Recombinant collagen and application thereof, collagen sponge and preparation method and application thereof

Abstract

The invention provides recombinant collagen and application thereof, collagen sponge and a preparation method and application thereof, and relates to the technical field of biological materials. The amino acid sequence of the recombinant collagen is SEQ ID NO: 1, the recombinant collagen is recombinant III-type collagen, the amino acid sequence is optimized, the recombinant collagen has activity similar to human III-type collagen, the core functional area of the collagen is fully reserved, the molecular weight of the recombinant collagen is closer to that of natural collagen, and the recombinant collagen has good cell proliferation and migration promoting capacity and can effectively promote wound healing. According to the amino acid sequence SEQ ID NO: 1, the recombinant collagen can be constructed through chemical synthesis or genetic engineering expression, the cost is low, the period is short, the operation is easy, commercial production is easier, and the problems of potential animal source disease infection caused by extraction of the III-type collagen depending on an animal source, immunological rejection or anaphylactic reaction caused by heterologous collagen and capacity limitation are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to recombinant collagen and its application, collagen sponge and its preparation method and application. Background Art

[0002] Collagen (or collagen) is a macromolecular protein commonly found in mammals, accounting for about 25% to 35% of the total protein in the body. In the human body, it is mainly found in the skin, bones, cartilage, ligaments and blood vessels. It is an extremely important structural protein in connective tissue, playing the role of connection, support, nutrition supply and protection in connective tissue. Collagen plays an important role in tissue engineering due to its good biocompatibility, in vivo degradability, good water permeability and air permeability. For example, collagen sponge is used for wound tissue repair to promote cell proliferation and migration to make wounds heal faster.

[0003] Type III collagen is mainly found in the skin or vascular endothelium of infants, the intestines, and the cartilage, vitreous body, and intervertebral discs of adults. Among them, type III collagen has a strong ability to synthesize, and can differentiate stem cells from the bone marrow into fibroblasts that help repair wounds. At present, type III collagen is extracted from animal sources. The extraction of animal-derived collagen is mainly industrialized through acid and enzyme methods, which are relatively cheaper, but there are problems such as animal-derived disease infection, heterologous collagen that may cause immune rejection or allergic reactions, and production capacity limitations. Summary of the invention

[0004] The problem solved by the present invention is how to solve the problems of animal-derived disease infection, immune rejection or allergic reaction caused by heterologous collagen, and production capacity limitation caused by extracting type III collagen from animal sources.

[0005] In order to solve the above problems, the present invention provides recombinant collagen and its application, collagen sponge and its preparation method and application.

[0006] In a first aspect, the present invention provides a recombinant collagen having an amino acid sequence of SEQ ID NO: 1.

[0007] In a second aspect, the present invention provides an application of the recombinant collagen as described above in the field of preparing collagen sponge.

[0008] In a third aspect, the present invention provides a method for preparing a collagen sponge, comprising the following steps:

[0009] S1: dissolving the recombinant collagen as described above in water to obtain a recombinant collagen aqueous solution;

[0010] S2: precooling the recombinant collagen aqueous solution;

[0011] S3: freeze-drying the pre-cooled recombinant collagen aqueous solution to obtain a recombinant collagen freeze-dried sample;

[0012] S4: The recombinant collagen freeze-dried sample is irradiated and cross-linked to obtain a collagen sponge.

[0013] Optionally, in S1, the recombinant collagen aqueous solution contains 1% to 10% by weight of recombinant collagen.

[0014] Optionally, in S2, the pre-cooling treatment temperature is 2 to 8°C and maintained for more than 4 hours.

[0015] Optionally, in S4, the radiation source for radiation crosslinking is selected from ultraviolet rays, gamma rays, X-rays, neutron beams, particle beams or electron beams, the radiation crosslinking dose is 20 to 40 kGy, and the radiation crosslinking temperature is 2 to 8°C.

[0016] Optionally, the thickness of the collagen sponge is greater than 1 mm.

[0017] Optionally, the method further includes step S5: cutting and sterilizing the collagen sponge.

[0018] In a fourth aspect, the present invention provides a collagen sponge, which is prepared by the method for preparing a collagen sponge as described in any one of the above items.

[0019] In a fifth aspect, the present invention provides a use of the collagen sponge as described above in the field of preparing hemostatic products and tissue filling.

