Preparation method of recombinant collagen hydrogel tissue adhesive for suture-free wound healing

Abstract

The invention discloses a preparation method of a recombinant collagen hydrogel tissue adhesive for suture-free wound healing, which comprises the following steps: 1, dissolving carboxymethyl chitosan and polylysine in deionized water, adjusting the pH value to be neutral, and adding glutamine transaminase to obtain a solution A; 2, dissolving the recombinant collagen and the four-arm-polyethylene glycol-aldehyde group in deionized water to obtain a solution B; and 3, uniformly mixing the solution A and the solution B to obtain the recombinant collagen hydrogel tissue adhesive for suture-free wound healing. According to the invention, the recombinant collagen is taken as a main component, the hydrogel is rapidly formed by utilizing the synergistic effect of various high-molecular polymers, and the hydrogel has the advantages of good adhesion, antibacterial property, promotion of wound collagen deposition, controllable biodegradation and the like, and the prepared dual-network hydrogel tissue adhesive can be injected into a deep wound and has a good application prospect. The suture line and the suturing nail are replaced to be applied to the aspect of wound suturing, and good application prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a method for preparing a recombinant collagen hydrogel tissue adhesive for sutureless wound healing. Background Art

[0002] Every year, millions of people suffer from various types of wounds, including those caused by accidental trauma and surgical incisions. After tissue damage, heavy bleeding and wound infection are the main causes of death. At present, the commonly used wound closure devices are staples, sutures, adhesives, sealants and mechanical devices. Among them, surgical sutures and staples play an important role in clinical surgery. However, their shortcomings such as time-consuming operation, poor appearance and unsatisfactory tissue integration make them no longer meet the growing demand for wound healing. In the past few years, tissue sealants and adhesives have gradually become alternatives to sutures and staples for suturing and sealing wounds or incisions. These materials have the advantages of less invasiveness, low professionalism, short application time, relatively ideal tissue integration and less tissue damage.

[0003] Commercial adhesives currently on the market are mainly divided into bio-based adhesives, organic synthetic adhesives and composite adhesives. Naturally occurring adhesives such as mussel byssus adhesives and fibrin adhesives have good biocompatibility and biodegradability, but their adhesive properties under physiological conditions are weak because their adhesion mainly relies on relatively weak physical interactions, usually 1 to 10 J / m 2 Although organic synthetic adhesives such as cyanoacrylate glue have high adhesion performance, the toxicity of cyanoacrylate limits its clinical application inside tissues.

[0004] Therefore, there is still a need for better tissue adhesives that have reliable bonding properties while having good biocompatibility, biodegradability, hemostasis and antibacterial activity, so as to serve as a convenient option for trauma emergency treatment and surgical incision bonding. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a recombinant collagen hydrogel tissue adhesive for sutureless wound healing in view of the deficiencies of the above-mentioned prior art. The method uses recombinant collagen as the main component and utilizes the synergistic effect between multiple high molecular polymers to achieve rapid formation of hydrogels and has the advantages of good adhesion, antibacterial properties, promotion of wound collagen deposition, and controllable biodegradation. The prepared double network hydrogel tissue adhesive can be injected into deep wounds, replacing sutures and staples in wound suturing, and has good application prospects.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing a recombinant collagen hydrogel tissue adhesive for sutureless wound healing, characterized in that it comprises the following steps:

[0007] Step 1: dissolving carboxymethyl chitosan and polylysine in deionized water at room temperature to obtain a mixed solution, adjusting the pH value of the mixed solution to neutral, and then adding glutamine aminotransferase to the mixed solution to obtain solution A;

[0008] Step 2, dissolving the recombinant collagen and four-arm-polyethylene glycol-aldehyde in deionized water to obtain solution B;

[0009] Step 3: Evenly mix the liquid A described in step 1 and the liquid B described in step 2 to obtain a recombinant collagen hydrogel tissue adhesive for sutureless wound healing.

[0010] The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing is characterized in that the mass percentage of carboxymethyl chitosan in the mixed solution in step one is 8.4% to 12%, and the mass percentage of polylysine is 6.7% to 9.5%.

[0011] The above-mentioned method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing is characterized in that in step one, the amount of glutamine transaminase is adjusted according to the mass of the recombinant collagen, and the amount of glutamine transaminase used per gram of recombinant collagen is not less than 0.08g.

