Biological glue and preparation method thereof

Abstract

The invention discloses biological glue and a preparation method thereof.The preparation method comprises the steps that gel and activation liquid are prepared, and the gel is prepared by evenly mixing I-type collagen freeze-dried powder, chitosan and a fibrinogen solution; and mixing the freeze-dried thrombin with a calcium chloride solution to prepare an activation solution. The meninx suturing device has the advantages that sliding in the meninx suturing process is effectively reduced, an operation is easier to carry out, the risk that the meninx or a repairing material slides after the operation can be reduced, and a wound is evenly healed; most of the components are type I collagen which is beneficial to meninx injury repair, and the components contain chitosan which has antibacterial and hemostatic effects.

Description

Technical Field

[0001] The present invention relates to the technical field of biological glue, and particularly to a biological glue for meningeal repair. Background Art

[0002] Diseases such as meningioma and meningitis, as well as traumas, etc. may cause serious problems such as cerebrospinal fluid leakage, intracranial infection, and brain tissue herniation. After surgical treatment for these problems, patients need to undergo meningeal repair surgery. Meningeal repair surgery usually adopts methods of surgical suture and material repair. Incomplete repair or insufficient material sealing may lead to cerebrospinal fluid leakage and increase the risk of infection. Meningeal repair surgery requires the use of biological glue to reduce risks such as cerebrospinal fluid leakage and meningeal slippage during and after surgery. However, the current biological glue has poor biocompatibility and adhesion effect, and the postoperative nursing risk for patients is relatively high. Summary of the Invention

[0003] The purpose of the present invention is to provide a biological glue and its preparation method to solve the problems in the prior art that the biological glue for meningeal repair has poor biocompatibility and adhesion effect, and high postoperative nursing risk.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] A preparation method of a biological glue, the biological glue includes a gel and an activation solution in independent packages, and the preparation steps include:

[0006] (1) Prepare the gel, the gel is prepared by uniformly mixing freeze-dried powder of type I collagen, chitosan and fibrinogen solution in a ratio of 5 - 25 g : 1 g : 20 mL;

[0007] (2) Prepare the activation solution, thrombin is mixed with calcium chloride solution to obtain a mixed solution with a thrombin concentration of 200 units / mL and a calcium chloride concentration of 50 mmol / L as the activation solution.

[0008] Further, the preparation process of the freeze-dried powder of type I collagen includes: chopping pigskin and putting it into a 0.75% sodium dodecylbenzenesulfonate solution for ultrasonic treatment, then washing the pigskin with pure water, adding 1% NaOH solution to the pigskin for degreasing, putting the degreased pigskin into an acetic acid solution containing pepsin for enzymatic hydrolysis, removing impurities to obtain type I collagen and making it into freeze-dried powder of type I collagen.

[0009] Further, the detailed process for preparing the freeze-dried type I collagen powder includes: cutting the pigskin into pieces and putting it into a 0.75% sodium dodecylbenzenesulfonate solution at a ratio of pigskin: 0.75% sodium dodecylbenzenesulfonate solution = 1 g: 2.5 mL, and performing ultrasonic treatment for 2 h; then washing the pigskin with pure water, adding a 1% NaOH solution to the pigskin at a ratio of pigskin: 1% NaOH solution = 1 g: 10 mL, and stirring for defatting for 4 h; putting the defatted pigskin into an acetic acid solution with a pH of 2.5 containing pepsin at a ratio of pigskin: acetic acid solution with a pH of 2.5 containing pepsin = 1 g: 10 mL for enzymatic hydrolysis, performing solid-liquid separation, salting out the liquid phase at a pH of 7.0 - 7.4, washing the precipitate to obtain type I collagen, making the obtained type I collagen into type I collagen particles, and freeze-drying the type I collagen particles to obtain the freeze-dried type I collagen powder.

[0010] Further, the particle size of the type I collagen particles is 100 - 200 μm.