[0020] The beneficial effects of the recombinant collagen and its application and the collagen sponge and its preparation method and application of the present invention are as follows: the recombinant collagen with the amino acid sequence of SEQ ID NO: 1 is a recombinant type III collagen, which has the activity of human type III collagen after optimizing the amino acid sequence, fully retains the core functional region of collagen, and has a molecular weight closer to that of natural collagen, has good cell proliferation and migration ability, and can effectively promote wound healing. According to the amino acid sequence SEQ ID NO: 1, chemical synthesis or genetic engineering expression can be used to construct recombinant collagen, which has low cost, short cycle, easy operation, and is easier to commercialize, solving the problems of potential animal-derived disease infection, immune rejection or allergic reaction caused by heterologous collagen, and production capacity limitation caused by extracting type III collagen from animal sources. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a flowchart of a method for preparing a collagen sponge according to an embodiment of the present invention. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be interpreted as being limited to the embodiments described herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are only for exemplary purposes and are not intended to limit the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention;

[0024] The term "including" and its variations used herein are open inclusions, i.e., "including but not limited to"; the term "based on" means "based at least in part on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given in the following description.

[0025] In the related art, the pores in a solid material such as collagen sponge include open pores and closed pores, wherein an open pore is a pore connected to the outer surface of the solid material at least at one end, which contributes to the liquid absorption performance of the solid material; a closed pore is a pore not connected to the outer surface of the solid material, which does not contribute to the liquid absorption performance of the solid material. Therefore, for solid materials, how to increase the number of open pores and their proportion in the total pores is a technical issue that technicians in this field always pay attention to.

[0026] In view of the problems existing in the above-mentioned related technologies, this embodiment provides recombinant collagen and its application, collagen sponge and its preparation method and application.

[0027]

[0028] In this embodiment, the recombinant collagen with the amino acid sequence of SEQ ID NO: 1 is a recombinant type III collagen. After optimizing the amino acid sequence, it has the activity of human type III collagen, fully retains the core functional region of collagen, and has a molecular weight closer to natural collagen. It has good cell proliferation and migration ability and can effectively promote wound healing. According to the amino acid sequence SEQ ID NO: 1, chemical synthesis or genetic engineering expression can be used to construct recombinant collagen, which has low cost, short cycle, easy operation, and is easier to commercialize. It solves the problems of potential animal-derived disease infection, immune rejection or allergic reaction caused by heterologous collagen, and production capacity limitation when extracting type III collagen from animal sources.

[0029] An embodiment of the present invention provides an application of the recombinant collagen as described above in the field of preparing collagen sponge.

[0030] In this embodiment, the collagen sponge prepared from recombinant collagen is used for wound tissue repair, has good ability to promote cell proliferation and migration, and can effectively promote wound healing.

[0031] like Figure 1 As shown, a method for preparing a collagen sponge provided by an embodiment of the present invention comprises the following steps:

[0032] S1: dissolving the recombinant collagen as described above in water to obtain a recombinant collagen aqueous solution;

[0033] S2: precooling the recombinant collagen aqueous solution;

[0034] S3: freeze-drying the pre-cooled recombinant collagen aqueous solution to obtain a recombinant collagen freeze-dried sample;

[0035] S4: The recombinant collagen freeze-dried sample is irradiated and cross-linked to obtain a collagen sponge.

[0036] Specifically, collagen sponge serves as a solid material, and the pores in the solid material include open pores and closed pores, wherein the open pores are pores connected to the outer surface of the solid material at least one end, which contribute to the liquid absorption performance of the solid material; the closed pores are pores not connected to the outer surface of the solid material, which do not contribute to the liquid absorption performance of the solid material.

[0037] In this embodiment, the recombinant collagen aqueous solution is first freeze-dried and then irradiated and cross-linked, which can achieve a higher porosity and water absorption multiple under the same pore-forming conditions, so that the collagen sponge can obtain a higher porosity and have better supporting cell reorganization performance, liquid absorption performance and hemostatic performance.

[0038] Optionally, in S1, the recombinant collagen aqueous solution contains 1% to 10% by weight of recombinant collagen.

[0039] In this optional embodiment, the recombinant collagen aqueous solution can achieve a higher porosity and water absorption multiple under the same pore-forming conditions when the concentration is 1% (by weight) to 10% (by weight), so that the collagen sponge has better supporting cell reorganization performance and liquid absorption performance.

[0040] Optionally, in S2, the pre-cooling treatment temperature is 2 to 8°C and maintained for more than 4 hours.

[0041] In this optional embodiment, when the recombinant collagen aqueous solution is pre-cooled to 2 to 8°C and maintained for more than 4 hours, a higher porosity and water absorption multiple can be achieved under the same pore-forming conditions, thereby making the collagen sponge have better supporting cell reorganization performance and liquid absorption performance.

