A strong collagen bandage material, preparation method and application thereof

Abstract

The present invention belongs to the field of electrochemical deposition technology and biomacromolecule assembly, and relates to a strong and tough collagen material that can be used as a temporary bandage, its preparation method and application. The collagen bandage of the present invention is composed of a collagen membrane with a composite Hofmeister ion having high strength and high toughness. The collagen bandage can recover to its original soft state and dynamically relax. The strong and tough collagen material of the present invention is prepared through two stages of assembly using improved EDP technology and soaking in Hofmeister ions. It can maintain a fixed shape, lift a weight of 1 kg, and will not break or tear when knotted and pulled. When placed in an animal body, it can gradually become soft and degrade, and can be used as a bandage in situations where temporary mechanical support is required, such as arterial ring contraction.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical deposition technology and biomacromolecule assembly, and relates to a strong and tough collagen material that can be used as a temporary bandage, a preparation method and application thereof. Background Art

[0002] Collagen is one of the most abundant proteins in vertebrates. As a key component of the extracellular matrix, collagen is the most widely distributed structural protein in animals and a key target for the comprehensive utilization of biomass resources. Due to its low immunogenicity, high biocompatibility, and ability to promote cell proliferation and wound healing, it has been widely used in various biomedical materials. Collagen has a triple-helical structure that, under the guidance of endogenous signals in the body, can undergo hierarchical and orderly assembly, starting from the triple-helical structure and progressing through the hierarchical assembly of collagen microfibrils, collagen fibrils, and collagen fibers, ultimately forming a tissue structure.

[0003] In vitro, solution casting is often used to process simple collagen into collagen biomaterials. This involves adjusting the collagen solution to neutrality, placing it in a mold, and incubating it at 37°C for a period of time to complete the hierarchical collagen assembly process. However, this method has several drawbacks: 1. It is difficult to process various heterogeneous collagen materials; 2. The process requires several hours, or even overnight, to complete; 3. The collagen fibers within the material are disordered, the collagen density is low, and the material appears opaque (Anelectrochemical fabrication process for the assembly of anisotropically oriented collagen bundles, Biomaterials 29 (2008) 3278–3288; Tenogenic Induction of Human MSCs by Anisotropically Aligned Collagen Biotextiles, Adv. Funct. Mater. 2014, 24, 5762–5770).

[0004] The most commonly used method for processing collagen involves first breaking it down into collagen molecules using solvents, then using this solution to prepare materials. While this method allows for a variety of collagen material forms, it is difficult to perfectly reproduce the aggregated structure of native collagen, resulting in a loss of collagen's inherently excellent mechanical properties. Therefore, exploring new collagen processing pathways to improve its mechanical properties is a challenge facing the comprehensive utilization of collagen at a high value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a bandage with high strength and toughness, which can recover to a soft state and dynamically relax in the body.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned bandage and its application as an artery constriction bandage.

[0007] In one aspect, the present invention provides a bandage comprising a short-range oriented collagen gel film that is capable of self-recovery and dynamic relaxation.

[0008] Optionally, the bandage is composed of a high-strength, high-toughness collagen membrane compounded with Hofmeister ions, and the collagen membrane can gradually recover from a high-strength state to a soft state in the environment. The high strength of high strength and high toughness refers to a breaking strength of not less than 2.0 MPa or a Young's modulus of not less than 9.0 MPa; the high toughness refers to a toughness value of not less than 0.5 MJ / M3.

[0009] Optionally, the environment in which the mechanical state of the collagen membrane changes is in a solution, such as pure water or a body fluid containing salts and (or) enzymes, or it can be a simulated human body fluid in the embodiment. In a preferred embodiment, the collagen membrane can be completely degraded in about 45 hours in water at 37°C containing collagenase (100U / mL). Optionally, the thickness of the bandage is 50-1000μm; preferably, it is 300-800μm. For example, the thickness of the bandage can be 100, 200, 300, 400, 500, 600, 700, 800, 900μm, etc.