[0011] Further, the process for preparing the fibrinogen solution includes: mixing freeze-dried fibrinogen and physiological saline at a ratio of 0.5 - 1.5 g: 10 mL, and completely dissolving the freeze-dried fibrinogen to obtain the fibrinogen solution.

[0012] The present invention also provides a biological glue prepared by the method described above.

[0013] The present invention also provides an application of the biological glue in the preparation of drugs for promoting wound healing.

[0014] The present invention also provides an application of the biological glue in the preparation of drugs for promoting the healing of meningeal suture wounds.

[0015] The advantages of the present invention are as follows: The biological glue has good biocompatibility and can be used in meningeal repair surgeries. The appropriate proportion of fibrinogen makes the biological glue have good adhesion, can bond and fix the meninges, effectively reduce the sliding during meningeal suture, make the surgery easier to perform, can reduce the risk of postoperative meningeal or repair material slippage, and enable uniform wound healing; most of the components are type I collagen, which is beneficial to the repair of meningeal injuries; the components contain chitosan, which has the effects of antibacterial and hemostasis. Specific Embodiments

[0016] The following specific examples will be used to illustrate the present invention in detail. Here, the illustrative embodiments and descriptions of the present invention are used to explain the present invention, but do not limit the present invention.

[0017] Example 1

[0018] This example provides a biological glue and its preparation method. The biological glue includes two components, a gel and an activating solution. The preparation method of the gel is as follows:

[0019] 1. Preparation of Type I collagen lyophilized powder:

[0020] Mix the minced pigskin and 0.75% sodium dodecylbenzenesulfonate (SDBS) solution at a ratio of 1 g: 2.5 mL, then ultrasonically treat for 2 h, and then wash the pigskin with pure water; mix the pigskin and 1% NaOH solution at a ratio of 1 g: 10 mL and stir on a magnetic stirrer for 4 h to degrease; mix the degreased pigskin and acetic acid solution with pepsin at a pH of 2.5 at a ratio of 1 g: 10 mL for enzymatic hydrolysis, and then separate the solid and liquid. The liquid phase is salting-out with sodium chloride at a pH of 7.0 - 7.4, and the precipitate is washed to obtain Type I collagen. The obtained Type I collagen is subjected to high-pressure homogenization to obtain Type I collagen particles with a particle size of about 100 - 200 μm, and the Type I collagen particles are freeze-dried to obtain Type I collagen lyophilized powder.

[0021] 2. Preparation of fibrinogen solution:

[0022] Add the lyophilized fibrinogen and physiological saline into a screw-cap bottle at a ratio of 1 g: 10 mL, place it on a horizontal shaker and shake until the lyophilized fibrinogen is completely dissolved to obtain a fibrinogen solution.

[0023] 3. Preparation of gel:

[0024] Mix the obtained Type I collagen lyophilized powder, chitosan and fibrinogen solution evenly at a ratio of 5 g: 1 g: 20 mL to obtain a gel.

[0025] The preparation method of the activating solution is as follows:

[0026] The activating solution is a mixed solution of thrombin and calcium chloride. The preparation process is: add the lyophilized thrombin and calcium chloride solution into a screw-cap bottle, place it on a horizontal shaker and shake until the thrombin is dissolved to obtain a mixed solution with a thrombin concentration of 200 units / ml and a calcium chloride concentration of 50 mmol / L as the activating solution.

[0027] Example 2

[0028] The difference between Example 2 and Example 1 is that the ratio of Type I collagen lyophilized powder, chitosan and fibrinogen solution is 10 g: 1 g: 20 mL.

[0029] Example 3

[0030] The difference between Example 3 and Example 1 is that the ratio of Type I collagen lyophilized powder, chitosan and fibrinogen solution is 15 g: 1 g: 20 mL.

[0031] Example 4

[0032] Example 4 is different from Example 1 in that the ratio of freeze-dried type I collagen powder, chitosan and fibrinogen solution is 20 g: 1 g: 20 mL.

