding lacuna.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example 1

According to the weight ratio of animal gelatin to cross-linking agent (acrylamide and trimethylpropane) of 95:5 in the ratio. Dissolving animal gelatin in deionized water, increasing temperature to accelerate dissolution, adding crosslinking agent, respectively adding 0.05M ferric ion and 0.05M divalent calcium ion at 200nm wavelength of 6000 μ W/cm²Stirring for 8 hours under the ultraviolet ray of intensity, filtering with filter paper, and washing for many times to wash off excessive cross-linking agent and metal ions, drying the filtered product in flowing air flow, harvesting the product after losing weight of more than 90%, grinding into tiny particles, sieving to make the particles of the product uniform, mixing the hemostatic material with 0.9% sodium chloride aqueous solution to form uniform yellowish colloid with semi-fluidity and granular texture.

Example 2

According to the weight ratio of animal gelatin to cross-linking agent (acrylamide and polyethylene glycol) of 99:1, in a ratio of 1. Dissolving animal gelatin in deionized water, increasing temperature to accelerate dissolution, adding crosslinking agent, respectively adding 0.05M ferric ion and 0.05M divalent calcium ion, and adding into gelatin solution at 30 deg.CWavelength of 0nm, 8000. mu.W/cm²Stirring for 12 hours under the ultraviolet ray of intensity, filtering with filter paper, and washing for many times to wash off excessive cross-linking agent and metal ions, drying the filtered product in flowing air flow, harvesting the product after losing weight of more than 90%, grinding into tiny particles, sieving to make the particles of the product uniform, mixing the hemostatic with 0.9% sodium chloride water solution to form uniform white jelly with semi-fluidity and particle texture.

Example 3

According to the weight ratio of animal gelatin to cross-linking agent (trimethylpropane) of 90: 10. Dissolving animal gelatin in deionized water, increasing temperature to accelerate dissolution, adding crosslinking agent, respectively adding 0.05M ferric ion and 0.05M divalent calcium ion at wavelength of 300nm and 6000 μ W/cm²Stirring for 16 hours under the ultraviolet ray of intensity, filtering with filter paper, and washing for many times to wash off excessive cross-linking agent and metal ions, drying the filtered product in flowing air flow, harvesting the product after losing weight of more than 90%, grinding into tiny particles, sieving to make the particles of the product uniform, mixing the hemostatic material with 0.9% sodium chloride aqueous solution to form uniform yellowish colloid with semi-fluidity and granular texture.

Example 4

According to the weight ratio of animal gelatin to cross-linking agent (bis-succinimide glutarate and polyethylene glycol) of 80:20 in proportion. Dissolving animal gelatin in deionized water, increasing temperature to accelerate dissolution, adding crosslinking agent at 300nm wavelength of 6000 μ W/cm²Stirring for 16 hours under the ultraviolet ray of intensity, filtering with filter paper, washing for many times to wash off excessive cross-linking agent, drying the filtered product in flowing air flow, harvesting the product after losing weight of more than 90%, grinding into tiny particles, sieving to make the particles of the product uniform, mixing the hemostatic material with 0.9% sodium chloride aqueous solution to form uniform yellowish jelly with semi-fluidity and particle texture.

Experimental example 1 measurement of crosslink Density

The crosslink density (Cd) refers to the number of chemical bonds formed between molecules in a unit space, and can be used to evaluate the effectiveness of Crosslinking agents and Crosslinking methods, i.e., the number and strength of chemical bonds generated between gelatin molecules, and to explain the differences in clinical applications of the crosslinked gelatin molecules generated under different Crosslinking conditions. Crosslink density is an industry accepted measure of colloidal properties, usually by indirect calculation of crosslink density through swelling or studies of colloidal rheology, i.e., studying colloidal viscosity, density, and material stability, which are time consuming, insensitive, and destructive to the sample. Recently, the low-frequency nuclear magnetic resonance technology becomes a more convenient and reliable crosslinking density measuring method, and the magnetic resonance method has the advantages of short time (within a few seconds), wide temperature range, good result repeatability, no chemical intervention in samples and the like, and can simultaneously measure physical and chemical crosslinking.

