CN115336410B

CN115336410B - Biological product DBT

Info

Publication number: CN115336410B
Application number: CN201410233597.4A
Authority: CN
Inventors: 万华印; 黄志霞; 李若冰; 万德夫; 付岩霖; 朱晋辉; 李敏
Original assignee: Guangzhou Zhongwei Biotechnology Co ltd
Current assignee: Guangzhou Zhongwei Biotechnology Co ltd
Priority date: 2014-05-28
Filing date: 2014-05-28
Publication date: 2018-09-14
Anticipated expiration: 2034-05-28

Abstract

The invention relates to the technical fields of biological materials, biological pharmacy, biological products, medical appliances and the like, in particular to a biological product protein plaster (Dan Bai Tie, DBT) which has main components from mammals and can be absorbed by biological organisms. The material contains main components such as fibrinogen, thrombin, albumin, XIII blood coagulation factor, collagen and the like, and auxiliary components such as sugar, sugar alcohol, amino acid, salt, vitamin, emulsifier and the like, wherein the auxiliary components have very important influence on the preparation, appearance, stability, efficacy and the like of DBT products. The DBT has good histocompatibility and obvious effect, and can be used for auxiliary hemostasis, adhesion prevention, wound closure, etc. in operation and emergency situations.

Description

Biological product DBT

Technical Field

The invention relates to the technical fields of biological pharmacy, biological products, biological materials, medical instruments and the like, in particular to a biological product DBT which is mainly derived from animals and can be absorbed by a biological organism, and the biological material can be used for the purposes of auxiliary hemostasis, wound closure and the like in the operation and emergency occasions.

Background

The hemostasis is rapid and thorough in the operation, the bleeding amount is reduced, the operation field is kept clear, and the hemostasis method is one of the cores of basic operation of the surgical operation. In various first-aid cases such as frequently-occurring traffic and accidents, the problems of rapid and effective hemostasis, patient pain relief, life saving and the like are also commonly encountered. Currently, the commonly used topical hemostatics include fibrin glue, thrombin powder and the like, and the hemostatic materials of medical instruments include gelatin sponge, oxidized cellulose, microfibril collagen, alginic acid fiber and the like. The local hemostatic material is widely used for surgical hemostasis and achieves certain hemostatic effect in clinical and animal experiments. The local hemostatic is used, so that the bleeding amount can be reduced, the operation is simplified, the operation time is shortened, and the wound healing can be promoted.

However, the currently used hemostatic materials still have many disadvantages in terms of hemostasis speed, adhesion effect, tissue compatibility, and ease of operation. Common collagen sponge, gelatin sponge and the like have poor viscosity and are easy to fall off when used for hemostasis, and the gelatin sponge has poor histocompatibility and is easy to induce granulation generation. Oxidized cellulose, alginic acid fiber, and the like are inferior in tissue compatibility to biomaterials composed of protein components. Fibrin glue is very loaded down with trivial details when using the operation, needs to prepare a certain time in advance, and in addition its adhesive strength is not good enough, and is poor to the wound surface result of use that the amount of bleeding is great.

The traditional wound hemostasis materials of our army are mainly first-aid kits, four-head bands, bandages and the like, which are not ideal in first-aid modes and first-aid effects, especially under the conditions of severe combat conditions and relatively weak backward delivery force, the incidence rate of war injury hemorrhagic shock is quite high, great difficulty is brought to later-stage treatment, and the death rate and the incidence rate of complications are increased.

The wound surface hemostatic materials used in war wounds for emergency treatment in the present army can be classified into two major categories, i.e., biological type and chemical type, such as dry fibrin dressing (DFSD, containing fibrinogen, thrombin, cellulose carrier, etc.), traumaDex (microporous polysaccharide particles), quikClot (specific zeolite powder), rapid delivery Hemostat (RDH, polysaccharide polymer extracted from algae), and HemCom dressing (polysaccharide polymer).

DFSD has been used in Iraq warfare, and the hemostatic effect of war wound is good, but the hemostatic effect of cellulose carrier and histocompatibility are not good as DBT of biological product, and fibrin also has the efficacy of promoting wound healing, preventing adhesion, etc.

QuikClot is a molecular sieve substance extracted from zeolite or similar silicate, and its hemostatic mechanism is to absorb the water from the blood flowing out of wound and concentrate the blood coagulation factor to exert hemostatic action, but its side effect is that the temperature rises and the fever is high when it is used. In addition, the material is not absorbable by the human body, and it takes time and cost to remove it.

The HemCon dressing can enable the wound to form firm and adhesive blood clots, and plays a role in stopping bleeding. The material is convenient to carry and use, but has poor histocompatibility, insufficient adhesive strength and no effect of promoting wound healing.

In the human coagulation mechanism, thrombin acts on fibrinogen to cleave 4 peptide bonds at the ends of 2 a α chains and 2B β chains, releasing 2 fibrinopeptides a and two fibrinopeptides B, and generating fibrin monomers. Fibrin monomers are spontaneously connected into unstable fibrin polymers, and under the action of XIII blood coagulation factors and calcium ions, amide transfer occurs in the soluble fibrin polymers to form stable covalent bonds, so that stable fibrin clots in transverse, longitudinal and cross connections are generated, and the blood coagulation process is finally completed.

