CN116173296A

CN116173296A - Double-protein elastic hydrogel with biological activity and preparation method thereof

Info

Publication number: CN116173296A
Application number: CN202310203025.0A
Authority: CN
Inventors: 冯龙宝; 郭瑞; 胡圣学
Original assignee: Guangzhou Bioscience Co ltd
Current assignee: Guangzhou Bioscience Co ltd
Priority date: 2023-03-03
Filing date: 2023-03-03
Publication date: 2023-05-30

Abstract

The invention provides a double-protein elastic hydrogel, which comprises sericin modified by methacrylic anhydride and collagen modified by methacrylic anhydride, wherein the swelling degree of the hydrogel is more than or equal to 1.42, and the compression modulus is more than or equal to 180.25kPa; the mass concentration of the methacrylic anhydride modified sericin is 5-20%, and the mass concentration of the methacrylic anhydride modified collagen is 2-6%. The swelling degree and compression modulus of the hydrogel can be regulated by regulating the mass concentration of sericin and collagen within a certain concentration range, the maximum swelling degree can reach 2.13, the maximum compression modulus can reach 253.10kPa, meanwhile, the hydrogel also contains a bioactive factor, namely platelet-rich plasma, so that the hydrogel has repair activity, is used for repairing chronic non-healable wounds, and can be used as a tissue repair material or a tissue engineering scaffold in the field of tissue engineering.

Description

Double-protein elastic hydrogel with biological activity and preparation method thereof

Technical Field

The invention belongs to the technical field of high molecular hydrogel, and in particular relates to double-protein elastic hydrogel with biological activity and a preparation method thereof

Background

Although graft technology has made great progress in skin wound care, the availability of donor skin to large wounds can be limited, resulting in severe scarring, deformity, and ineffective vascularization remains a challenge. Furthermore, for elderly people over 65 years old, the circulatory system may decline with age, which may affect the healing ability of the skin. Topical application of growth factors is a clinically challenging potential therapy that promotes normal healing processes and improves angiogenesis.

Numerous animal studies have demonstrated the feasibility of this approach, suggesting that topical application of growth factors such as transforming growth factor β (TGF-b), platelet Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (BFGF), and Epidermal Growth Factor (EGF) may accelerate wound closure. However, in addition to PDGF, human studies assessing the local application of individual growth factors are largely disappointing, suggesting that wound healing may rely on the "synergistic effect" of multiple growth factors. Furthermore, both the exuding and proteolytic environment of the wound may lead to rapid degradation of topically applied growth factors, which would require multiple re-use.

Conventional dressings use a single growth factor loaded and the growth factor degrades quickly in the dressing. Traditional natural hydrogel dressing has poor mechanical properties and cannot be flexibly applied to wound surfaces. Thus, a more effective wound healing therapy may be by providing a combination of growth factors to promote healing.

Disclosure of Invention

In order to solve the technical problems, the invention provides a double-protein elastic hydrogel with bioactivity and a preparation method thereof. The double-protein elastic hydrogel can be more similar to a structure similar to a natural cytoplasmic matrix, and can more effectively realize the slow release of growth factors in PRP.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a double-protein elastic hydrogel comprises sericin modified by methacrylic anhydride and collagen modified by methacrylic anhydride, wherein the swelling degree of the hydrogel is more than or equal to 1.42, and the compression modulus is more than or equal to 180.25kPa.

Preferably, the hydrogel has a swelling degree of 1.42 to 2.13 and a compression modulus of 180.25 to 253.40kPa.

Preferably, the mass concentration of the methacrylic anhydride modified sericin is 5-20%, and the mass concentration of the methacrylic anhydride modified collagen is 2-6%. More preferably, the mass concentration of the methacrylic anhydride modified sericin is 15%, and the mass concentration of the methacrylic anhydride modified collagen is 4%. The present inventors have demonstrated by way of example that within this concentration range, the swelling degree and compression modulus of the hydrogel can be adjusted by adjusting the mass concentration of sericin and collagen, and particularly when the mass concentration of sericin is 15% and the mass concentration of collagen is 4%, a hydrogel having an optimal swelling degree and compression modulus is obtained.

