A recombinant collagen sponge and a method of manufacturing the same

Abstract

The patent application discloses a kind of recombinant collagen sponge and its manufacturing method.The manufacturing method of the recombinant collagen sponge includes precooling treatment to aqueous solution of recombinant collagen, so that its temperature is 2-5 DEG C, and keep 2 hours or more;Immediately after the precooling aqueous solution of recombinant collagen is irradiated crosslinking;The aqueous solution of recombinant collagen after irradiation crosslinking is freeze-dried, so that the recombinant collagen sponge is obtained;The thickness of the recombinant collagen sponge is greater than 1mm;Wherein, the amino acid sequence of the recombinant collagen is the amino acid sequence obtained by repeating the short amino acid sequence shown in SEQ ID NO:1 52 times.In the same pore-forming condition, the recombinant collagen sponge can obtain higher porosity, and has better liquid absorption performance (hemostatic performance).

Description

Technical Field

[0001] This application belongs to the field of biomaterials, specifically relating to a recombinant collagen sponge that can be absorbed by the body and a method for manufacturing the same. Background Technology

[0002] Collagen is the most widely distributed, abundant, and diverse family of fibrous proteins in animals, accounting for more than 30% of the total protein in the human body, found in various organs and tissues. As a major component of the extracellular matrix, collagen is considered one of the most useful biomaterials. Collagen has low antigenicity, is biodegradable, easily absorbed, non-toxic, and has good biocompatibility. It also has hemostatic properties and plays an important role in tissue repair. For example, using collagen sponges in artificial skin transplantation at wound sites, its porous structure suitable for cell growth and the role of collagen itself in tissue repair can promote cell proliferation and accelerate tissue healing.

[0003] Collagen sponges are primarily used for hemostasis in internal wound oozing areas, emergency hemostasis, and surgical hemostasis, or for filling cavities and wounds. To fully utilize the hemostatic effect of collagen sponges, a certain volume of collagen sponge is required to absorb and collect more fluid at the wound site. The hemostatic performance of collagen sponges is closely related to their porosity and water absorption rate; the higher the porosity and water absorption rate, the better the hemostatic effect of the product.

[0004] Generally speaking, pores in solid materials include open pores and closed pores. An open pore is a pore that is connected to the outer surface of the solid material at least at one end, and it contributes to the liquid absorption performance of the solid material. A closed pore is a pore that is not connected to the outer surface of the solid material, and it does not contribute to the liquid absorption performance of the solid material.

[0005] Therefore, for solid materials, how to increase the number of openings and their proportion in the total number of openings is a technical problem that those skilled in the art have always been concerned about. Summary of the Invention

[0006] In view of the above-mentioned technical problems existing in the prior art, the purpose of the present invention is to provide a new recombinant collagen sponge and its manufacturing method. Under the same pore-forming conditions, the recombinant collagen sponge can obtain higher porosity and has better liquid absorption performance (hemostatic performance).

[0007] The inventors conducted in-depth research to solve the above-mentioned technical problems and found that: the type I recombinant collagen obtained by repeating the short amino acid sequence shown in SEQ ID NO:1 (G AP GAPG SQ G AP GLQ) 52 times can self-assemble to a certain extent at low temperature (2-5℃) to form polymers. The formation of these polymers makes the pores of the collagen sponge made from the type I recombinant collagen more interconnected, and more pores can be formed, thereby improving its porosity and liquid absorption performance (hemostatic performance).

[0008] That is, the present invention includes:

[0009] 1. A method for manufacturing a recombinant collagen sponge, comprising:

[0010] Step A: Pre-cool the recombinant collagen aqueous solution to a temperature of 2-5℃ and maintain this temperature for at least 2 hours;

[0011] Step B: Immediately subject the pre-cooled recombinant collagen aqueous solution to irradiation crosslinking;

[0012] Step C: The irradiated cross-linked recombinant collagen aqueous solution is freeze-dried to obtain the recombinant collagen sponge; the thickness of the recombinant collagen sponge is greater than 1 mm;

[0013] The amino acid sequence of the recombinant collagen is obtained by repeating the short amino acid sequence shown in SEQ ID NO:1 (GAPGAPG SQ G AP GLQ) 52 times.

[0014] 2. The manufacturing method according to claim 1, wherein the recombinant collagen aqueous solution contains 0.5 to 10% by weight, preferably 1 to 8% by weight, more preferably 1 to 4% by weight of the recombinant collagen.