[0033] Example 5

[0034] Example 5 is different from Example 1 in that the ratio of freeze-dried type I collagen powder, chitosan and fibrinogen solution is 25 g: 1 g: 20 mL.

[0035] Example 6

[0036] This example provides a biological glue and its preparation method. The biological glue includes two components: gel and activation solution. The preparation method of the gel is as follows:

[0037] 1. Preparation of freeze-dried type I collagen powder: Refer to Example 1.

[0038] 2. Preparation of fibrinogen solution:

[0039] Add freeze-dried fibrinogen and physiological saline to a screw-cap bottle at a ratio of 0.5 g: 10 mL, and place it on a horizontal shaker to shake until all the fibrinogen is dissolved.

[0040] 3. Preparation of gel:

[0041] Mix the obtained freeze-dried type I collagen powder, chitosan and fibrinogen solution evenly at a ratio of 20 g: 1 g: 20 mL to obtain the biological glue gel.

[0042] The preparation method of the activation solution refers to Example 1.

[0043] Example 7

[0044] Example 7 is different from Example 6 in that the ratio of freeze-dried fibrinogen to physiological saline is 0.75 g: 10 mL.

[0045] Example 8

[0046] Example 8 is different from Example 6 in that the ratio of freeze-dried fibrinogen to physiological saline is 1.25 g: 10 mL.

[0047] Example 9

[0048] Example 9 is different from Example 6 in that the ratio of freeze-dried fibrinogen to physiological saline is 1.5 g: 10 mL.

[0049] Viscosity test

[0050] Take fresh pigskin, scrape off the fat on the inner surface and wash it clean. Apply the gels of the prepared biological glue to the corresponding inner surfaces of the pigskin, with a thickness of about 1 mm. Then spray the activation liquid on the surface of the gel. After standing for about 30 s, attach the inner surface of another piece of pigskin to the pigskin coated with the biological glue. After standing for 30 minutes, test the bonding strength of the two pieces of pigskin. For each example of the biological glue, three parallel experiments are conducted to test the bonding strength. The average bonding strength of the parallel experiments for each example is shown in Table 1.

[0051] Table 1 Viscosity Test Table

[0052]

[0053] As can be seen from Examples 1 to 5, the higher the proportion of type I collagen in the biological glue, the lower the bonding strength of the biological glue. The adhesion of this biological glue mainly comes from fibrinogen. The higher the proportion of type I collagen, the lower the proportion of fibrinogen. As can be seen from Examples 4, 6, and 7, the higher the proportion of fibrinogen in the biological glue, the higher the bonding strength. However, the proportion of fibrinogen in Examples 4, 8, and 9 has no obvious effect on the bonding strength. When the ratio of fibrinogen to normal saline is 1 g:10 mL, the solubility of fibrinogen in normal saline is close to saturation, and the ratio of fibrinogen to normal saline is 1 g:10 mL is the best.

[0054] Wound Healing Test

[0055] Take large dogs, prepare 5 wounds of about 3×3 mm on the dura mater of the large dogs' intracranial cavity. Apply the gels of the biological glue of Examples 1 to 5 to the 5 wounds of the large dogs' dura mater respectively. After spraying the activation liquid on the surface of the gel, stand for 30 s, and attach the biological dura mater repair material to the wound. A total of 3 large dogs are used for 3 groups of parallels. Dissect the 3 large dogs 20 days after the operation to observe the repair degree of the meningeal wound.