[0012] The above-mentioned method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing is characterized in that the content of recombinant collagen in the B solution in step 2 is adjusted according to the time required for wound healing, the mass percentage of recombinant collagen is not less than 8%, and the mass percentage of four-arm-polyethylene glycol-aldehyde group is 8% to 12%.

[0013] The method for preparing the above-mentioned recombinant collagen hydrogel tissue adhesive for sutureless wound healing is characterized in that the mass percentage of the recombinant collagen in the B liquid is 8% to 15%.

[0014] The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing is characterized in that the recombinant collagen in step 2 is CF-1552 type I recombinant collagen, and the molecular weight of the four-arm polyethylene glycol-aldehyde group is 10,000.

[0015] The above-mentioned method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing is characterized in that the volume ratio of liquid A to liquid B in step three is 1:1.

[0016] The method for preparing the above-mentioned recombinant collagen hydrogel tissue adhesive for sutureless wound healing is characterized in that in step three, liquid A and liquid B are mixed evenly using a three-way valve and a syringe.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. The present invention utilizes recombinant collagen (especially CF-1552 type I recombinant collagen from the Institute of Biomedicine, School of Chemical Engineering, Northwestern University, which contains a large amount of glutamine) to form an amide bond with polylysine (EPL) through an enzymatic reaction, and through the formation of a double network, while retaining its low immunogenicity, the adhesive is endowed with advantages such as strong adhesion, antibacterial properties, self-healing properties, injectability and controllable biodegradability that are different from other protein adhesives.

[0019] 2. The present invention utilizes a combination of an enzymatic reaction mediated by transglutaminase (TGase) and a Schiff base reaction to rapidly form a wound-adaptive tissue adhesive through a first network and impart certain mechanical properties to it, and then enhances its adhesion properties and controls its degradation rate through a second network. The formed double network structure solves the shortcomings of a single collagen network, such as a long gelation time, poor mechanical properties, lack of fatigue toughness, excessive swelling, and a fast degradation rate, and the instability, poor adhesion, flexibility, and toughness of a single Schiff base network.

[0020] 3. The present invention is the first to use carboxymethyl chitosan (CMCS) and polylysine (EPL) to provide amino-synthesized hydrogel adhesives. The synergistic effect of CMCS and EPL is used to solve the shortcomings of the hydrogel formed by CMCS and aldehyde groups, which has no adhesion and the hydrogel formed by EPL and aldehyde groups, which has a long gelling time.

[0021] 4. The present invention uses four-arm-polyethylene glycol-aldehyde as a polymer to provide a large number of aldehyde groups for the formation of the hydrogel Schiff base network, forming reversible Schiff base bonds with amino groups to give the hydrogel injectability and self-healing properties, while enhancing the adhesion of the hydrogel to the tissue.

[0022] 5. The present invention preferably utilizes a three-way valve and a syringe to mix liquid A and liquid B, which can quickly mix the two solutions evenly and realize the injectability of the tissue adhesive by connecting the syringe to the three-way valve, so as to achieve a better effect of closing deep wounds.

[0023] 6. The preparation method of the present invention is simple and does not require any chemical cross-linking agent. The prepared recombinant collagen hydrogel tissue adhesive has high biological compatibility and good reproducibility, can tightly adhere to wounds, and has the advantages of strong adhesion, strong antibacterial ability and low immunogenicity. It can replace sutures and staples to promote the repair of deep wounds caused by surgical incisions and accidental trauma, and has good application prospects.

[0024] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a schematic diagram of the process for preparing a hydrogel tissue adhesive.

[0026] Figure 2 This is a diagram showing the appearance of liquid A and liquid B of the present invention and a mixture of the two at room temperature.

[0027] Figure 3 This is the FTIR spectrum of each network in the hydrogel tissue adhesive prepared in the present invention.

[0028] Figure 4 The SEM images of the networks in the hydrogel tissue adhesive prepared in the present invention are shown in FIG.

[0029] Figure 5 The following are the test results of the adhesion performance of the hydrogel tissue adhesive prepared in the present invention.

[0030] Figure 6 The self-healing test results of the hydrogel tissue adhesive prepared in the present invention.