The magnetic resonance method calculates the cross-linking density by measuring the relaxation time of hydrogen protons in macromolecular cross-linking substances, in a low-frequency magnetic field, an external radio-frequency pulse excites the hydrogen protons in the cross-linking substances to enable the hydrogen protons to reach a stable high-energy state, the whole process is called a relaxation process from the stop of the external radio-frequency pulse to the recovery of the hydrogen protons to a magnetic moment state before excitation, namely a recovery process, the required time is called the relaxation time, the larger the cross-linking density of the macromolecular cross-linking substances is, namely the more cross-linking bonds are in a unit volume, the stronger the binding force of the hydrogen protons is, the shorter the relaxation time of the hydrogen protons is, and a nuclear magnetic method cross-linking density instrument can calculate the number of the cross-linking bonds in a specified spatial range by measuring the relaxation time of the hydrogen protons in combination with calculation software set according to different cross-linking substances in.

Because the hemostatic material accelerates and enhances the hemostatic process by virtue of the cross-linked reticular macromolecular polypeptide structure, the effect of accelerating and enhancing the hemostatic process is stronger when the number of cross-links in a unit volume is more, but if the number of cross-links in the unit volume is too much, the fluidity of the hemostatic material mixed with the 0.9% sodium chloride aqueous solution is reduced. For this reason, we examined the amount of the crosslinking agent and the kind of the crosslinking agent.

(I) examination of the amount of crosslinking agent

The present invention is illustrated by the following test examples. The samples involved are defined as follows:

0.1% cross-linked gelatin: the weight ratio of the animal gelatin to the cross-linking agent in the preparation process is 99.9: 0.1;

0.5% cross-linked gelatin: the weight ratio of the animal gelatin to the cross-linking agent in the preparation process is 99.5: 0.5;

1% cross-linked gelatin: the weight ratio of the animal gelatin to the cross-linking agent in the preparation process is 99: 1;

5% cross-linked gelatin: the weight ratio of the animal gelatin to the cross-linking agent in the preparation process is 95: 5;

10% cross-linked gelatin: the weight ratio of the animal gelatin to the cross-linking agent in the preparation process is 90: 10;

20% cross-linked gelatin: the weight ratio of the animal gelatin to the cross-linking agent in the preparation process is 80: 20;

30% cross-linked gelatin: the weight ratio of the animal gelatin to the cross-linking agent in the preparation process is 70: 30.

(1) The purpose is as follows: the cross-linking density of cross-linked gelatins prepared with different percentages of cross-linking agent was evaluated.

(2) Test samples: using gelatin and polyethylene glycol, 6 kinds of dried fine powders of crosslinked gelatin, 0.1% of crosslinked gelatin, 1% of crosslinked gelatin, 5% of crosslinked gelatin, 10% of crosslinked gelatin, 20% of crosslinked gelatin and 30% of crosslinked gelatin, and 1 kind of dried fine powders of blank control (pure gelatin) were prepared by the preparation method described in example 1.

(3) The test method comprises the following steps: 5 parts of each test sample, 1g of each dry fine powder, were put into the bottom of the measuring tube, and measured in a nuclear magnetic resonance crosslinking densitometer (low field nuclear magnetic resonance crosslinking densitometer VTNMR20-010V-T, Nippon Newman electronics Co., Ltd.) at room temperature (22 ℃ C.) to read the values.

(4) The results of the specific experiments are shown in tables 1, 2 and 3.