Preferably, the hydrogel further comprises a cell growth factor.

Preferably, the cell growth factor is platelet rich plasma. Platelet-rich plasma (PRP) was chosen as the active substance in the present invention because the fibrin matrix generated after Platelet activation can provide a scaffold for tissue ingrowth to aid tissue repair.

The invention also provides a method for preparing the double-protein hydrogel, which comprises the following steps:

s1: preparing modified sericin: cutting silkworm cocoon, and using Na ₂ CO ₃ Soaking and boiling the solution, and removing impurities to obtain silk collagen liquid; dripping methacrylic anhydride into silk collagen solution, stirring, centrifuging, removing impurities, dialyzing the solution, and freeze-drying the solution obtained by dialysis to obtain methacrylic anhydrideAcidified sericin;

s2: preparation of modified collagen: after the collagen powder is dissolved, methacrylic anhydride is added for reaction, then dialysis is carried out, and finally, the solution obtained by dialysis is freeze-dried, thus obtaining the methacrylic sericin;

s3: preparing modified sericin/collagen hydrogel: dissolving the methacrylic acid sericin prepared in the step S1, the methacrylic acid collagen prepared in the step S2 and the LAP photoinitiator in water, and performing ultraviolet irradiation to obtain the methacrylic acid modified sericin/collagen double-protein hydrogel.

Preferably, the dialysis bag used in step S1 has a molecular weight cut-off of 3500Da.

Preferably, the dialysis bag used in step S2 has a molecular weight cut-off of 1000Da.

Preferably, the ultraviolet irradiation time is 30s; the mass concentration of the photoinitiator is 0.1%.

The present invention also provides a method for preparing a double-protein hydrogel having biological activity, the method comprising the steps of:

s1: preparing modified sericin: cutting silkworm cocoon, and using Na ₂ CO ₃ Soaking and boiling the solution, and removing impurities to obtain silk collagen liquid; dripping methacrylic anhydride into the silk collagen solution, stirring, centrifuging, removing impurities, dialyzing the solution, and finally freeze-drying the solution obtained by dialysis to obtain the methacrylic acid sericin;

s2: preparation of modified collagen: after the collagen powder is dissolved, methacrylic anhydride is added for reaction, then dialysis is carried out, and finally, the solution obtained by dialysis is freeze-dried, so that the methacrylic collagen is obtained;

s3: dissolving the methacrylic acid sericin prepared in the step S1, the methacrylic acid collagen prepared in the step S2 and the photoinitiator in deionized water, adding platelet-rich plasma, and performing ultraviolet irradiation to obtain the double-protein hydrogel with bioactivity.

The invention also provides application of the hydrogel as a tissue repair material or a tissue engineering scaffold in the field of tissue engineering.

The beneficial effects of the invention are as follows: the invention provides a double-protein elastic hydrogel and a preparation method thereof, the swelling degree and compression modulus of the hydrogel can be regulated by regulating the mass concentration of sericin and collagen in a certain concentration range, the maximum swelling degree can reach 2.13, the maximum compression modulus can reach 253.10kPa, meanwhile, the hydrogel also contains a biological activity factor-platelet-rich plasma, the hydrogel is endowed with repair activity, and the hydrogel can be used for repairing chronic non-healable wounds and can be used as a tissue repair material or a tissue engineering scaffold in the field of tissue engineering.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a polymer of modified sericin (SerMA).

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of a polymer of modified collagen (ColMA).

FIG. 3 is a scanning electron microscope image of hydrogels prepared in comparative example 1 and example 8; wherein A is a scanning electron microscope image of the hydrogel prepared in comparative example 1, and B is a scanning electron microscope image of the hydrogel prepared in example 8.

FIG. 4 is a graph showing the in vitro growth factor release performance of example 13.

FIG. 5 is a graph showing cell viability of examples 8 and 13 and comparative example 1.

Detailed Description

In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the technical scheme of the present invention is described in detail below with reference to the specific embodiments and the accompanying drawings.