In this patent study, the following patents were retrieved and compared:

CN02804097.X suspension containing fibrinogen, thrombin and alcohol and method for coating carrier therewith

Preparation method of CN200810167199.1 instant fibrinogen and thrombin combined preparation

CN03126852.8 rapid biological hemostasis sealing device and manufacturing process thereof

CN2621681Y rapid hemostatic dressing material

CN1234425Y absorbable fibrin hemostatic plaster preparation method

CN03142013.3 instant fibrinogen preparation resistant to dry heat treatment

CN201110103540.9 fibrin hemostatic plaster and its preparation method

Method for preparing CN200810046747.5 human fibrinogen preparation

Among the above patents, similar to the main components of the biological product DBT of this study, there are "suspension containing fibrinogen, thrombin and alcohol and method for coating carrier thereof" CN02804097.X, "CN 03126852.8 rapid biological hemostatic closure device and process for manufacturing the same", "CN 201110103540.9 fibrin hemostatic patch and process for manufacturing the same", and "CN 1234425Y an absorbable fibrin hemostatic patch" and the like. The hemostatic materials disclosed in these patents all contain fibrinogen, thrombin and dressing. The fibrinogen contained in cn02804097.X suspension containing fibrinogen, thrombin and alcohol and method of coating the carrier is specifically human fibrinogen, and other patents include human and other mammalian fibrinogen. The preparation method adopted by the suspension containing fibrinogen, thrombin and alcohol and the method for coating the suspension on the carrier in CN02804097 and the preparation method of the absorbable fibrin hemostatic plaster in CN1234425Y is a method for uniformly mixing the fibrinogen and the thrombin by ethanol and then coating the mixture on the carrier. CN03126852.8 rapid biological hemostatic sealing device and its manufacturing process, CN201110103540.9 fibrin hemostatic plaster and its manufacturing method, except the low temperature organic solvent mixing and coating method, also have the layered freeze drying method.

The biological product DBT of this study contains, as major components, fibrinogen, thrombin and collagen from human and other mammalian sources. The main difference between this study and the above invention is the composition of the auxiliary ingredients used. Specifically, the biological DBT biomaterial of the present study contains auxiliary components such as albumin and emulsifiers.

Albumin is a major plasma protein, which can be extracted from human plasma or other mammalian plasma, and is itself an important blood product. Albumin has a higher inactivation temperature relative to fibrinogen and thrombin. The albumin is added, so that a certain heat stabilization auxiliary effect on the fibrinogen and the thrombin is achieved, and the quality guarantee period of the product is prolonged. In addition, the addition of a proper amount of albumin in the thrombin solution is helpful for the preparation process, and the fibrinogen layer, the thrombin layer and the collagen layer can be bonded without layering. If albumin is not added, the fibrinogen layer is easy to fall off, and a complete product cannot be formed, so that the action effect of the fibrinogen-modified fibrinogen is greatly influenced.

Some fibrin hemostatic products in the market also contain albumin, such as albumin added to component 1 and component 3 of porcine fibrin adhesive, and the albumin plays a role of a heating protective agent and a freeze-drying excipient. However, the product form and the action mode of the biological product DBT are obviously different from those of the biological product DBT in the research, and the porcine fibrin adhesive is a freeze-dried powder preparation bottled by glass, and is formed by a special spraying device after being respectively dissolved when in use. The foreign human fibrin plaster Tachosil contains human serum albumin, but the raw material source and the preparation method thereof are obviously different from the research. The Tachosil is prepared by freeze-drying fibrinogen and thrombin, pulverizing, mixing with low-temperature anhydrous ethanol, and coating on collagen sponge. The albumin contained in the biological product DBT in the research aims to promote the adhesion of a three-layer structure, so that the hemostatic effect is enhanced, and no other report or application is found.

The preparation process of the biological DBT adopts a layered freezing and vacuum drying method, but due to the difference of the relative molecular weight, viscosity, thickness, water content and the like of fibrinogen and collagen, the defects of easy product breakage, difficult peeling from a freeze-drying mold box, low yield, poor hemostatic effect and the like exist during the production of the conventional formula and the freeze-drying method.

After a proper amount of emulsifier is added into the DBT component of the biological product, the freeze-drying problem can be effectively solved, the yield and the qualification rate of finished products are improved by one to two times, and the using effect is also obviously improved. Common emulsifying agents recorded in Chinese pharmacopoeia include polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, polyoxyl (40) stearate, sucrose stearate, sodium lauryl sulfate, triethanolamine, soybean lecithin, egg yolk lecithin, hydrogenated castor oil, poloxamer, span 20, span 40, span 60, span 80, and span 85. Polysorbate 80, polysorbate 60, egg yolk lecithin, sucrose stearate and the like also have the function of a solubilizer, and a proper amount of the compound is allowed to be applied to injection.

The biological product DBT of the research is a very effective local hemostatic material and can be used for the purposes of auxiliary hemostasis, wound closure and the like in the operation and emergency occasions. Has the characteristics of easy use, easy carrying, rapid hemostasis, good histocompatibility, complete absorption by organisms, small side effect and the like.

The biological product DBT contains main components such as fibrinogen, thrombin, albumin, XIII blood coagulation factor, collagen and the like and auxiliary components such as sugar, sugar alcohol, amino acid, salt, vitamin, emulsifier and the like, and the formula composition and the proportion of the auxiliary components have very important influence on the preparation, the appearance, the stability, the efficacy and the like of the product. According to the research, the DBT biological product with good stability, attractive and complete appearance and remarkable effect is obtained by optimizing the formula composition and proportion of the product.

Disclosure of Invention

The aim of the research is to provide a biological product DBT which has good stability, beautiful and complete appearance and obvious drug effect.

The biological product DBT contains fibrinogen, thrombin, albumin, XIII blood coagulation factor, collagen and other main components derived from mammalian plasma, and auxiliary components containing an emulsifier.