TABLE 1 Cross-Linked Density test results for hemostats prepared with different amounts of cross-linking agent

TABLE 2 Cross-linking Density mean Difference univariate variance analysis results

	Sum of squares (ss)	Degree of freedom (df)	Root Mean Square (MS)	F	P
						Between groups	0.288	6	0.048	455.580	0.000
In group	0.003	28	0.000
						Total of	0.291	34

TABLE 3 Cross-linking Density mean difference Tukey test results

(5) And (4) conclusion: the crosslinking density of the gelatin is in direct proportion to the percentage content of the crosslinking agent, the crosslinking density of the pure gelatin is the lowest, the crosslinking density of all the crosslinked gelatins prepared by the crosslinking agent is obviously higher than that of the pure gelatin, the crosslinking density of the crosslinked gelatin is gradually increased along with the increase of the percentage content of the crosslinking agent, and the crosslinking density of 1 percent of the crosslinked gelatin is obviously higher than that of 0.1 percent of the crosslinked gelatin; the crosslink density of the 5% crosslinked gelatin is also significantly higher than the crosslink density of the 0.1% crosslinked gelatin; the cross-link density of 10% cross-linked gelatin is significantly higher than the cross-link density of 0.1% cross-linked gelatin and 1% cross-linked gelatin; the crosslinking density of the 20% crosslinked gelatin is significantly higher than the crosslinking density of 0.1% crosslinked gelatin, 1% crosslinked gelatin and 5% crosslinked gelatin; the crosslink density of the 30% crosslinked gelatin was significantly higher than the crosslink density of 0.1% crosslinked gelatin, 1% crosslinked gelatin, 5% crosslinked gelatin, and 10% crosslinked gelatin. Meanwhile, in the tested sample, the crosslinking density of 1% crosslinked gelatin and 5% crosslinked gelatin has no significant difference, and the crosslinking density of 5% crosslinked gelatin and 10% crosslinked gelatin has no significant difference; there was no significant difference in the crosslink density between the 10% crosslinked gelatin and the 20% crosslinked gelatin, and between the 20% crosslinked gelatin and the 30% crosslinked gelatin.

(II) examination of the type of crosslinking agent

The invention is illustrated by the following test examples in which the weight ratio of animal gelatin to cross-linking agent is 95:5, except that the cross-linking agent employed is one or more of acrylamide, trimethylpropane, polyethylene glycol and bis-succinimidyl glutarate.

Group 1: animal gelatin and cross-linking agent (trimethylpropane)

Group 2: animal gelatin and cross-linking agent (acrylamide)

Group 3: animal gelatin and crosslinking agent (bis-succinimidyl glutarate)

Group 4 animal gelatin and crosslinking agent (acrylamide and trimethylpropane)

Group 5: animal gelatin and cross-linking agent (acrylamide and polyethylene glycol)

Group 6: animal gelatin and crosslinking agent (polyethylene glycol and bis-succinimidyl glutarate)

Group 7: animal gelatin and crosslinking agent (trimethylpropane, polyethylene glycol and disuccinimidyl glutarate)

TABLE 4 Cross-Linked Density test results for hemostats prepared with different classes of cross-linking agents

TABLE 5 mean difference univariate variance analysis results of cross-linking density of hemostats prepared with different kinds of cross-linking agents

	Sum of squares (ss)	Degree of freedom (df)	Root Mean Square (MS)	F	P
						Between groups	0.001	6	0.000	1.870	0.121
In group	0.004	28	0.000
						Total of	0.005	34

The experimental results show that the cross-linking agent is one or more of acrylamide, trimethylpropane, polyethylene glycol and disuccinimide glutarate, the cross-linking density of the cross-linked gelatin prepared by various cross-linking agents and combinations of the cross-linking agents is not obviously different (P is 0.121), the cross-linking density is 0.3200-0.4000, the cross-linking effect is good, and the prepared hemostatic can form a jelly with semi-fluidity, viscosity and adhesiveness in a short time and can keep the semi-fluidity for a long time.

Experimental example 2 measurement of hemostatic Properties

According to the actual requirement of clinical operation, the operation hemostat requires that the powdery cross-linked gelatin can form jelly with semi-fluidity, viscosity and adhesiveness in a short time after being mixed with 0.9% sodium chloride aqueous solution, and can keep the semi-fluidity for a longer time, namely, the colloid stability can be kept in a reasonable time range, if the colloid stability is poor, especially for long-time operation, the colloid in the operation is ineffective and instruments applied by the hemostat are blocked.