[0031] Figure 7 The injectability test results of the hydrogel tissue adhesive prepared in the present invention are shown in FIG.

[0032] Figure 8 The cytotoxicity test results of the hydrogel tissue adhesive prepared in the present invention are shown.

[0033] Fig. 9 The antibacterial test results of the hydrogel tissue adhesive prepared in the present invention.

[0034] Fig.10 The wound closure and healing test results of the hydrogel tissue adhesive prepared in the present invention are shown. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with embodiments. In the following examples, the molecular weight of carboxymethyl chitosan is 544, and the molecular weight of polylysine is 2000-5000, both of which are purchased from Aladdin; the enzyme activity of transglutaminase (TGase) is 1000u / g, which is purchased from Solebao; the recombinant collagen is CF-1552 type I recombinant collagen, which is derived from the Institute of Biomedicine, School of Chemical Engineering, Northwest University. CF-1552 type I recombinant collagen is generated by reverse transcription of mRNA of human collagen to generate cDNA, and then expressed in Escherichia coli BL21. Its structure and acquisition method can refer to the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method", or the document Recombinant collagen for the pair of skin wounds and photo-aging damage, Regenerative Biomaterials, 2024, 11, rbae108; the molecular weight of four-arm-polyethylene glycol-aldehyde group is 10000, which is purchased from McLean. In the description of the present invention, unless otherwise specified, all reagents used are commercially available.

[0036] The preparation method of the recombinant collagen hydrogel tissue adhesive for sutureless wound healing of the present invention is as follows: Figure 1 As shown, the specific steps include:

[0037] Step 1: dissolving carboxymethyl chitosan and polylysine in deionized water at room temperature to obtain a mixed solution, adjusting the pH value of the mixed solution to neutral, and then adding glutamine aminotransferase to the mixed solution to obtain solution A; the mass percentage of carboxymethyl chitosan in the mixed solution is 8.5% to 12%, and the mass percentage of polylysine is 6.7% to 9.5%; adjusting the amount of glutamine aminotransferase according to the mass of the recombinant collagen in step 2, and using no less than 0.08g of glutamine aminotransferase per gram of recombinant collagen;

[0038] Step 2, dissolving recombinant collagen and four-arm-polyethylene glycol-aldehyde in deionized water to obtain liquid B; the content of recombinant collagen in the liquid B is adjusted according to the time required for wound healing, the mass percentage of recombinant collagen is not less than 8%, preferably 8% to 15%, and the mass percentage of four-arm-polyethylene glycol-aldehyde is 8% to 12%;

[0039] Step 3: Use a three-way valve and a syringe to mix the liquid A in step 1 and the liquid B in step 2 in a volume ratio of 1:1 to obtain a recombinant collagen hydrogel tissue adhesive for sutureless wound healing. The reversible Schiff base bond formed by the amino group and the aldehyde group is formed within ten seconds to form the first network of the hydrogel tissue adhesive; then glutamine and the amino group are used to slowly form an amide bond through the action of glutamine transaminase to form the second network of the hydrogel tissue adhesive.

[0040] Example 1

[0041] The preparation method of the recombinant collagen hydrogel tissue adhesive for sutureless wound healing of this embodiment specifically comprises the following steps:

[0042] Step 1: Dissolve 0.10 g of carboxymethyl chitosan and 0.08 g of polylysine in 1 mL of deionized water at room temperature, stir and mix at a speed of 400 rpm for 2 h to dissolve them to obtain a mixed solution, adjust the pH value of the mixed solution to neutral, and then add 0.008 g of TG enzyme (glutamine aminotransferase) to the mixed solution, and mix to obtain liquid A;

[0043] Step 2: Dissolve 0.1 g of recombinant collagen and 0.10 g of four-arm polyethylene glycol-aldehyde in 1 mL of deionized water, and adjust the pH to neutral to obtain solution B;

[0044] Step 3: Use a three-way valve and a syringe to evenly mix the liquid A described in step 1 and the liquid B described in step 2 in a volume ratio of 1:1 to obtain a recombinant collagen hydrogel tissue adhesive for sutureless wound healing.