[0042] Optionally, in S4, the radiation source for radiation crosslinking is selected from ultraviolet rays, gamma rays, X-rays, neutron beams, particle beams or electron beams, the radiation crosslinking dose is 20 to 40 kGy, and the radiation crosslinking temperature is 2 to 8°C.

[0043] In this optional embodiment, the recombinant collagen can self-assemble to a certain extent at 2 to 8°C to form polymers. These polymers are fixed due to the subsequent radiation cross-linking and do not disaggregate even when the temperature returns to room temperature. The presence of these polymers improves the connectivity between the pores of the collagen sponge and can form more open pores, thereby increasing its porosity and achieving better support for cell reorganization and hemostasis properties.

[0044] Optionally, the thickness of the collagen sponge is greater than 1 mm.

[0045] Optionally, the method further includes step S5: cutting and sterilizing the collagen sponge.

[0046] In this optional embodiment, a sterile collagen sponge is provided.

[0047] In a fourth aspect, the present invention provides a collagen sponge, which is prepared by the method for preparing a collagen sponge as described in any one of the above items.

[0048] In a fifth aspect, the present invention provides a use of the collagen sponge as described above in the field of preparing hemostatic products and tissue filling.

[0049] The present invention is further described below in conjunction with specific embodiments.

[0050] Example 1

[0051] 1. Weigh 10 g of type III recombinant collagen with an amino acid sequence of SEQ ID NO: 1, and dissolve it in 90 g of water to obtain a recombinant collagen aqueous solution with a weight concentration of 10%;

[0052] 2. Filter the recombinant collagen solution with a 0.22 μm filter membrane to remove insoluble substances, fill the filtered recombinant collagen aqueous solution into a specific mold, and precool the recombinant collagen aqueous solution at 4°C for 4 hours;

[0053] 3. Freeze-drying the pre-cooled recombinant collagen aqueous solution using a vacuum freeze dryer to obtain a recombinant collagen freeze-dried sample;

[0054] 4. The recombinant collagen freeze-dried sample was subjected to irradiation cross-linking, the irradiation cross-linking dose was 40 kGy, the irradiation cross-linking temperature was 4° C., and a collagen sponge was obtained, the thickness of the collagen sponge was 1.5 mm.

[0055] Example 2

[0056] 1. Weigh 2 g of type III recombinant collagen with an amino acid sequence of SEQ ID NO: 1, and dissolve it in 98 g of water to obtain a recombinant collagen aqueous solution with a weight concentration of 2%;

[0057] 2. Filter the recombinant collagen solution with a 0.22 μm filter membrane to remove insoluble substances, fill the filtered recombinant collagen aqueous solution into a specific mold, and precool the recombinant collagen aqueous solution at 4°C for 4 hours;

[0058] 3. Freeze-drying the pre-cooled recombinant collagen aqueous solution using a vacuum freeze dryer to obtain a recombinant collagen freeze-dried sample;

[0059] 4. The recombinant collagen freeze-dried sample was subjected to irradiation cross-linking, the irradiation cross-linking dose was 20 kGy, the irradiation cross-linking temperature was 4° C., and a collagen sponge was obtained, and the thickness of the collagen sponge was 1.5 mm.

[0060] Comparative Example 1

[0061] 1. Weigh 10 g of type III recombinant collagen with an amino acid sequence of SEQ ID NO: 1, and dissolve it in 90 g of water to obtain a recombinant collagen aqueous solution with a weight concentration of 10%;

[0062] 2. Filter the recombinant collagen solution with a 0.22 μm filter membrane to remove insoluble substances, fill the filtered recombinant collagen aqueous solution into a specific mold, and precool the recombinant collagen aqueous solution at 4°C for 2 hours;

[0063] 3. The pre-cooled recombinant collagen aqueous solution is irradiated and cross-linked, the irradiation cross-linking dose is 40 kGy, and the irradiation cross-linking temperature is 4° C.;

[0064] 4. Freeze-dry the irradiated cross-linked recombinant collagen using a vacuum freeze dryer to obtain a collagen sponge with a thickness of 1.5 mm.

[0065] Comparative Example 2

[0066] 1. Weigh 10 g of type III recombinant collagen with an amino acid sequence of SEQ ID NO: 1, and dissolve it in 90 g of water to obtain a recombinant collagen aqueous solution with a weight concentration of 10%;

[0067] 2. Filter the recombinant collagen solution with a 0.22 μm filter membrane to remove insoluble substances, fill the filtered recombinant collagen aqueous solution into a specific mold, and precool the recombinant collagen aqueous solution at 8° C. for 4 hours;

[0068] 3. The pre-cooled recombinant collagen aqueous solution is irradiated and cross-linked, the irradiation cross-linking dose is 40 kGy, and the irradiation cross-linking temperature is 4° C.;

[0069] 4. Freeze-dry the irradiated cross-linked recombinant collagen using a vacuum freeze dryer to obtain a collagen sponge with a thickness of 1.5 mm.