[0010] On the other hand, the preparation method of the collagen bandage of the present invention includes two stages: "EDP technology assembly and Hofmeister ion soaking":

[0011] Prepare a collagen solution and use electrochemical deposition technology (Electro-deposition EDP) to prepare a short-range oriented collagen gel film that can be peeled off from the electrode;

[0012] Soak in salt solution for 8-60 hours.

[0013] Salt can precipitate proteins from aqueous solutions, an effect known as the Hofmeister effect. The mechanism is that direct interactions between salt ions and macromolecules and their hydration shells result in the extraction of water from the protein, leading to protein folding and precipitation. Hofmeister ions can be used to enhance the mechanical properties of membranes by strengthening weak interactions within the E-Col network.

[0014] Optionally, the preparation method comprises the following steps:

[0015] S1, preparation of collagen solution: adding acetic acid to the collagen solution to completely dissolve the collagen, adjusting the pH value of the final solution to 1.5-4.0, removing impurities and concentrating to obtain a collagen solution with a concentration in the range of 1-20 mg / ml;

[0016] S2, adding a standard hydrogen peroxide solution to the collagen solution obtained in step S1 to a final volume percentage of 5%-17% in the solution, stirring, removing bubbles, and placing at 0-10°C for later use;

[0017] S3, placing the anode and cathode parallel to each other in an electrolytic cell with the distance between the electrodes controlled to be 0.5-3.0 cm, and slowly adding the collagen solution prepared in step S2 into the electrolytic cell;

[0018] S4, performing electrochemical deposition for 8-60 minutes to obtain a collagen gel film that can be directly peeled off from the cathode;

[0019] S5, soaking the collagen gel membrane prepared in step S4 in the first salt solution for 8-60 hours.

[0020] Optionally, the first salt solution is selected from a solution containing a soluble salt of Hofmeister ions.

[0021] This invention utilizes a two-step process: improved EDP technology for collagen assembly and Hofmeister ion soaking, to produce a high-strength, high-toughness collagen membrane. The E-Col collagen membrane, produced using the improved EDP technology, relies primarily on non-covalent bonding, such as hydrogen bonds and hydrophobic interactions, to assemble. This ensures that the internal structure of the resulting E-Col collagen membrane possesses dynamic remodeling capabilities (due to the ease of breakage and regeneration caused by non-covalent bonds).

[0022] The electrochemically assembled collagen membrane E-Col, when not treated, has a breaking strength of 0.13 MPa, a Young's modulus of 0.32 MPa, and a toughness of 0.19 MJ / M 3 The conventional idea is to use chemical or physical cross-linking to improve the mechanical properties of the material. When 0.5% w / v glutaraldehyde is used to chemically cross-link the collagen membrane, the breaking strength can be increased to 5.22 MPa, the Young's modulus is increased to 11.39 MPa, and the toughness value is 1.27 MJ / M 3 Using 1mg / ml riboflavin UV cross-linked collagen membrane, the breaking strength of the material can be increased to 1.33MPa, the Young's modulus is increased to 1.24MPa, and the toughness value is 0.23MJ / M 3. However, compared with the Hofmeister ion strengthening technology of the present invention, the degree of improvement in its mechanical properties is limited. More importantly, due to the introduction of a large number of covalent bonds inside the collagen membrane, the mechanical properties of the cross-linked collagen E-Col will not have dynamic recovery capabilities. Using the preparation method of the present invention, the fracture strength of the collagen membrane can reach 5.85MPa and above, the maximum Young's modulus can reach 16.42MPa and above, and the maximum toughness value can reach 3.33MJ / M 3 and above.

[0023] Optionally, in step S1, the mass of the added collagen raw material is adjusted so that the concentration of the final collagen solution obtained is 5-10 mg / ml.

[0024] Optionally, the concentration of hydrogen peroxide added in step S2 is 20-150 μl / ml.

[0025] Optionally, the electrode distance in step S3 is 1.0-2.0 cm.

[0026] Optionally, in step S4, electrochemical deposition is performed in a constant current mode or a constant voltage mode.

[0027] Optionally, in step S5, the concentration of the salt solution is 0.1-4M.

[0028] Optionally, in step S1, glacial acetic acid is added dropwise to the collagen solution to promote complete dissolution of the collagen.