According to clinical investigation, the ideal semi-fluid clinical operation hemostat has effective performances of gelling time of 5-10 minutes, gelling stability of 8-12 hours, and low or high dosage of cross-linking agent, and the prepared semi-fluid hemostat may have poor performance.

(1) The purpose is as follows: the gelling time and gelling stability of the crosslinked gelatin prepared by the crosslinking agents with different percentage contents are examined.

(2) Test samples: dried fine powders of 7 kinds of crosslinked gelatin (gelatin and polyethylene glycol), 0.1% crosslinked gelatin, 0.5% crosslinked gelatin, 1% crosslinked gelatin, 5% crosslinked gelatin, 10% crosslinked gelatin, 20% crosslinked gelatin, and 30% crosslinked gelatin.

(3) The test method comprises the following steps: 5 parts of each test sample were taken, each 1g of dry fine powder.

And (3) measuring gelling time: mixing 1g of the powdery hemostatic with 5-7 ml of 0.9% sodium chloride aqueous solution, spreading on a smooth plane, inclining for 30 degrees, if no obvious flowing phenomenon exists, forming gel, and measuring the time required from mixing with the 0.9% sodium chloride aqueous solution to forming gel;

and (3) determining the gelling stability: the powder hemostat 1g is mixed with 5-7 ml of 0.9% sodium chloride aqueous solution, and then the mixture is spread on a smooth plane, if the texture is wet and uniform and the drying and blocking phenomena are avoided, the powder hemostat is an effective hemostat. The hold time from gelling to the point at which the gel appeared to dry into a mass was determined.

(4) The results of the experiments are shown in Table 6.

TABLE 6 gelation time and gelation stability of gelatin with crosslinking agent

(5) And (4) conclusion: according to the experimental result, in order to keep the gelling time of the cross-linked gelatin within 5-10 minutes and the gelling stability within 8-12 hours, the gelling time of the hemostatic prepared under the conditions is reasonable and the gelling stability is maintained for a long time, and 1% of the cross-linked gelatin, 5% of the cross-linked gelatin, 10% of the cross-linked gelatin and 20% of the cross-linked gelatin can meet the ideal clinical surgical requirements.

The properties of the hemostats prepared in examples 1-4 above were further determined as shown in Table 7:

TABLE 7 gelation time and gelation stability Studies of gelatin with crosslinking agent

According to the experimental results, the hemostatic prepared by the invention has reasonable gelling time and longer gelling stability maintaining time, and is more suitable for clinical application.

Experimental example 3 animal experiments

The absorbability, histocompatibility, safety and efficacy of the hemostat of the present invention are illustrated by the following experiments.

Animal experiment I, absorption time and tissue compatibility experiment

The experimental animals were 24 adult SD rats, half male and half female, each weighing about 200-250g, and randomly divided into 4 groups of 6 animals each. Each group consisted of 4 experimental animals, 2 control animals, the experimental samples were the pale yellow colloidal semifluid prepared as described in examples 1-4 above (experimental group), and the control samples were sterile 0.9% saline (control group).

The experimental method comprises the following steps: after 10% chloral hydrate is injected into abdominal cavity of rat for anesthesia, fixing the rat on an operation table, shaving the operated area on the back of the rat, and disinfecting the rat. An approximately 2cm insertion opening was cut in the midline of the dorsal spine, the right side of the insertion opening was bluntly separated with scissors, 0.4ml of the test specimen was inserted into the right muscle layer, sutured with a sterile thread, and 1ml of sterile 0.9% saline was injected below the left side of the insertion opening as a control. Subjecting the experimental animal to animal experimentPutting back into a cage, feeding normally after the anaesthesia and revival, and monitoring body temperature, wound and general reaction. One group of animals was euthanized in the first week, the second week, and the remaining animals were euthanized in the third week after the operation. With CO₂After the rat is euthanized, a cut of about 3-4cm is cut at the left side of the midline of the spine of the back of the rat by scissors (the implanted opening is opened by about 0.5cm), the upper end and the lower end of the cut are transversely extended by 3cm to the left and the right to form a rectangular flap which is turned open, the implanted areas of the experimental sample and the control sample are fully exposed, the implanted materials and the tissue conditions are observed by naked eyes, the tissues of the implanted areas are taken, 10 percent formalin is used for fixing, and pathological section staining HE is carried out.