Example 1

The embodiment provides a preparation method of a double-protein elastic hydrogel, which comprises the following steps:

s1: modified sericin (SerMA) was prepared, specifically as follows:

40g of cocoons were cut into small pieces and washed with deionized water. The cocoons were then treated with 400mL of 0.02M Na ₂ CO ₃ The solution is soaked and boiled for 1h. The boiled solution was removed every half hour and then passed throughFiltering with filter cloth to remove impurities to obtain silk collagen liquid. 300mL of the sericin solution was taken, and 6mL of methacrylic anhydride was slowly added dropwise to the sericin solution with a burette. After stirring at room temperature for 6 hours, the supernatant was centrifuged at 6000r/min for 8min, and then filtered again through a filter cloth (22-25 μm) to remove impurities. Dialyzing in dialysis bag (molecular weight 3500 Da) for 3-5 days. Finally, the mixture is frozen and dried to obtain the methacrylated sericin (SerMA).

S2: modified collagen (ColMA) was prepared as follows:

10g of collagen is weighed and dissolved in 200mL of distilled water, after the dissolution at 37 ℃, 6mL of methacrylic anhydride is added for reaction for 5 hours at 37 ℃, the distilled water is dialyzed for 2 to 3 days, and the interception molecular weight of a dialysis bag is 1000Da. Freeze-drying at-80deg.C to obtain methacrylated collagen (ColMA).

S3: the preparation method of the SerMA/ColMA double-protein hydrogel comprises the following specific operations:

dissolving SerMA prepared in the step S1, colMA prepared in the step S2 and LAP photoinitiator in deionized water, and irradiating with ultraviolet light for 30S to obtain the methacrylic acid SerMA/ColMA double-protein hydrogel, wherein the mass concentration of SerMA is 5%, the mass concentration of ColMA is 2%, and the mass concentration of LAP photoinitiator is 0.1%.

Example 2

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 2, wherein the mass concentration of SerMA is 5% and the mass concentration of ColMA is 4%.

Example 3

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 3, wherein the mass concentration of SerMA is 5% and the mass concentration of ColMA is 6%.

Example 4

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 4, wherein the mass concentration of SerMA is 10% and the mass concentration of ColMA is 2%.

Example 5

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 5, wherein the mass concentration of SerMA is 10% and the mass concentration of ColMA is 4%.

Example 6

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 6, wherein the mass concentration of SerMA is 10% and the mass concentration of ColMA is 6%.

Example 7

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 7, wherein the mass concentration of SerMA is 15% and the mass concentration of ColMA is 2%.

Example 8

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 8, wherein the mass concentration of SerMA is 15% and the mass concentration of ColMA is 4%.

Example 9

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 9, wherein the mass concentration of SerMA is 10% and the mass concentration of ColMA is 6%.

Example 10

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 10, wherein the mass concentration of SerMA is 20% and the mass concentration of ColMA is 2%.

Example 11

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 11, wherein the mass concentration of SerMA is 20% and the mass concentration of ColMA is 4%.

Example 12

The only difference between this example and example 1 is that the methacrylated SerMA/ColMA double protein hydrogel prepared in example 12, wherein the mass concentration of SerMA is 20% and the mass concentration of ColMA is 6%.

Comparative example 1

The only difference between comparative example 1 and example 8 is that comparative example 1 contained SerMA alone and ColMA alone, and the total mass of the hydrogels prepared in comparative example 1 was the same.

Comparative example 2

The only difference between comparative example 2 and example 8 is that comparative example 2 contained only ColMA and no SerMA, and the total mass of the hydrogels prepared in comparative example 2 was the same.

Table 1: the mass concentration ratio of the hydrogels SerMA and ColMA prepared in examples 1 to 12 and comparative examples 1 and 2

The hydrogels prepared in examples 1 to 12 and comparative examples 1 and 2 were subjected to performance testing as follows:

1. nuclear magnetic test

3-5mg of the methacrylated sericin obtained in the step S1 and the methacrylated collagen obtained in the step S2 in the example 1 are respectively weighed, dissolved in a proper amount of deuterated heavy water, then put into a clean nuclear magnetic tube, nuclear magnetic structure measurement is carried out by using a nuclear magnetic resonance spectrometer under the room temperature condition, and map analysis is carried out by using MestReNova software.