[0015] 3. The manufacturing method according to item 1, wherein the irradiation crosslinking temperature in step B is 2 to 8°C.

[0016] 4. The method according to item 1, wherein the irradiation crosslinking dose in step B is 10-100 kGy, preferably 20-80 kGy, more preferably 40-60 kGy.

[0017] 5. The manufacturing method according to claim 1, wherein the radiation source for radiation crosslinking is selected from ultraviolet rays, gamma rays, X-rays, neutron beams, particle beams, or electron beams.

[0018] 6. The manufacturing method according to item 1, wherein in step A, the recombinant collagen aqueous solution is pre-cooled to a temperature of 2-5°C and maintained for more than 4 hours.

[0019] 7. The manufacturing method according to item 1, wherein the thickness of the recombinant collagen sponge is 20 mm or less, preferably 10 mm or less, and more preferably 2 to 5 mm.

[0020] 8. The manufacturing method according to item 1 further includes the step of cutting the recombinant collagen sponge obtained after freeze-drying into a specified size and sterilizing it.

[0021] 9. A recombinant collagen sponge prepared by any one of items 1 to 8.

[0022] 10. The use of the recombinant collagen sponge prepared by any one of claims 1 to 8 in the preparation of hemostatic products. The hemostatic products are preferably suitable for hemostasis of wounds, burns, surgical wounds, and filling of residual cavities.

[0023] Based on the present invention, the recombinant collagen can be obtained through genetic engineering expression, which effectively reduces the potential immunogenicity and viral transmission risk of the product; under the same pore-forming conditions, the recombinant collagen sponge can obtain higher porosity and has better liquid absorption performance (hemostatic performance). Attached Figure Description

[0024] Figure 1 This is a transmission electron microscope image taken immediately after the collagen solution in Example 2 was pre-cooled.

[0025] Figure 2 Transmission electron microscopy (TEM) image of the collagen solution in Comparative Example 2 taken immediately after pre-cooling. Detailed Implementation

[0026] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0027] Type I recombinant collagen (i.e., the HLC protein mentioned in the aforementioned Chinese patent application publication CN115947828A) was prepared according to Example 2.

[0028] Example 1: Preparation of Recombinant Collagen Sponge 1

[0029] Accurately weigh 1g of the above-mentioned type I recombinant collagen and 99g of water for injection, dissolve them thoroughly to obtain a 1% by weight recombinant collagen solution.

[0030] The recombinant collagen solution was filtered through a 0.22 μm filter membrane to remove insoluble substances.

[0031] The filtered recombinant collagen solution was filled into a specific mold and pre-cooled at 4°C for 6 hours.

[0032] The irradiation dose was set to 20 kgy, and the pre-cooled recombinant collagen solution was immediately cross-linked at 5 ± 3 °C using an electron accelerator.

[0033] Set the freeze-drying parameters and use a vacuum freeze dryer to freeze-dry the cross-linked recombinant collagen solution.

[0034] The freeze-dried recombinant collagen sponge was cut using relevant equipment to obtain recombinant collagen sponges of different sizes. The thickness of the recombinant collagen sponge was 1.5 mm. This is recombinant collagen sponge 1.

[0035] Example 2: Preparation of Recombinant Collagen Sponge 2

[0036] A recombinant collagen sponge was prepared as in Example 1, except that the concentration of type I recombinant collagen was 4% by weight, the pre-cooling temperature was 4°C, the pre-cooling time was 4h, and the irradiation crosslinking dose was 40kgy. This recombinant collagen sponge is Example 2.

[0037] Comparative Example 1

[0038] A recombinant collagen sponge was prepared in the same manner as in Example 2, except that the pre-cooling time was 0.5 h. This recombinant collagen sponge is the comparative example, recombinant collagen sponge 1.

[0039] Comparative Example 2

[0040] A recombinant collagen sponge was prepared in accordance with Example 2, except that the pre-cooling temperature was 8°C, and the recombinant collagen sponge was prepared as the comparative example recombinant collagen sponge 2.

[0041] Using the recombinant collagen sponges obtained in Examples 1-2 and Comparative Examples 1-2, the porosity of the recombinant collagen sponges was determined using a high-performance fully automated mercury porosimeter, referring to GB / T 21650.1-2008 "Mercury Porosimetry and Gas Adsorption Method for Determination of Pore Size Distribution and Porosity of Solid Materials - Part 1: Mercury Porosimetry". It should be noted that the porosity determined by this method represents the ratio of the total open volume of the recombinant collagen sponge to its apparent volume.