The biological product DBT further comprises one or more of sugar, sugar alcohol, amino acid, salt, vitamins, etc.

The DBT of the biological product has the main component of fibrinogen content of 1-15 mg/cm ² Thrombin 1-100 IU/cm ² 0.5-20 mg/cm of collagen ² 0.1-4 mg/cm of albumin ² 0.01-0.5 IU/cm of the blood coagulation factor XIII ² 。

The main component of the biological product DBT is preferably fibrinogen content of 2-10 mg/cm ² Thrombin 2-80 IU/cm ² 1-15 mg/cm of collagen ² 0.2-2 mg/cm of albumin ² The blood coagulation factor XIII is 0.02-0.2 IU/cm ² 。

The main component of the biological product DBT is preferably fibrinogen content of 3-8 mg/cm ² Thrombin 4-60 IU/cm ² 2-10 mg/cm of collagen ² 0.3-1 mg/cm of albumin ² 0.03-0.15 IU/cm of the blood coagulation factor XIII ² 。

The biological product DBT contains sugar (including one or more of glucose, sucrose, and trehalose) 1 × 10 ^-6 ～8×10 ^-5 mol/cm ² (ii) a The sugar alcohol comprises one or more of xylitol, mannitol, and sorbitol, and has content of 1 × 10 ^-6 ～1×10 ^-4 mol/cm ² (ii) a The salt comprises one or more of sodium chloride, calcium chloride, and trisodium citrate at a content of 1 × 10 ^-7 ～1.5×10 ^-4 mol/cm ² (ii) a The amino acid comprises one or more of lysine, arginine, glycine, histidine, alanine, and glutamic acid, and the content is 0.5 × 10 ^-6 ～2×10 ^-4 mol/cm ² (ii) a The vitamins include vitamin C, vitamin E, vitamin K, and vitamin B ₂ Rutin and vitamin B ₆ One or more of them, the content is 2 x 10 ^-9 ～3.2×10 ^-7 mol/cm ² (ii) a The emulsifier comprises one or more of polysorbate, span, sugar ester and phospholipid, and the content of the emulsifier is 0.1-50 mu g/cm ² 。

The biological product DBT preferably comprises the following auxiliary components: the sugar comprises one or more of glucose, sucrose and trehalose, and has content of 2 × 10 ^-6 ～4×10 ^-5 mol/cm ² (ii) a The sugar alcohol comprises one or more of xylitol, mannitol, and sorbitol, and has content of 3 × 10 ^-6 ～5×10 ^-5 mol/cm ² (ii) a The salt comprises one or more of sodium chloride, calcium chloride, and trisodium citrate, and has a content of 5 × 10 ^-7 ～3×10 ^-5 mol/cm ² (ii) a The amino acid comprises one or more of lysine, arginine, glycine, histidine, alanine, and glutamic acid, and the content is 2 × 10 ^-6 ～2×10 ^-5 mol/cm ² (ii) a The vitamins include vitamin C, vitamin E, vitamin K, and vitamin B ₂ Rutin and vitamin B ₆ One or more of them, the content is 2 x 10 ^-8 ～1×10 ^-7 mol/cm ² (ii) a The emulsifier comprises one or more of polysorbate, span, sugar ester and phospholipid, and the content is 0.5-25 mu g/cm ² 。

The biological product DBT preferably further comprises the following auxiliary components: the sugar comprises one or more of glucose, sucrose and trehalose, and has content of 3 × 10 ^-6 ～3×10 ^-5 mol/cm ² (ii) a The sugar alcohol comprises one or more of xylitol, mannitol, and sorbitol, and has content of 4 × 10 ^-6 ～2×10 ^-5 mol/cm ² (ii) a The salt comprises one or more of sodium chloride, calcium chloride, and trisodium citrate at a content of 1 × 10 ^-6 ～1×10 ^-5 mol/cm ² (ii) a The amino acid comprises one or more of lysine, arginine, glycine, histidine, alanine, and glutamic acid, and the content is 4 × 10 ^-6 ～1×10 ^-5 mol/cm ² (ii) a The vitamins include vitamin C, vitamin E, vitamin K, and vitamin B ₂ Rutin and vitamin B ₆ One or more of them, the content is 3X 10 ^-8 ～5.5×10 ^-8 mol/cm ² (ii) a MilkThe agent comprises one or more of polysorbate, span, sugar ester and phospholipid, and the content is 1-20 mu g/cm ² 。

The biological DBT is preferably composed of: fibrinogen 8mg/cm ² Thrombin 5IU/cm ² Collagen 8mg/cm ² Albumin 0.58mg/cm ² 0.05IU/cm of blood coagulation factor XIII ² Sucrose 4.38X 10 ^-6 mol/cm ² (1.5mg/cm ² ) Mannitol 1.21X 10 ^-5 mol/cm ² (2.2mg/cm ² ) Sodium chloride 5.13X 10 ^-6 mol/cm ² (0.3mg/cm ² ) Calcium chloride 2.70X 10 ^-8 mol/cm ² (3μg/cm ² ) Trisodium citrate 1.70X 10 ^-6 mol/cm ² (0.5mg/cm ² ) Glycine 3.99X 10 ^-6 mol/cm ² (0.3mg/cm ² ) Histidine 1.29X 10 ^-6 mol/cm ² (0.2mg/cm ² ) Vitamin E1.16X 10 ^-9 mol/cm ² (0.5μg/cm ² ) Vitamin B ₂ 3.99×10 ^-8 mol/cm ² (15μg/cm ² ) Rutin 7.5X 10 ^-10 mol/cm ² (0.5μg/cm ² ) Polysorbate 80. Mu.g/cm ² 。

The biological product DBT is prepared from mammals including human, monkey, pig, cattle, sheep, horse, and dog.