From the nuclear magnetic resonance analyses of fig. 1 and 2, it was found that characteristic functional groups (δ=5.5 and 5.8 ppm) of the methacrylate appeared on the products of step S1 and step S2, and thus it was confirmed that SerMA and ColMA were successfully produced.

2. Scanning electron microscope test

Comparative example 1 and example 8 the prepared hydrogels were freeze-dried and then sprayed with gold, and then observed under a scanning electron microscope. The test conditions were: 5kV electron beam.

As can be seen from FIG. 3, the hydrogels prepared in comparative example 1 (FIG. 3-A) and example 8 (FIG. 3-B) exhibited three-dimensional porous network structures. The pore size was reduced and the pore density was increased for the 15% SerMA/4% ColMA of example 8 compared to the pure 15% SerMA hydrogel. The results indicate that the addition of ColMA promotes densification of the hydrogel.

3. Swelling ratio test

Placing the hydrogel in a water bath at 37deg.C for 15min, demolding, weighing its initial weight, and recording as W ₀ Soaking the hydrogel in PBS buffer solution with pH of 7.4, taking out the hydrogel at regular intervals, rapidly sucking water on the surface of the hydrogel with slightly wetted filter paper, weighing the hydrogel until the weight of the hydrogel is basically unchanged, and recording the weight of the hydrogel when the hydrogel is saturated with water as W _t The swelling ratio of the hydrogel was calculated according to the formula.

When the hydrogel is used as a wound dressing, tissue fluid flowing out of a wound can be absorbed through the water absorption swelling property, and the moist environment of the wound surface can be maintained. The proper swelling performance can avoid wound infection and inflammation caused by excessive accumulation of secretion, and can provide nutrient substances for cells, thereby being beneficial to growth and propagation of cells and promoting wound healing. The results of the swelling degree of the hydrogels of comparative example 1 and examples 1 to 12 are shown in Table 2.

Table 2: swelling degree of hydrogels prepared in examples 1 to 12 and comparative examples 1 and 2

Grouping	Swelling degree
		Example 1	1.52
Example 2	1.65
		Example 3	1.58
Example 4	1.65
		Example 5	1.76
Example 6	1.72
		Example 7	1.78
Example 8	2.13
		Example 9	1.62
Example 10	1.43
		Example 11	1.58
Example 12	1.42
		Comparative example 1	1.13
ComparisonExample 2	1.02

As is clear from Table 2, the swelling degree of the SerMA hydrogel alone in comparative example 1 was 1.13, the swelling degree of the ColMA hydrogel alone in comparative example 2 was 1.02, and the swelling degree of the hydrogels of examples 1 to 12 was not less than 1.42, wherein example 8 was as high as 2.13. Therefore, the swelling degree of the hydrogel can be remarkably improved by compounding SerMA and ColMA. Meanwhile, as is clear from examples 1 to 12, the concentration ratio of SerMA to ColMA can affect the swelling degree of the hydrogel. In examples 1 to 12, the swelling degree of the double-protein hydrogel increased with an increase in the concentration of ColMA when the concentration of SerMA was constant, but the swelling degree of the hydrogel showed a turning point when the concentration of ColMA increased to 4%, and then no increase in the concentration of ColMA was observed, and the swelling degree was rather decreased when the concentration of ColMA increased to 6%. Further, it can be seen from Table 2 that the swelling degree of the double-protein hydrogel increased with an increase in SerMA concentration when the concentration of ColMA was constant, but the swelling degree of the hydrogel turned when the concentration of SerMA increased to 20%, and then increased without an increase in SerMA concentration, and the swelling degree decreased when the concentration of SerMA increased to 20%. Therefore, the concentration of SerMA and ColMA in the double-protein hydrogel can influence the swelling degree of the hydrogel, and the swelling degree of the hydrogel can be improved only in a certain concentration range.

4. Compression performance test

The hydrogel sample was placed directly under a gel strength dedicated probe which squeezed the hydrogel until it ruptured, and the force required to rupture the hydrogel was recorded and defined as the compressive strength (or compressive modulus) of the hydrogel. The ideal hydrogel should have good mechanical properties to maintain its convenience and integrity in use. The compression properties of the hydrogels prepared in comparative examples 1 and 2 and examples 1 to 12 are shown in Table 3.