[0045] Example 2

[0046] The preparation method of the recombinant collagen hydrogel tissue adhesive for sutureless wound healing of this embodiment specifically comprises the following steps:

[0047] Step 1: dissolve 0.12 g of carboxymethyl chitosan and 0.09 g of polylysine in 1 mL of deionized water at room temperature, stir and mix at a speed of 300 rpm for 2 h to dissolve them to obtain a mixed solution, adjust the pH value of the mixed solution to neutral, and then add 0.008 g of TG enzyme to the mixed solution, and mix to obtain liquid A;

[0048] Step 2: Dissolve 0.1 g of recombinant collagen and 0.12 g of four-arm polyethylene glycol-aldehyde in 1 mL of deionized water, and adjust the pH to neutral to obtain solution B;

[0049] Step 3: Use a three-way valve and a syringe to evenly mix the liquid A described in step 1 and the liquid B described in step 2 in a volume ratio of 1:1 to obtain a recombinant collagen hydrogel tissue adhesive for sutureless wound healing.

[0050] Example 3

[0051] The preparation method of the recombinant collagen hydrogel tissue adhesive for sutureless wound healing of this embodiment specifically comprises the following steps:

[0052] Step 1: Dissolve 0.15 g of carboxymethyl chitosan and 0.10 g of polylysine in 1 mL of deionized water at room temperature, stir and mix at a speed of 200 rpm for 3 h to dissolve them to obtain a mixed solution, adjust the pH value of the mixed solution to neutral, and then add 0.01 g of TG enzyme to the mixed solution, and mix to obtain liquid A;

[0053] Step 2: Dissolve 0.1 g of recombinant collagen and 0.12 g of four-arm polyethylene glycol-aldehyde in 1 mL of deionized water, and adjust the pH to neutral to obtain solution B;

[0054] Step 3: Use a three-way valve and a syringe to evenly mix the liquid A described in step 1 and the liquid B described in step 2 in a volume ratio of 1:1 to obtain a recombinant collagen hydrogel tissue adhesive for sutureless wound healing.

[0055] Example 4

[0056] The preparation method of the recombinant collagen hydrogel tissue adhesive for sutureless wound healing of this embodiment specifically comprises the following steps:

[0057] Step 1: dissolving 0.12 g of carboxymethyl chitosan and 0.10 g of polylysine in 1 mL of deionized water at room temperature, stirring and mixing at a speed of 200 rpm for 3 h to dissolve the mixture to obtain a mixed solution, adjusting the pH value of the mixed solution to neutral, and then adding 0.008 g of TG enzyme to the mixed solution, and mixing to obtain liquid A;

[0058] Step 2: Dissolve 0.1 g of recombinant collagen and 0.15 g of four-arm polyethylene glycol-aldehyde in 1 mL of deionized water, and adjust the pH to neutral to obtain solution B;

[0059] Step 3: Use a three-way valve and a syringe to evenly mix the liquid A described in step 1 and the liquid B described in step 2 in a volume ratio of 1:1 to obtain a recombinant collagen hydrogel tissue adhesive for sutureless wound healing.

[0060] Example 5

[0061] The preparation method of the recombinant collagen hydrogel tissue adhesive for sutureless wound healing of this embodiment specifically comprises the following steps:

[0062] Step 1: dissolve 0.15 g of carboxymethyl chitosan and 0.12 g of polylysine in 1 mL of deionized water at room temperature, stir and mix at a speed of 200 rpm for 3 h to dissolve them to obtain a mixed solution, adjust the pH value of the mixed solution to neutral, and then add 0.016 g of TG enzyme to the mixed solution, and mix to obtain liquid A;

[0063] Step 2: Dissolve 0.2 g of recombinant collagen and 0.15 g of four-arm polyethylene glycol-aldehyde in 1 mL of deionized water, and adjust the pH to neutral to obtain solution B;

[0064] Step 3: Use a three-way valve and a syringe to evenly mix the liquid A described in step 1 and the liquid B described in step 2 in a volume ratio of 1:1 to obtain a recombinant collagen hydrogel tissue adhesive for sutureless wound healing.

[0065] Performance evaluation:

[0066] Add solution A and solution B into the vial, mix well and invert to observe the formation of hydrogel morphology. Figure 2 ,As can be seen from the figure, after the two liquids are mixed evenly, they can change from liquid to gel at room temperature.