The mammal of the biological product DBT is pig.

The biological product DBT is prepared by adopting a layered freeze vacuum drying process.

The biological product DBT can be used for auxiliary hemostasis in operation and emergency: including hemostasis of various wounds and parenchymal organ sections, hemostasis of surgical field bleeding during operation, tissue defect sealing, tissue adhesion prevention and wound healing promotion.

Drawings

FIG. 1 is a schematic diagram of the beginning of sponge degradation and absorption due to the infiltration of a large amount of inflammatory cells in the biological DBT under a 40-fold optical microscope after 7 days of operation.

FIG. 2 is a schematic diagram of partial degradation and absorption of DBT of a biological product and the interface of the DBT and liver tissue with granuloma foreign body but with less collagen fiber deposition under a 40-fold optical microscope at 14 days after operation.

FIG. 3 is a schematic diagram of biological DBT mostly degraded and absorbed and regenerated liver cells are visible under a 200-fold optical microscope after 20 days of operation.

As shown in fig. 1 to 3, the biological product DBT can be degraded and absorbed in piglets, and generally, the biological product DBT can be completely degraded after 4 to 8 weeks depending on individual differences and use conditions.

Detailed Description

The present invention will be described in detail with reference to the following embodiments in order to explain the technical content, objects and effects of the invention in detail.

Example 1

Extracting fibrinogen, thrombin, albumin, etc. from pig plasma according to conventional mature blood product preparation method, and extracting collagen from pig tendon.

Respectively dissolving collagen, fibrinogen, thrombin and the like, and adding auxiliary components to ensure that the contents of the auxiliary components respectively reach:

the 3 solutions are freeze-dried into the biological product DBT by adopting a layered freeze-vacuum drying method, and the freeze-dried biological product DBT has uniform appearance thickness and does not have the phenomena of crack, delamination, disc sticking and the like. And taking out the biological product DBT, and cutting, sealing and packaging according to the specification.

Example 2

Referring to the formulation and the freeze-drying process in example 1, the formulation composition was adjusted, and the biological product DBT product without adding polysorbate 80 to the fibrinogen solution, without adding mannitol to the thrombin solution, and without adding albumin to the thrombin solution was freeze-dried, respectively, and the results of comparing the freeze-drying conditions of the products of different formulations are shown in the following table.

Table 1 comparison of four lyophilized products of different formulations

As can be seen from table 1, the emulsifier (polysorbate 80) has a great influence on the plate sticking of the product, and the plate sticking is easily caused without adding polysorbate 80, which is especially obvious when using a mold box made of stainless steel or glass. After the disc sticking occurs, the biological product DBT cannot be completely taken out for shearing, so that the yield is low. The thrombin is not added with mannitol and albumin, so the fibrinogen layer and the collagen layer can not be bonded with the thrombin to form an integral three-layer structure. When mannitol is not added, the situation is particularly serious, and a qualified finished product cannot be obtained.

Example 3

Fibrinogen, thrombin, albumin and the like are extracted from human plasma according to a conventional mature blood product preparation method, and collagen is extracted from pig skin.

Dissolving fibrinogen, thrombin, albumin, collagen and the like respectively, and adding auxiliary components to make the contents of the auxiliary components respectively reach the following contents according to the surface area of a biological product DBT: fibrinogen 1.0mg/cm ² Thrombin 2IU/cm ² Collagen 5.5mg/cm ² Albumin 0.25mg/cm ² Sucrose 1mg/cm ² Trehalose 0.5mg/cm ² Mannitol 1.25mg/cm ² Sodium chloride 0.5mg/cm ² 0.1mg/cm of calcium chloride ² Trisodium citrate 1.1mg/cm ² The amino acid comprises lysine 0.05mg/cm ² 0.2mg/cm glycine ² Vitamin E1. Mu.g/cm ² Vitamin B ₂ 10μg/cm ² Rutin 4 mu g/cm ² Triethanolamine 5. Mu.g/cm ² 。

And (3) carrying out vacuum freeze drying on the prepared solution, and shearing, sealing and packaging the freeze-dried biological product DBT to obtain a finished product.

Example 4

Fibrinogen, thrombin, albumin, etc. are extracted from human plasma according to a conventional mature blood product preparation method, and collagen is extracted from bovine tendon.

Dissolving fibrinogen, thrombin, albumin, collagen and the like respectively, and adding auxiliary components to make the contents of the auxiliary components respectively reach the following contents according to the surface area of a biological product DBT: fibrinogen 4.5mg/cm ² Thrombin 3IU/cm ² Collagen 2.4mg/cm ² Albumin 0.5mg/cm ² 0.01IU/cm of blood coagulation factor XIII ² Sucrose 2.1mg/cm ² Trehalose 1.5mg/cm ² Mannitol 1.5mg/cm ² Sorbitol 1.5mg/cm ² Sodium chloride 1.1mg/cm ² 0.5mg/cm calcium chloride ² Trisodium citrate 1.25mg/cm ² The amino acid comprises lysine 0.17mg/cm ² 0.25mg/cm of glycine ² Histidine 0.18mg/cm ² Vitamin C0.12 μ g/cm ² Vitamin E014 mu g/cm ² Vitamin B ₂ 5μg/cm ² Span 80. Mu.g/cm ² 。

And carrying out layered combination vacuum freeze drying on each component solution of the biological product DBT material, wherein the appearance of the freeze-dried biological product DBT is complete and uniform without large cracks, delamination and other phenomena, and the freeze-dried biological product DBT is cut, sealed and packaged to obtain a finished product.