The experimental results are as follows: the experimental animal after the operation has good state and no abnormal behavior; the body temperature is stable and no hyperpyrexia phenomenon exists; the operation incision is well healed, and abnormal conditions such as bleeding and the like do not exist. The first group of animals were euthanized in the first week after the operation, the implanted area was observed with naked eyes after dissection, no experimental sample residue was found in 6 animals, the tissue was free from scabbing and obvious inflammatory reaction, and the experimental sample implanted tissue (experimental group) was not significantly different from the normal saline subcutaneous injection tissue (control group). In the second postoperative week, the second group of animals were euthanized, no material residue was found in 6 animals, no scab and no obvious abnormality were observed in the tissues, and no significant difference was observed between the tissue implanted with the experimental sample (experimental group) and the tissue injected subcutaneously with physiological saline (control group). And (3) after the third week of operation, animals in the third group and the fourth group are euthanized, no material residue is seen subcutaneously in 12 animals, no obvious inflammation-like reaction exists, and the implanted tissue of the experimental sample (the experimental group) and the subcutaneous tissue of the normal saline (the control group) have no obvious difference. After pathological section HE staining, obvious interstitial small blood vessel hyperplasia, cell tissue infiltration, occasional granuloma and no special tissue are observed on both sides after one week, two weeks and three weeks of operation, and the implanted tissue (experimental group) of the experimental sample and the subcutaneous tissue (control group) of the normal saline do not have obvious difference.

Animal experiment II, animal safety and effectiveness experiment

The experimental animals were 40 adult SD rats, male, each weighing about 220-250g, randomly divided into 5 groups of 8 animals, 6 animals in each group, 2 control animals, and the experimental samples were light yellow colloidal semi-fluid prepared in examples 1-4.

The experimental method comprises the following steps: after 10% chloral hydrate is injected into abdominal cavity of rat for anesthesia, fixing the rat on an operation table, shaving and disinfecting the abdominal operation area. A surgical opening of about 2-3cm is cut by scissors along the midline of the abdomen of the upper abdomen of a rat, the upper right lobe of the liver is fully exposed, a wound surface is cut on the surface of the liver by scissors until active bleeding occurs, an experimental sample (example 1-4) is applied on the wound surface of the liver of an experimental animal, the hemostasis time is recorded, only the hemostasis time is recorded for a control group of animals, and then the surgical opening is sutured with a sterile suture in a double layer manner to close the abdomen. After operation, the animals are returned to the cages, fed normally after the animals are anaesthetized and revived, and the body temperature, the wound and the general reaction are monitored. One group of animals was euthanized on days 2, 4, 7, 9, and 12 post-surgery with CO₂After euthanizing the rats, the abdominal operation area was dissected. The lower abdomen of the rat is transversely cut by scissors, and two ends of the lower abdomen are extended upwards to form a rectangular skin flap which is turned upwards to fully expose organs in the abdominal cavity. And (3) observing the conditions of the surface wound, organs and abdominal wall of the liver and the residual condition of the experimental sample by naked eyes, taking the tissues of the area of the experimental sample applied to the surface of the liver wound, fixing the tissues by 10% formalin, and carrying out HE (Hee staining) on pathological sections.