[0067] The infrared absorption spectrum of the freeze-dried hydrogel tissue adhesive sample was measured using a Fourier transform infrared spectrometer (FT-IR). The test conditions were as follows: the test resolution was 4 cm -1 , the scanning times are 32 times, and the test wave number range is 400~4000cm -1 The results are shown in Figure 3 As can be seen from the figure, PEG-EPL / CMCS has a peak at 1690-1640 cm -1 (C=N) exhibits weak infrared absorption, while EPL-CF has a strong infrared absorption at 1670 cm -1 The absorption peak of C=O bond is shown at 3430cm -1 The infrared spectrum of PEG-EPL / CMCS-CF shows that the absorption peaks of PEG-EPL and EPL-CF are at 1660-1690 cm -1 and 3240cm -1 The two characteristic peaks at , namely amide and imine, indicate that two networks have been formed.

[0068] The freeze-dried hydrogel tissue adhesive was cut into samples with a thickness of about 2 mm, adhered to the sample stage with conductive glue, and then the hydrogel was gold-sprayed by a particle sputtering instrument and its microscopic morphology was observed by scanning electron microscopy (SEM). Figure 4 ,As can be seen from the figure, the hydrogel tissue adhesive has a porous three-dimensional ,space network, and smaller pores than the Schiff base single network hydrogel.

[0069] The adhesion performance test results of the hydrogel tissue adhesive prepared in Examples 1, 4, and 5 of the present invention were performed using a universal material compressor. Before each test, 1 mL of hydrogel tissue adhesive was injected into pig skin (50 mm long, 5 mm wide, and 2 mm thick), the test rate was set to 1 mm / min, and a lap shear test was performed to detect the adhesion performance. In addition, the compressive strength of the hydrogel was analyzed, and the hydrogel was prepared in a cylindrical mold (10 mm in diameter and 5 mm in height). The compression rate was 1 mm / min. Each test was repeated at least three times. For the cyclic compression test, the cylindrical hydrogel (10 mm in diameter and 5 mm in height) was cyclically compressed to 50% strain and returned to the initial height. See the results. Figure 5 As can be seen from the figure, as the proportion of aldehyde groups in the hydrogel system increases, the adhesion strength between the hydrogel and the pig skin also increases, with the highest adhesion strength reaching 58.7 kPa. In addition, the hydrogel can adhere tightly to various tissues, including the liver, heart, and kidney, and achieve firm incision closure.

[0070] According to the method of the present invention, two different colors of hydrogel tissue adhesives were prepared using dyes, the hydrogels were cut along the symmetry line with a sterile scalpel, and then the two different colors of hydrogels were aligned along the incision. After a period of time, their self-healing properties were observed by stretching. The results are shown in Figure 6 ,As can be seen from the figure, due to the reversibility of the Schiff base bond formed between the aldehyde group and the amino group, the hydrogel can maintain its integrity under gravity and can be stretched like a complete hydrogel.

[0071] A rotational rheometer was used to evaluate the rheological properties of the hydrogel at room temperature. The prepared hydrogel tissue adhesive samples were placed on the rotary rheometer frustum, and the injectability of the PEG-EPL-CF hydrogel was tested by strain sweep rheology. In addition, the self-healing properties of the hydrogel were evaluated at the microscopic level by step strain rheology testing between 5% and 500% strain. The pre-gel solution was strain swept to verify the linear response. The gel point was determined as the time when the torsional modulus (G′) exceeded the loss modulus (G″). Oscillatory rheological measurements were performed under linear viscoelastic conditions at 37°C. The results are shown in Figure 7 As can be seen from the figure, when a strain of 318% is applied to the hydrogel tissue adhesive, its internal network collapses and appears as a viscous liquid, indicating that the hydrogel tissue adhesive of the present invention is injectable.

[0072] To determine the in vitro cytocompatibility of the hydrogel tissue adhesive, the hydrogel was co-cultured with cell culture medium for 12 h and then 5 A 100 μL L929 cell suspension (100 μL) was added to a 96-well plate. After 12 hours, the culture medium was aspirated and the hydrogel extract (100 μL) was added and incubated together. After 24, 48, and 72 hours, 10 μL CCK-8 solution was added. After incubation at 37°C in the dark for 2 hours, the absorbance value was recorded at 450 nm using a microplate reader.