After the biological product DBT is stored for 3 months at 37 ℃, the color appearance has no difference compared with the initial reference product, the use effect is completely normal, after the biological product DBT is stored for 6 months at 37 ℃, the color appearance has slight change compared with the initial reference product, and the solidification time and the bonding strength have no obvious difference when in use.

Example 5

Extracting fibrinogen, thrombin, albumin, etc. from sheep plasma according to conventional mature blood product preparation method, and extracting collagen from horse skin.

Dissolving fibrinogen, thrombin, albumin, collagen, etc., respectively, and adding auxiliary components to make their contents reach DBT content of biological productThe surface area is: fibrinogen 6.5mg/cm ² Thrombin 5IU/cm ² Collagen 3.6mg/cm ² Albumin 0.75mg/cm ² 0.02IU/cm of blood coagulation factor XIII ² Glucose 0.6mg/cm ² 3mg/cm of sucrose ² Trehalose 1.5mg/cm ² Mannitol 2mg/cm ² Sorbitol 1.5mg/cm ² Sodium chloride 1.5mg/cm ² 0.6mg/cm calcium chloride ² Trisodium citrate 1.75mg/cm ² The amino acid comprises lysine 0.17mg/cm ² Glycine 0.25mg/cm ² Histidine 0.18mg/cm ² Vitamin C0.12 μ g/cm ² Vitamin E0.14. Mu.g/cm ² Vitamin B ₂ 0.24μg/cm ² Rutin 1 μ g/cm ² Phospholipid 10. Mu.g/cm ² 。

The biological product DBT material is subjected to vacuum freeze drying and combined together to form a spongy structure, the appearance of the freeze-dried biological product DBT is complete and uniform, large cracks, delamination and other phenomena are avoided, the yield reaches 96.3%, and the freeze-dried biological product DBT is cut, sealed and packaged to obtain a finished product.

The packaged biological product DBT is subjected to a stability test under high illumination with the illumination intensity of 5000 +/-500 Lx and a high humidity environment with the relative humidity of about 75 +/-5%. After being stored for 3 months, the color appearance is not different from that of an initial reference substance, the fibrinogen content and the coagulable protein content which are main components are not changed, and the using effect is completely normal; after 6 months of storage, the color appearance is slightly changed compared with the initial reference product, and the setting time and the adhesive strength are not obviously different when the color-changing adhesive is used.

Example 6

Extracting fibrinogen, thrombin, albumin, etc. from dog plasma according to conventional mature blood product preparation method, and extracting collagen from sheep tendon.

Respectively dissolving fibrinogen, thrombin, albumin, collagen and the like, and adding auxiliary components to ensure that the contents of the auxiliary components respectively reach the following contents according to the surface area of a biological product DBT: fibrinogen 5.0mg/cm ² Thrombin 3.6IU/cm ² Collagen eggWhite 3.0mg/cm ² Albumin 0.45mg/cm ² 0.02IU/cm of blood coagulation factor XIII ² 3mg/cm of sucrose ² Trehalose 1.5mg/cm ² Mannitol 2mg/cm ² Sodium chloride 1.2mg/cm ² 0.6mg/cm calcium chloride ² Trisodium citrate 1.4mg/cm ² The amino acid comprises glycine 0.25mg/cm ² And histidine 0.18mg/cm ² Vitamin C0.12 μ g/cm ² Vitamin E0.4. Mu.g/cm ² Vitamin B ₂ 5.4μg/cm ² Rutin 1.2 mu g/cm ² Sucrose stearate 8.5. Mu.g/cm ² 。

The biological product DBT material is subjected to vacuum freeze drying and combined together to form a spongy structure, the appearance of the freeze-dried biological product DBT is complete and uniform, large cracks, delamination and other phenomena are avoided, the yield reaches 95.1%, and the freeze-dried biological product DBT is cut, sealed and packaged to obtain a finished product.

Example 7

Fibrinogen, thrombin, albumin, etc. are extracted from bovine plasma according to a conventional mature blood product preparation method, and collagen is extracted from bovine cartilage.

Dissolving fibrinogen, thrombin, albumin, collagen and the like respectively, and adding auxiliary components to make the contents of the auxiliary components respectively reach the following contents according to the surface area of a biological product DBT: fibrinogen 4.3mg/cm ² Thrombin 2IU/cm ² Collagen 2.2mg/cm ² Albumin 0.15mg/cm ² 3mg/cm of sucrose ² Mannitol 2mg/cm ² Sodium chloride 1.5mg/cm ² 0.6mg/cm calcium chloride ² Trisodium citrate 1.75mg/cm ² The amino acid comprises lysine 0.17mg/cm ² Glycine 0.25mg/cm ² Vitamin E0.14. Mu.g/cm ² Vitamin B ₂ 0.24μg/cm ² Rutin 0.84 mu g/cm ² Polysorbate 60. Mu.g/cm ² 。

Example 8

Fibrinogen, thrombin, albumin, etc. are extracted from human plasma according to a conventional mature blood product preparation method, and collagen is extracted from horse tendon.