The experimental results are as follows: all experimental animals revive after the operation, the state is good, abnormal behaviors do not exist, the operation incision is good in healing, and abnormal conditions such as bleeding do not exist. The animals are euthanized and dissected, and then are observed by naked eyes, no obvious foreign matters are seen in all organs in the abdominal cavity of the experimental animal, the wound surface on the surface of the liver has no exudation and bleeding, light white scars are formed, the abdominal wall and all organs have no adhesion, the surgical wound surface on the surface of the liver of a control animal is smooth, has no exudation and bleeding, light white scars are formed, the abdominal wall and all organs have no adhesion, and the experimental animal and the control animal have no obvious difference. Pathological observation shows that the lobular structures of liver tissues are clear, the blood vessels are congested, interstitial edema exists, small amount of inflammatory cell infiltration of lymphocytes can be seen in individual convergent areas, punctate necrosis around central veins and in the lobules occasionally appears, and the rest is not special after 2, 4, 7, 9 and 12 days of operation. Liver tissue in the area to which the test sample was applied (experimental group) was not significantly different from liver tissue in the area to which the test sample was not applied (control group).

Bleeding times and statistics are shown in table 8.

TABLE 8 measurement results of bleeding time

Summary of animal experimental results: (1) the experimental sample has no systemic and local toxicity to animals, and no inflammation and anaphylactic reaction, (2) the total absorption time of the experimental sample in the animal body is 1-2 weeks, (3) the experimental sample has no toxicity to animal viscera, and no inflammation and anaphylactic reaction, and (4) the experimental sample obviously shortens the bleeding time of the animal liver operation incision.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. An absorbable semi-flowable cross-linked polypeptide biological surgical hemostat, comprising: the hemostatic material comprises 80-99 wt% of gelatin and 1-20 wt% of cross-linking agent, wherein the cross-linking agent comprises: acrylamide and trimethylpropane or acrylamide and polyethyleneglycol or disuccinimide glutarate and polyethyleneglycol or trimethylpropane, wherein the crosslinking density Cd of the hemostat meets the following requirements: cd is more than or equal to 0.3200 and less than or equal to 0.4000.

2. The absorbable semi-flowable crosslinked polypeptide as claimed in claim 1, wherein: the weight percentage of the gelatin is 90-99%, and the weight percentage of the cross-linking agent is 1-10%.

3. The absorbable semi-flowable crosslinked polypeptide as claimed in claim 2, wherein: the weight percentage of the gelatin is 95-99 percent, and the weight percentage of the cross-linking agent is 1-5 percent.

4. The absorbable semi-flowable crosslinked polypeptide as claimed in claim 1, wherein: the gelling time of the hemostatic is 5-10 minutes, and the gelling stability is maintained for 8-12 hours.

5. The absorbable semi-flowable crosslinked polypeptide as claimed in claim 1, wherein: the hemostat also comprises iron ions and calcium ions, and the concentration of the iron ions and the calcium ions is 0.05M.

6. A method of preparing an absorbable semi-flowable crosslinked polypeptide biosurgical hemostat of claim 1, wherein the method comprises the steps of: dissolving gelatin in deionized water; adding a cross-linking agent after dissolution, and stirring for 8-16 hours under the irradiation of ultraviolet rays; filtering, washing for many times to wash off the excessive cross-linking agent; drying the washed product in flowing air flow, collecting the product after over 90% weight loss, grinding into fine particles, sieving to make the particles uniform, and mixing the hemostatic material with small amount of 0.9% sodium chloride water solution to form uniform, semi-flowing, white or light yellow colloid with granular texture.

7. The method of claim 6, wherein: dissolving gelatin in deionized water; after dissolving, adding a cross-linking agent, ferric ions and divalent calcium ions, and stirring for 8-16 hours under the irradiation of ultraviolet rays; filtering, washing for many times to wash off redundant cross-linking agent and metal ions; drying the washed product in flowing air flow, collecting the product after over 90% weight loss, grinding into fine particles, sieving to make the particles uniform, and mixing the hemostatic material with small amount of 0.9% sodium chloride water solution to form uniform, semi-flowing, white or light yellow colloid with granular texture.

8. The production method according to claim 6 or 7, characterized in that: the intensity of ultraviolet irradiation is 200nm-300nm,6000μW-8000μW/cm²。