[0073]

[0074] OD sample: culture medium containing cells, CCK8, toxic substances; OD control: culture medium containing cells, CCK8, no toxic substances; OD blank: culture medium without cells and toxic substances, CCK8. Results are shown in Figure 8 The results show that the cytotoxicity of the hydrogel tissue adhesive of the present invention is less than 80%, and has good biocompatibility.

[0075] To evaluate the antibacterial properties of the hydrogel tissue adhesive, 100 μL of 10 6 CFU / mL of bacterial solution was applied for use in the inhibition zone experiment. A hydrogel tissue adhesive was placed at the bottom of the test tube and a bacterial suspension (10 6 CFU / mL) was added to the hydrogel tissue adhesive at different volume percentages (10%, 20%, 30%). The test tube was incubated at 37°C for 12 hours. After imaging, the supernatant was transferred to a 96-well plate and the OD value at 600nm was measured using an ELISA reader (the specimen without hydrogel was used as the control group). At the same time, the bacterial solution after co-culture was spread, and the sterilization rate was calculated by the number of colonies after 12 hours. The results are shown in Fig. 9 The results showed that the hydrogel tissue adhesive of the present invention had good antibacterial efficacy, and the MBCs (bactericidal rate > 99.9%) of the hydrogel tissue adhesive against Staphylococcus aureus and Escherichia coli were 20% (v / v) and 30% (v / v), respectively.

[0076] To further investigate the potential of hydrogels for wound closure, a full-thickness skin incision model was used in SD rats (male, incision length = 2 cm). Solutions A and B were mixed and injected into the incision, which was then clamped with fingers for 2 minutes. Surgical sutures, cyanoacrylate glue, and Schiff base single network hydrogel were used for comparison. The results are shown in Fig.10The results showed that the hydrogel tissue adhesive of the present invention has a good effect of bonding wounds. The double network hydrogel group can promote collagen deposition at the wound and heal faster than the Schiff base single network hydrogel group. In contrast, the wounds in the suture group healed slowly and were prone to inflammation, while the cyanoacrylate glue was very brittle and the incisions were easily split secondary.

[0077] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a recombinant collagen hydrogel tissue adhesive for sutureless wound healing, characterized in that: The following steps are involved: Step 1: dissolving carboxymethyl chitosan and polylysine in deionized water at room temperature to obtain a mixed solution, adjusting the pH value of the mixed solution to neutral, and then adding glutamine aminotransferase to the mixed solution to obtain solution A; Step 2, dissolving the recombinant collagen and four-arm-polyethylene glycol-aldehyde in deionized water to obtain solution B; Step 3: Evenly mix the liquid A described in step 1 and the liquid B described in step 2 to obtain a recombinant collagen hydrogel tissue adhesive for sutureless wound healing.

2. The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing according to claim 1, characterized in that: In the mixed solution in step 1, the mass percentage of carboxymethyl chitosan is 8.4% to 12%, and the mass percentage of polylysine is 6.7% to 9.5%.

3. The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing according to claim 1, characterized in that: In step 1, the amount of transglutaminase is adjusted according to the mass of the recombinant collagen, and no less than 0.08 g of transglutaminase is used per gram of recombinant collagen.

4. The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing according to claim 1, characterized in that: The content of recombinant collagen in the B solution in step 2 is adjusted according to the time required for wound healing, the mass percentage of recombinant collagen is not less than 8%, and the mass percentage of four-arm-polyethylene glycol-aldehyde is 8% to 12%.

5. The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing according to claim 4, characterized in that: The mass percentage of the recombinant collagen in the B solution is 8% to 15%.

6. The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing according to claim 1, characterized in that: The recombinant collagen in step 2 is CF-1552 type I recombinant collagen, and the molecular weight of the four-arm polyethylene glycol-aldehyde group is 10,000.

7. The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing according to claim 1, characterized in that: In step 3, the volume ratio of liquid A to liquid B is 1:

1.

8. The method for preparing the recombinant collagen hydrogel tissue adhesive for sutureless wound healing according to claim 1, characterized in that: In step 3, liquid A and liquid B are mixed evenly using a three-way valve and a syringe.

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