Dissolving fibrinogen, thrombin, albumin, collagen and the like respectively, and adding auxiliary components to make the contents of the auxiliary components respectively reach the following contents according to the surface area of a biological product DBT: fibrinogen 15mg/cm ² Thrombin 10IU/cm ² Collagen 7mg/cm ² Albumin 0.45mg/cm ² Sucrose 2.5mg/cm ² Trehalose 1mg/cm ² Mannitol 2.5mg/cm ² Sodium chloride 1.5mg/cm ² 0.6mg/cm calcium chloride ² Trisodium citrate 3.75mg/cm ² The amino acid comprises lysine 0.35mg/cm ² 0.6mg/cm of glycine ² Vitamin E1. Mu.g/cm ² Vitamin B ₂ 10μg/cm ² Rutin 4 mu g/cm ² Polysorbate 80. Mu.g/cm ² 。

Example 9

The use effect of the biological product DBT is as follows: animal experiments are carried out on the biological product DBT obtained in the embodiment, and the influence of the DBT on wound bleeding of livers and spleens of Tibet miniature pigs under normal pressure is observed.

The Tibetan miniature pig is selected as a research object, the liver and spleen of the miniature pig are injured by a direct trauma method, and then a biological product DBT with a proper size is used for treatment. The test was divided into 4 groups, i.e., model group, biological product DBT group, emulsifier-free biological product DBT group and albumin-free biological product DBT group, 6 animals per group, 3% pentobarbital sodium was anesthetized by intravenous injection. The liver injury standard is 0.5cm deep, 5.0cm long and 2.0cm wide; the spleen injury criteria were 0.5cm deep, 5.0cm long and 2.0cm wide. For single administration, the liver dose was 2.4X 7.0 (cm) and the spleen dose was 4.8X 6.0 (cm). Directly sticking the biological product DBT on the damaged surface, and lightly pressing for a plurality of minutes by using wet saline gauze.

After the liver and spleen are wounded, the groups apply hemostatic medicines (weighed) slightly larger than the area of the wound surface to the wound surface immediately, gauze (weighed) is covered to absorb all the outflowing blood, the gauze is pressed lightly, the hemostatic condition is observed for 1 time every 4-5 seconds, the bleeding time is determined when the wound surface does not bleed any more (the bleeding time is measured in 20min if the bleeding time is more than 20 min), the weight of the yarn blocks and the hemostatic medicines at the moment is weighed, and the bleeding amount is calculated. Bleeding amount (ml) = [ post-hemostasis weight (g) -pre-hemostasis weight (g) ]/specific blood gravity (1.050 g/ml).

The results are shown in the following table.

TABLE 2 influence of the biological product DBT on the bleeding volume and bleeding time of the liver wound of normal pressure miniature pig

Note: the comparison is made with the set of models, ^* p＜0.05， ^** p is less than 0.01; compared with the DBT group of the biological products, ^&& p＜0.01

the results in the table show that 3 biological products DBT with different formulas have hemostatic effect on liver wounds, and the amount of bleeding is reduced remarkably (p is less than 0.01) compared with the model group, but the effect of reducing the amount of bleeding of the biological product DBT without emulsifier and albumin is not as good as that of reducing the amount of bleeding of the biological product DBT, and the biological products DBT and the biological product DBT are different remarkably (p is less than 0.01). Compared with a model group, the biological product DBT can remarkably shorten the bleeding time of the hepatic wound (p is less than 0.01), and the biological product DBT without an emulsifier and albumin can remarkably shorten the bleeding time of the hepatic wound (p is less than 0.05). The biological product DBT without emulsifier and albumin has no obvious difference (p is less than 0.01) compared with the biological product DBT group. In addition, in the test process, the biological product DBT is obviously closely combined with the wounded surface of the liver and the spleen compared with the biological product DBT without an emulsifier and albumin, and is not easy to fall off.

TABLE 3 influence of biological product DBT on spleen bleeding amount and bleeding time of normal pressure piglets

the results in the table show that 3 kinds of biological products DBT with different formulas have hemostatic effect on spleen wounds, and have obvious reduction of bleeding amount compared with a model group (p is less than 0.01), but the biological products DBT without emulsifier and albumin have no effect of reducing the bleeding amount as compared with the biological products DBT, and have obvious difference compared with the biological products DBT group (p is less than 0.01). Compared with the model group, the biological product DBT can remarkably shorten the bleeding time of the spleen wound (p is less than 0.01), and the biological product DBT without an emulsifier and albumin can remarkably shorten the bleeding time of the spleen wound (p is less than 0.05). The biological product DBT without the emulsifier and the albumin shortens the spleen wound and facial bleeding time more than the biological product DBT, but has no obvious difference compared with the biological product DBT group.

Example 10

In order to examine the dose-effect relationship of the DBT hemostatic effect of the biological product, a Beagle canine kidney injury model is prepared by adopting a direct wound method, then the biological product DBT with a proper size is used for treatment, and the experiment is divided into 4 groups, namely a model group, a biological product DBT low, medium and high dose groups, and 6 dogs in each group. The criteria for kidney injury were 1/3 of the left kidney excised. The product obtained in example 1 is used as a medium-dose biological product DBT, the biological product DBT product with the content of each component being three times of the medium dose is a high-dose biological product DBT, and the biological product DBT product with the content of each component being one third of the medium dose is a low-dose biological product DBT. The dose in each dose group was: 1 piece/piece. The administration method comprises the following steps: the biological product DBT is soaked in normal saline for about 15s and then is attached to the wound surface of the Beagle dog, and then the wound surface is lightly pressed for a plurality of minutes by gauze. The administration frequency is as follows: single administration.

TABLE 4 influence of biological product DBT on the amount and bleeding time of wound mountain blood of Beagle dog kidney

Note: the comparison is made with the set of models, ^** p＜0.01

the results in the table show that the biological product DBT has good hemostatic effect in the high, medium and low dose groups, can obviously reduce the bleeding amount of the kidney wound surface of the Beagle dog and shorten the bleeding time, and has very obvious difference in the bleeding amount and the bleeding time compared with the model group (the) ^** p is less than 0.01), and the quantity-effect relationship is obvious.