Table 3: compression modulus of hydrogels prepared in examples 1 to 12 and comparative examples 1 and 2

As is clear from Table 3, the compressive strength of the SerMA hydrogel alone in comparative example 1 was 150.39kPa, the compressive strength of the ColMA hydrogel alone in comparative example 2 was 145.25kPa, and the compressive strength of the hydrogels of examples 1 to 12 were not less than 150.39, wherein example 8 was up to 253.40kPa. Therefore, the compression strength of the hydrogel can be remarkably improved by compounding SerMA and ColMA. Meanwhile, as is clear from examples 1 to 12, the concentration ratio of SerMA to ColMA can affect the compressive strength of the hydrogel. In examples 1 to 12, the compressive strength of the double-protein hydrogel increased with an increase in the concentration of ColMA when the concentration of SerMA was constant, but when the concentration of ColMA was increased to 4%, the compressive strength of the hydrogel became a turning point, and then the concentration of ColMA was not increased, and when the concentration of ColMA was increased to 6%, the compressive strength was rather decreased. In addition, it can be seen from table 3 that the compressive strength of the double-protein hydrogel increased with increasing SerMA concentration when the concentration of ColMA was constant, but the compressive strength of the hydrogel turned when the concentration of SerMA increased to 20%, and then increased without increasing SerMA concentration, and the compressive strength decreased when the concentration of SerMA increased to 20%. Thus, the concentration of SerMA and ColMA in the double-protein hydrogel can influence the compression strength of the hydrogel, and the compression strength of the hydrogel can be improved only in a certain concentration range.

From tables 2 and 3, it is clear that the combination of SerMA and ColMA has a synergistic effect, and neither of comparative examples 1 and 2 can achieve the purpose of improving the swelling degree and compressive strength of the hydrogel. The concentration ratio of SerMA to ColMA can influence not only the swelling degree but also the compression strength of the hydrogel, and the inventors show through examples 1-12 that the compression modulus and the swelling degree of SerMA/ColMA double-protein hydrogel can be adjusted by adjusting the concentration of SerMA and ColMA. Therefore, the double-protein hydrogel with the maximum compression modulus of more than or equal to 180.25kPa and the swelling degree of more than or equal to 1.42 can be obtained within the mass concentration range of 5-20% of SerMA and the mass concentration range of 2-4% of ColMA, and the biological material has potential biomedical application prospect.

Example 13

The embodiment provides a double-protein hydrogel with bioactivity, which comprises the following specific operations:

s1: modified sericin (SerMA) was prepared, specifically as follows:

40g of cocoons were cut into small pieces and washed with deionized water. The cocoons were then treated with 400mL of 0.02M Na ₂ CO ₃ The solution is soaked and boiled for 1h. Taking out the boiled solution every half hour, and filtering to remove impurities by filter cloth to obtain silk collagen liquid. 300mL of the sericin solution was taken, and 6mL of methacrylic anhydride was slowly added dropwise to the sericin solution with a burette. After stirring at room temperature for 6 hours, the supernatant was centrifuged at 6000r/min for 8min, and then filtered again through a filter cloth (22-25 μm) to remove impurities. Dialyzing in dialysis bag (molecular 3500 Da) for 3-5 days. Finally, the mixture is frozen and dried to obtain the methacrylated sericin (SerMA).

S2: modified collagen (ColMA) was prepared as follows:

S3: the preparation method of SerMA/ColMA/PRP hydrogel comprises the following specific steps:

dissolving SerMA prepared in the step S1 and ColMA and LAP photoinitiator prepared in the step S2 in deionized water, adding platelet rich plasma (wherein the volume ratio of PRP to hydrogel solution is 1:10, PDGF-BB, TGF-beta 1 and EGF in PRP are 17440.18 +/-82.53 pg/mL,8643.05 +/-69.72 pg/Ml and 885.68 +/-5.31 pg/mL respectively), and irradiating with ultraviolet light for 30S to obtain SerMA/ColMA/PRP hydrogel, wherein the mass concentration of SerMA is 15%, the mass concentration of ColMA is 4%, and the mass concentration of LAP photoinitiator is 0.1%.