[0042] In addition, the liquid absorbency of the recombinant collagen sponge was determined using the following method: Approximately 20 mg of the product was accurately weighed and recorded as m1. It was then immersed in a beaker containing water at 20℃±1℃, and gently rubbed with fingers until completely soaked and all air was removed, taking care not to damage the sponge. After absorbing sufficient water, a corner was gently held with small tweezers and removed from the water. The sponge was then gently held on the water surface for 1 minute to dissipate the water, and weighed again, recording the result as m2. The absorbency was calculated using the following formula. Five samples were randomly selected, and the average water absorption ratio was calculated.

[0043] A=(m2-m1) / m1

[0044] In the formula:

[0045] A-------Sample water absorption ratio

[0046] m1-------Mass of the sample before wetting, in grams (g);

[0047] m2 ------- The mass of the sample after it has been wetted, in grams (g);

[0048] The main experimental conditions of all the above embodiments and comparative examples, as well as the test results of the porosity and water absorption ratio of the prepared recombinant collagen sponges, are summarized in Table 1.

[0049] Table 1. Preparation conditions and related indicators of recombinant collagen sponge

[0050]

[0051] The test results in Table 1 show that by maintaining the HLC protein solution at 2–5°C for more than 2 hours, higher porosity and water absorption ratio can be achieved under the same pore-forming conditions, resulting in better liquid absorption performance (hemostatic properties) for the recombinant collagen sponge. After studying this phenomenon, the inventors believe that the reason may be that the HLC can self-assemble to a certain extent at 2–5°C to form polymers. These polymers are fixed by subsequent irradiation cross-linking and do not depolymerize even when the temperature returns to room temperature. The presence of these polymers improves the connectivity between the pores of the collagen sponge, enabling the formation of more open pores, thereby increasing its porosity and liquid absorption performance (hemostatic properties).

[0052] Example 5: Study on the low-temperature self-assembly performance of HLC

[0053] Example 3 of Chinese Patent Application Publication CN115947828A confirms that the HLC does not self-assemble at room temperature. However, further research by the inventors shows that the HLC can undergo a certain degree of self-assembly at low temperatures (see Chinese Patent Application 202410092488.9). Figure 1 and Figure 2 (Figures 9 and 10 in Chinese Patent Application 202410092488.9) are transmission electron microscope (TEM) images taken immediately after pre-cooling of the collagen solution in Example 2 and Comparative Example 2, respectively. It can be seen that under the experimental conditions of the examples, the HLC underwent a certain degree of self-assembly. It should be noted that... Figure 1 The middle arrow points to the protein filaments formed by the self-assembly of HLC.

Claims

1. A method for manufacturing a recombinant collagen sponge, comprising: Step A: Pre-cool the recombinant collagen aqueous solution to a temperature of 2-5℃ and maintain this temperature for at least 2 hours; Step B: Immediately subject the pre-cooled recombinant collagen aqueous solution to irradiation crosslinking; Step C: The irradiated cross-linked recombinant collagen aqueous solution is freeze-dried to obtain the recombinant collagen sponge; the thickness of the recombinant collagen sponge is greater than 1 mm; The amino acid sequence of the recombinant collagen is obtained by repeating the short amino acid sequence shown in SEQ ID NO: 1 (GAPGAPG SQ GA PG LQ) 52 times. The recombinant collagen aqueous solution contains 1-8% by weight of the recombinant collagen.

2. The manufacturing method according to claim 1, wherein, The irradiation crosslinking temperature in step B is 2~8℃.

3. The method according to claim 1, wherein, The irradiation crosslinking dose in step B is 10~100kGy.

4. The method according to claim 1, wherein, The irradiation crosslinking dose in step B is 40~60kGy.

5. The manufacturing method according to claim 1, wherein, The radiation source for the radiation crosslinking is selected from ultraviolet rays, gamma rays, X-rays, neutron beams, particle beams, or electron beams.

6. The manufacturing method according to claim 1, wherein, In step A, the recombinant collagen aqueous solution is pre-cooled to a temperature of 2-5°C and maintained for more than 4 hours.

7. The manufacturing method according to claim 1, wherein, The thickness of the recombinant collagen sponge is less than 20 mm.

8. The manufacturing method according to claim 1, wherein, The thickness of the recombinant collagen sponge is 2-5 mm.

9. The manufacturing method according to claim 1, further comprising the step of cutting the recombinant collagen sponge obtained after freeze-drying into a specified size and sterilizing it.

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