Example 11

Degradation and absorption of the biological product DBT in pig bodies:

after the piglets were weighed, they were anesthetized by intraperitoneal injection with 1ml/kg (30 mg/kg) of 3% sodium pentobarbital. After satisfactory anesthesia, the piglets are fixed on an operating table in the supine position, the abdominal hairs are cut off, and depilatory is used for depilation. Disinfecting the operative field with an iodine disinfectant and laying a disinfectant towel. The median incision was abdominal and the liver and spleen of the rats were exposed. An area of 3X 4cm was created on the surface of the liver and spleen, respectively ² And adhering a biological product DBT with a corresponding size to the wound surface of artificial blood seepage with the depth of 2mm, lightly pressing the wound surface with dry gauze for 5 minutes respectively, and observing the hemostasis condition of the wound surface and the adhesion condition of the dressing and the wound surface. Then, the anterior lobe of the left liver and the lower splenic pole are respectively cut off, a biological product DBT with corresponding size is attached to the surface of the wound, hemostasis is respectively carried out by lightly pressing the upper surface of dry gauze for 5 minutes, and the hemostasis condition of the surface of the wound and the adhesion condition of the dressing and the surface of the wound are observed. Independently feeding the piglets after operation, and observing the survival condition; after the operation, the abdominal cavity is dissected on 7, 14 and 20 days, and the adhesion of the abdominal cavity, infection, internal hemorrhage and the dressing to the wound surface are observed.

Example 12

The biological product DBT has hemostatic effect when being applied to partial resection of human liver.

20 liver sections which can not achieve 'lip-closing' suture after the right part of the liver of general surgery is excised are selected and randomly divided into a biological product DBT group and a blank control group. The liver section of the biological product DBT group is firstly sewn and pricked to cause active hemorrhage, the biological product DBT with the corresponding size is attached to the wound surface, the biological product DBT is lightly pressed on the surface of the wound surface for 5 minutes by using dry gauze respectively, and the hemostasis condition of the wound surface and the adhesion condition of the dressing and the wound surface are observed. Active bleeding is sewed on the liver section of the control group, and residual blood and tissue fluid on the liver section are sucked by dry gauze and then are not treated. The two groups all use the large compression liver section to stop bleeding, and two abdominal cavity drainage tubes are arranged on the liver section (upper and lower parts on the right side).

And (4) visual observation: the density and speed of the blood exudation of the liver section after the biological product DBT is used are obviously reduced compared with the blood exudation before the biological product DBT is used. Drainage quantity: compared with the self-hemostasis group, the drainage volume of the biological product DBT group in the first 3 days after the operation is reduced by 85%, 70% and 42% respectively. Conclusion the biological product DBT has obvious hemostatic effect on the blood seepage of the damaged section of the liver, and the blood loss (abdominal drainage) of a patient is obviously reduced after the biological product DBT is used, thereby being beneficial to the recovery after the operation.

Example 13

Experimental study of biological product DBT to prevent postoperative adhesions of abdominal cavity.

20 rabbits with unlimited male and female bodies and a body weight of 1.5-2.1kg are randomly divided into two groups, namely a normal saline control group and a biological product DBT group. The animals were subjected to partial cecal resection, the cecal section was resected by 5cm, the severed end was ligated, and the stump was embedded by purse-string suture. After the operation of the control group, 5ml of normal saline is sprayed on the wound surface; biological product DBT group the appropriate size biological product DBT was used for each wound. The abdominal cavity is sutured conventionally, and gentamicin 40000 unit is injected intravenously after operation to prevent infection. The animals were sacrificed 7 days after the operation, and the number of serious adhesions (formation of adhesion bands with a diameter of more than 5mm or adhesion conglomerates such as intestinal tracts and the like) occurred on three wounds (peritoneum of the inferior wall layer of the incision, embedded end of the cecum and stripped area of the ileal membrane) in the abdominal cavity was observed, and the results are shown in the following table.

TABLE 5 comparison of the incidence of adhesions in two groups of animals

The difference between the two groups is obvious, and the biological product DBT is effective in preventing postoperative abdominal cavity adhesion.

Example 14

Application of biological product DBT in treating diabetic foot.

20 orthopedic hospitalized diabetic foot patients were selected and randomly divided into a biological DBT treatment group and a conventional treatment group according to disease classification. Adopts the treatment measures of sugar control, blood circulation promotion, anti-inflammation, debridement and dressing change, general support and the like, and the biological product DBT covers the wound surface with the biological product DBT with proper size when the biological product DBT is used for dressing change. The results after 6 weeks of treatment are shown in the following table:

TABLE 6 therapeutic Effect of Bioagent DBT on diabetic foot

The biological product DBT can obviously improve the cure rate of the diabetic foot.

Claims

1. A biological product DBT is characterized by containing fibrinogen which is a main component derived from mammal plasma and has the content of 1-15 mg/cm ² The content of thrombin is 1-100 IU/cm ² Albumin content 0.1-4 mg/cm ² The content of the blood coagulation factor XIII is 0.01-0.5 IU/cm ² And the content of collagen from skin, cartilage and tendon of mammal is 0.5-20 mg/cm ² (ii) a And the content of the emulsifier is 0.1-50 mu g/cm ² The auxiliary component (4).