The hydrogel prepared in example 13 was subjected to effect detection as follows:

1. in vitro growth factor Release Properties

In an in vitro release study, the hydrogel prepared in example 13 was placed in 1mL of PBS solution (pH 7.4), incubated at 37 ℃, PBS supernatant was collected at each time point and replaced with the same volume of fresh PBS. The collected PBS was stored at-80 ℃. The amount of Platelet Derived Growth Factor (PDGF) was measured by enzyme-linked immunosorbent assay (ELISA) and the cumulative release rate was calculated.

Because of the special space structure of the hydrogel, different medicines can be loaded, and the hydrogel has the function of slowly releasing the medicines. As can be seen from fig. 1, a faster release of PDGF was observed at the initial 24h, probably because PDGF was released at the hydrogel surface and shallow layers. After burst release, PDGF on the hydrogel surface and in the superficial layer is released to completion and the release rate decreases. And the slow release is continued after 24 hours. It is presumed that this is due to three reasons: firstly, the internal movement of the hydrogel and the outside reach the release balance; and then slow degradation of the hydrogel, resulting in release of PDGF from the hydrogel; as surface and superficial PDGF is released, the internal PDGF concentration in the hydrogel is low and the release driving force becomes weak.

2. Cell viability

L929 fibroblasts in the form of a fusiform or triangle are selected for cell implantation. Cells were counted using a cell counting plate and diluted to a certain concentration. The cell planting density in this test was 3×10 ⁴ cells/well, re-planted in the corresponding well of the 24-well culture plate with hydrogel placed therein, and then cultured in a carbon dioxide incubator at 37 ℃ for a certain period of time, the cultured cells were taken out of the corresponding well plate, 50ul of CCK8 solution was added to each well, and negative control (blank medium) was set, and cultured in the cell incubator for 30min to 60min. And taking out the culture plate according to the judgment of the color change, and transferring and sucking the liquid in the corresponding hole into a 96-well plate. And the absorbance (OD) was measured at a wavelength of 450nm with a microplate reader, and the data was recorded and calculated. As described in comparative example 8, example 13 and comparative example 1The prepared dressing was subjected to cell viability test, and the test results are shown in fig. 2.

Cell viability as shown in figure 2, the relative viability of cells prepared in comparative example 1 containing only SerMA hydrogel was comparable to the control, indicating that the SerMA hydrogel group was not cytotoxic. Whereas the hydrogel cell viability of example 8 (15% SerMA/4% ColMA) and example 13 (15% SerMA/4% ColMA/PRP) was significantly higher than that of the control group. This is mainly due to the good biocompatibility of collagen, and the three-dimensional porous structure of hydrogels can promote cell proliferation. And the PRP contains a plurality of cell growth factors, so that the cell proliferation can be synergistically promoted.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A double-protein elastic hydrogel is characterized by comprising sericin modified by methacrylic anhydride and collagen modified by methacrylic anhydride, wherein the swelling degree of the hydrogel is more than or equal to 1.42, and the compression modulus is more than or equal to 180.25kPa.

2. The double-protein hydrogel according to claim 1, wherein the mass concentration of the methacrylic anhydride modified sericin is 5 to 20% and the mass concentration of the methacrylic anhydride modified collagen is 2 to 6%.

3. The dual protein hydrogel of claim 2, wherein said hydrogel further comprises a cell growth factor.

4. The dual protein hydrogel of claim 3, wherein said cell growth factor is platelet rich plasma.

5. A method for preparing the double protein hydrogel according to any one of claims 1 to 3, comprising the steps of:

6. The method according to claim 5, wherein the dialysis bag used in the step S1 has a molecular weight cut-off of 3500Da.

7. The method according to claim 5, wherein the dialysis bag used in step S2 has a molecular weight cut-off of 1000Da.

8. The method of claim 5, wherein the ultraviolet irradiation time is 30s; the mass concentration of the photoinitiator is 0.1%.

9. A method of preparing the dual protein hydrogel of claim 4, comprising the steps of:

10. Use of the hydrogel according to any one of claims 1 to 4 as a tissue repair material or tissue engineering scaffold in the field of tissue engineering.