[0029] Optionally, in step S2, the centrifugal speed is 6000-8000 rpm / min.

[0030] Optionally, in step S3, both electrodes are perpendicular or parallel to the bottom of the electrolytic cell.

[0031] Optionally, in step S4, the deposition time is 500-2000 seconds.

[0032] Optionally, in step S5, the concentration of the salt solution is 0.5-2 M; or the soaking time is 10-40 hours.

[0033] Different ions have significant differences in their effects on protein solubility. Anions have a more significant effect than cations, and the anions themselves have different hydration effects (i.e., they take away the hydration water of macromolecules). Anions that can usually change protein solubility include CO3 2- 、SO4 2- 、S2O3 2- 、H2PO4 - 、NO3 - 、CH3COO - ClO4 - 、F - 、Cl- Br -、 SCN - , I - However, not all Hofmann ions and anions are suitable for the collagen membrane of the present invention. For example, when the gel membrane is soaked in NaCl solution, the gel membrane remains soft and has no obvious mechanical enhancement; when the gel membrane is soaked in NaI solution, the gel membrane swells; and NaSCN solution directly dissolves the gel membrane. Considering all factors, CO3 2- or SO4 2- The effect will be better and more obvious.

[0034] Optionally, a second salt solution is used instead of the first salt solution, the second salt solution being selected from a salt solution containing CO3 2- or SO4 2- For example, one or more of ammonium sulfate, sodium sulfate or sodium carbonate.

[0035] Optionally, the collagen solution in step S1 is prepared as follows: accurately weigh collagen and ultrapure water in a ratio of 400 mg type I collagen to 40 mL ultrapure water, dropwise add glacial acetic acid and stir thoroughly to completely dissolve the collagen, and adjust the pH value of the final solution to 3.0-4.0; Wcut off =7.0 kDa dialysis bags were placed in an aqueous solution containing glacial acetic acid and dialyzed at 0-5°C for 3 days to remove small molecular impurities; after dialysis, a viscous collagen liquid was obtained.

[0036] Optionally, in step S2, 50-100 μl / ml of hydrogen peroxide is added to the collagen solution described in step S1, and the mixture is stirred evenly. The mixture is centrifuged at 5000-10000 rpm / min at 0-5°C to remove bubbles. The centrifuged collagen solution is stored in an ice-water mixed bath to prevent decomposition of the hydrogen peroxide.

[0037] Optionally, in step S3, a titanium sheet is selected as the cathode and a platinum wire or sheet is selected as the anode, and the collagen solution prepared in step S2 is carefully added to the electrolytic cell slowly to prevent bubbles caused by excessive viscosity of the solution.

[0038] Optionally, in step S4, the electrode is then connected to an electrochemical workstation, a cathode voltage is applied, and constant current deposition is performed with a current density of 5-10 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , deposition time 1000-2000 seconds.

[0039] Optionally, in step S5, the collagen gel membrane peeled from the electrode is placed in 1-2 M Na2CO3 for treatment for 12-24 hours.

[0040] In another aspect, the present invention provides a use of the bandage, wherein the bandage can be used as an artery constriction bandage.

[0041] The collagen bandage prepared by the present invention can dissolve on its own and gradually relax in the body or in simulated body fluids (SBF). In some interventional surgeries (such as pulmonary artery decompression surgery), a medical bandage is tied around the artery to temporarily restrict blood flow and protect vulnerable downstream areas from hypertension. After the operation, the bandage should be able to provide sustained vasoconstriction in the short term, but over time, as heart function gradually recovers, the bandage should gradually relax to allow normal blood flow (the timing depends on clinical details). Therefore, the ideal material for such a medical bandage should have the ability to dynamically relax in an in vivo environment. The E-Col membrane of the present invention can meet this mechanical property requirement because when the E-Col membrane of the present invention is placed in the body, salt can gradually seep out of the collagen fiber network, and the mechanical strength of the E-Col will gradually decrease, thereby weakening the constriction effect on the implant site.