2. The bioproduct DBT of claim 1, wherein the principal component is preferably a fiber eggThe content of the white raw material is 2-10 mg/cm ² Thrombin 2-80 IU/cm ² 1-15 mg/cm of collagen ² 0.2-2 mg/cm of albumin ² The blood coagulation factor XIII is 0.02-0.2 IU/cm ² 。

3. Bioproduct DBT as in claim 1, characterized in that the main component is preferably a fibrinogen content of 3-8 mg/cm ² Thrombin 4-60 IU/cm ² 2-10 mg/cm of collagen ² 0.3-1 mg/cm of albumin ² 0.03-0.15 IU/cm of the blood coagulation factor XIII ² 。

4. The bioproduct DBT of claim 1, wherein the adjunct further comprises one or more of a sugar, a sugar alcohol, an amino acid, a salt, and a vitamin.

5. The bioproduct DBT of claim 4, wherein the sugar comprises one or more of glucose, sucrose and trehalose in an amount of 1 x 10 ^-6 ～8×10 ^-5 mol/cm ² (ii) a The sugar alcohol comprises one or more of xylitol, mannitol, and sorbitol, and has content of 1 × 10 ^-6 ～1×10 ^-4 mol/cm ² (ii) a The salt comprises one or more of sodium chloride, calcium chloride, and trisodium citrate at a content of 1 × 10 ^-7 ～1.5×10 ^-4 mol/cm ² (ii) a The amino acid comprises one or more of lysine, arginine, glycine, histidine, alanine, and glutamic acid, and the content is 0.5 × 10 ^-6 ～2×10 ^-4 mol/cm ² (ii) a The vitamins include vitamin C, vitamin E, vitamin K, and vitamin B ₂ Rutin and vitamin B ₆ One or more of them, the content is 2 x 10 ^-9 ～3.2×10 ^-7 mol/cm ² (ii) a The emulsifier comprises one or more of polysorbate, span, sugar ester and phospholipid, and the content is 0.1-50 mu g/cm ² 。

6. The bioproduct DBT of claim 5, whereinIn that the auxiliary components are preferably: the sugar comprises one or more of glucose, sucrose and trehalose, and has content of 2 × 10 ^-6 ～4×10 ^-5 mol/cm ² (ii) a The sugar alcohol comprises one or more of xylitol, mannitol, and sorbitol, and has content of 3 × 10 ^-6 ～5×10 ^-5 mol/cm ² (ii) a The salt comprises one or more of sodium chloride, calcium chloride, and trisodium citrate, and has a content of 5 × 10 ^-7 ～3×10 ^-5 mol/cm ² (ii) a The amino acid comprises one or more of lysine, arginine, glycine, histidine, alanine, and glutamic acid, and the content is 2 × 10 ^-6 ～2×10 ^-5 mol/cm ² (ii) a The vitamins include vitamin C, vitamin E, vitamin K, and vitamin B ₂ Rutin and vitamin B ₆ One or more of them, the content is 2 x 10 ^-8 ～1×10 ^-7 mol/cm ² (ii) a The emulsifier comprises one or more of polysorbate, span, sugar ester and phospholipid, and the content is 0.5-25 mu g/cm ² 。

7. The bioproduct DBT of claim 5, characterized in that the adjunct ingredients are preferably: the sugar comprises one or more of glucose, sucrose and trehalose, and has content of 3 × 10 ^-6 ～3×10 ^-5 mol/cm ² (ii) a The sugar alcohol comprises one or more of xylitol, mannitol, and sorbitol, and has content of 4 × 10 ^-6 ～2×10 ^-5 mol/cm ² (ii) a The salt comprises one or more of sodium chloride, calcium chloride, and trisodium citrate at a content of 1 × 10 ^-6 ～1×10 ^-5 mol/cm ² (ii) a The amino acid comprises one or more of lysine, arginine, glycine, histidine, alanine, and glutamic acid, and the content is 4 × 10 ^-6 ～1×10 ^-5 mol/cm ² : the vitamins include vitamin C, vitamin E, vitamin K, and vitamin B ₂ Rutin and vitamin B ₆ One or more of them, the content is 3X 10 ^-8 ～5.5×10 ^-8 mol/cm ² (ii) a The emulsifier comprises one or more of polysorbate, span, sugar ester and phospholipid, and the content is 1-20 mu g/cm ² 。

8. The bioproduct DBT of claim 1, wherein the component is preferably: fibrinogen 8mg/cm ² Thrombin 5IU/cm ² Collagen 8mg/cm ² Albumin 0.58mg/cm ² 0.05IU/cm of blood coagulation factor XIII ² Sucrose 4.38X 10 ^-6 mol/cm ² Mannitol 1.21X 10 ^-5 mol/cm ² Sodium chloride 5.13X 10 ^-6 mol/cm ² Calcium chloride 2.70X 10 ^-8 mol/cm ² Trisodium citrate 1.70X 10 ^-6 mol/cm ² Glycine 3.99X 10 ^-6 mol/cm ² Histidine 1.29X 10 ^-6 mol/cm ² Vitamin E1.16X 10 ^-9 mol/cm ² Vitamin B ₂ 3.99×10 ^-8 mol/cm ² Rutin 7.5X 10 ^-10 mol/cm ² Polysorbate 80. Mu.g/cm ² 。

9. The bioproduct DBT of claim 1, wherein the mammal comprises a human, monkey, pig, cow, sheep, horse, dog.

10. The bioproduct DBT of claim 1, wherein the mammal is a pig.

11. The bioproduct DBT of any of claims 1-10, characterized by using a layered freeze vacuum drying process.

12. The biologic DBT of claim 1, which is useful for assisting in hemostasis during surgery and emergency procedures, including hemostasis of various wounds, cuts of parenchymal organs, hemostasis of surgical field bleeding during surgery, sealing of defective tissue, preventing tissue adhesions, and promoting wound healing.