[0070] Effect Example

[0071] The products prepared in Example 1 and Example 2 and Comparative Example 1 and Example 2 were respectively tested for the porosity of the collagen sponge using a high-performance fully automatic mercury intrusion instrument in accordance with GB / T 21650.1-2008 Determination of pore size distribution and porosity of solid materials by mercury intrusion and gas adsorption method Part 1: Mercury intrusion method. The test results are shown in Table 1. It should be noted that the porosity measured by this method represents the ratio of the total volume of open pores in the collagen sponge to its apparent volume.

[0072] In addition, the following method was used to determine the liquid absorption of collagen sponge: take about 20 mg of this product, weigh it accurately, record it as m1, immerse it in a beaker filled with 20℃±1℃ water, rub it gently with your fingers until it is completely soaked and all the air is removed, and be careful not to damage it. After absorbing enough water, gently clamp one corner with small tweezers and take it out of the water. Hold the tweezers lightly on the water surface to drain for 1 minute, weigh it again, record it as m2, and calculate it according to the following formula 1. A total of 5 samples were randomly selected and the average water absorption multiple was calculated.

[0073]

[0074] In formula 1:

[0075] A: water absorption multiple of the sample;

[0076] m1: the mass of the sample before wetting, in grams (g);

[0077] m2: the mass of the sample after being soaked, in grams (g);

[0078] The test results of the porosity and water absorption multiple of the collagen sponges prepared in Example 1, Example 2 and Comparative Example 1, Example 2 are summarized in Table 1.

[0079]

[0080] From the porosity and water absorption test results in Table 1, it can be seen that the sample is freeze-dried first and then irradiated and cross-linked, which is beneficial to the protein having a more dispersed pore structure. The aqueous solution of recombinant collagen is first freeze-dried and then cross-linked at a concentration of 2% (weight) to 10% (weight) and pre-cooled at 2 to 5°C for more than 4 hours. Under the same pore-forming conditions, higher porosity and water absorption multiples can be achieved, so that the recombinant collagen sponge has better supporting cell reorganization performance and liquid absorption performance. After studying this phenomenon, it is believed that the reason may be that recombinant collagen can self-assemble to a certain extent at 2 to 5°C to form polymers. These polymers are fixed by the subsequent irradiation cross-linking, and even if the temperature returns to room temperature, they will not depolymerize. The presence of these polymers makes the connectivity between the pores of the collagen sponge better, and can form more open pores, thereby improving its porosity to obtain better supporting cell reorganization performance and hemostasis performance.

[0081] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A recombinant collagen, characterized in that: The amino acid sequence is SEQ ID NO:

1.

2. Use of the recombinant collagen as claimed in claim 1 in the preparation of collagen sponge.

3. A method for preparing a collagen sponge, characterized in that: The following steps are involved: S1: dissolving the recombinant collagen as claimed in claim 1 in water to obtain a recombinant collagen aqueous solution; S2: precooling the recombinant collagen aqueous solution; S3: freeze-drying the pre-cooled recombinant collagen aqueous solution to obtain a recombinant collagen freeze-dried sample; S4: irradiating and cross-linking the recombinant collagen freeze-dried sample to obtain the collagen sponge.

4. The method for preparing collagen sponge according to claim 3, characterized in that: In S1, the recombinant collagen aqueous solution contains 1% to 10% by weight of the recombinant collagen.

5. The method for preparing collagen sponge according to claim 3, characterized in that: In S2, the pre-cooling treatment temperature is 2 to 8°C and maintained for more than 4 hours.

6. The method for preparing collagen sponge according to claim 3, characterized in that: In S4, the radiation source of the radiation cross-linking is selected from ultraviolet rays, gamma rays, X-rays, neutron beams, particle beams or electron beams, the radiation cross-linking dose is 20 to 40 kGy, and the radiation cross-linking temperature is 2 to 8°C.

7. The method for preparing collagen sponge according to claim 3, characterized in that: The thickness of the collagen sponge is greater than 1 mm.

8. The method for preparing collagen sponge according to claim 3, characterized in that: The method further comprises step S5: cutting and sterilizing the collagen sponge.

9. A collagen sponge, characterized in that: The collagen sponge is prepared by the preparation method of any one of claims 3 to 8.

10. Use of the collagen sponge as claimed in claim 9 in the preparation of hemostatic products and tissue filling.

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