[0056] The meningeal wounds of the 3 large dogs have not fully recovered. The meningeal wounds coated with the biological glue of Example 4 and Example 5 are nearly completely healed, without obvious scars or depressions, and the new tissues are similar to the surrounding normal meningeal structures. The effects of the two examples are similar. Most of the meningeal wounds of Example 3 are healed, without obvious scars or depressions, and the new tissues are similar to the surrounding normal meningeal structures. About half of the meningeal wounds of Example 2 are healed, and there is a slight depression in the center of the meningeal repair material. About half of the meningeal wounds of Example 1 are healed, and there is a depression in the center of the meningeal repair material, which is more obvious and deeper than that of Example 2. Considering the repair degree of the dura mater of the 3 large dogs comprehensively, the repair degrees of Example 4 and Example 5 are similar. The repair degree of Example 4 is better than that of Example 3, the repair degree of Example 3 is better than that of Example 2, and the repair degree of Example 2 is better than that of Example 1. As known from Table 1, the product technical effects of Examples 4, 8, and 9 are equivalent.

[0057] It can be seen from the degree of dural repair in large dogs that as the proportion of type I collagen increases, the healing of the large dog meninges is faster. However, in the data of Examples 4-5, as the proportion of type I collagen increases, the decrease in the healing time is not obvious.

[0058] The technical solutions provided by the embodiments of the present invention have been introduced in detail above. Specific examples are used herein to elaborate on the principles and implementation manners of the embodiments of the present invention. The descriptions of the above embodiments are only applicable to helping understand the principles of the embodiments of the present invention. At the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A method for preparing a biological glue, characterized in that: The biological glue comprises independently packaged gel and activation liquid, and the preparation steps include: (1) preparing a gel, wherein the gel is prepared by uniformly mixing type I collagen freeze-dried powder, chitosan and fibrinogen solution in a ratio of 5-25 g: 1 g: 20 mL; (2) Prepare an activation solution by mixing thrombin with a calcium chloride solution to obtain a mixed solution having a thrombin concentration of 200 units / mL and a calcium chloride concentration of 50 mmol / L, which is the activation solution.

2. A method for preparing a biological glue according to claim 1, characterized in that: The preparation process of the type I collagen freeze-dried powder includes: chopping pig skin and placing it in a 0.75% sodium dodecylbenzene sulfonate solution for ultrasonic treatment, then washing the pig skin with pure water, adding a 1% NaOH solution to the pig skin, defatting, placing the defatted pig skin in an acetic acid solution containing pepsin for enzymatic hydrolysis, removing impurities to obtain type I collagen and preparing type I collagen freeze-dried powder.

3. A method for preparing a biological glue according to claim 2, characterized in that: The detailed preparation process of the type I collagen freeze-dried powder includes: according to the ratio of pig skin: 0.75% sodium dodecylbenzene sulfonate solution = 1g: 2.5mL, the pig skin is chopped and then placed in the 0.75% sodium dodecylbenzene sulfonate solution for ultrasonic treatment for 2h; then the pig skin is washed with pure water, according to the ratio of pig skin: 1% NaOH solution = 1g: 10mL, 1% NaOH solution is added to the pig skin, and the pig skin is stirred for degreasing for 4h; according to the ratio of pig skin: acetic acid solution containing pepsin with a pH of 2.5 = 1g: 10mL, the defatted pig skin is placed in an acetic acid solution containing pepsin with a pH of 2.5 for enzymolysis, solid-liquid separation, liquid phase salting out at a pH of 7.0-7.4, the precipitate is washed to obtain type I collagen, the obtained type I collagen is made into type I collagen particles, the type I collagen particles are freeze-dried to obtain type I collagen freeze-dried powder.

4. A method for preparing a biological glue according to claim 3, characterized in that: The particle size of the type I collagen particles is 100 to 200 μm.

5. A method for preparing a biological glue according to claim 1 or 4, characterized in that: The preparation process of the fibrinogen solution comprises: mixing freeze-dried fibrinogen with physiological saline at a ratio of 0.5-1.5 g:10 mL, and completely dissolving the freeze-dried fibrinogen to obtain the fibrinogen solution.

6. A bioglue prepared by the method described in any one of claims 1 to 5.

7. Use of the biological glue as claimed in claim 6 in preparing a medicine for promoting wound healing.

8. Use of the biological glue as claimed in claim 7 in preparing a drug for promoting meningeal suture wound healing.

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