[0042] Simulated body fluid SBF is a liquid that simulates the composition and pH of human body fluids. In a preferred embodiment of the present invention, a product of Guangzhou Yazhi Biotechnology Co., Ltd. is used, with the product number PH1820.

[0043] Optionally, the bandage is used as an artery constriction bandage in the following manner:

[0044] Identify the location where the artery undergoes circumferential constriction;

[0045] The collagen bandage of the present invention is passed around the position where the artery is to be constricted, and a sliding hydrogel knot is formed;

[0046] Adjust the ring contraction and remove any excess bandage.

[0047] The sliding hydrogel knot can be tied by taking one end of the hydrogel band as an axis, tying a surgical knot around the axis at the other end, passing the axis through the center of the surgical knot to form a basic knot, and then tightening the knot.

[0048] The present invention uses a first salt solution ion soaking method to deprive the bound water inside the collagen through strong hydration, thereby creating a hydrophobic microenvironment inside the collagen, which can enhance the H bonds and hydrophobic interactions between the collagen microfibers (water will interfere with H bonds and hydrophobic interactions), thereby greatly improving the mechanical strength and toughness of the collagen membrane. Experiments have shown that the strong and tough collagen material of the present invention can maintain a fixed shape, lift a weight of 1 kg, and will not break or tear when knotted and pulled. In the in vivo environment, with the dissolution of Hofmeister salt, the mechanical properties of the collagen membrane will decrease, and the restrictive effect on blood flow will be lost, and it will eventually be degraded by collagenase in the body. Therefore, it can be used as an in vivo "arterial ring constriction" bandage to provide a temporary effect of restricting blood flow rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a collagen gel membrane.

[0050] Among them, a layer of collagen gel film appeared on the cathode, such as Figure 1 As shown in (a), Figure 1 Middle (b) shows that the E-Col collagen material has a very uniform appearance and is highly transparent in both dry and wet states.

[0051] Figure 2 This is a test diagram of the mechanical enhancement phenomenon of the E-Col membrane based on the Hofmeister effect.

[0052] Figure 2 As shown in (a), the E-Col membrane and the S-Col membrane were cut into rectangular strips with a length of 30 mm and a width of 10 mm, and then immersed in a (NH4)2SO4 (2 mol / L) solution for 24 h. Figure 2 (b) shows that the network of E-Col collagen membrane can show obvious hardening phenomenon under the stimulation of the classic Hofmeister salt - ammonium sulfate, and the mechanical properties of the gel membrane are significantly enhanced. Figure 2 Figures (cd) show that after treating the E-Col network with (NH4)2SO4 (2M, 24h), the transparent E-Col gel film can withstand a load of 1 kg while maintaining the network's flexibility. The film can be knotted without breaking. In contrast, S-Col treated with the same method showed less strengthening effect. The treated film could not withstand a load of 500 g and suffered brittle fracture. Figure 2 (e) shows the state of the material after E-Col gel film was immersed in different Hofmeister salts for 24 hours.

[0053] Figure 3 This is a test chart of the change in water content after the E-Col membrane is treated with different Hofmeister salt concentrations.

[0054] E-Col and S-Col membranes were immersed in a series of (NH₄)₂SO₄ solutions with varying concentrations at room temperature for 24 hours. The water content of the gels was then measured. The water content of both gels decreased to some extent after immersion, with the decrease becoming more pronounced with increasing (NH₄)₂SO₄ concentrations. No significant difference in water content was observed between the two collagen membranes after treatment.

[0055] Figure 4 This is a diagram showing the quantitative characterization of the mechanical properties of the E-Col membrane based on the Hofmeister effect.

[0056] The collagen gel film was immersed in ammonium sulfate solution of different concentrations (1M, 2M, 2.5M, 4M) at room temperature for 12 hours to strengthen the hydrophobic and H-bond interactions. The mechanical properties of the collagen film at room temperature were studied using an Electro-Force3200 biodynamic tester. The sample was stretched using a clamp at a strain rate of 10 mm / min. The Young's modulus of the sample (megapascals; MPa) was calculated using the slope of the initial linear region of the stress-strain curve. The integral area of ​​the tensile stress-strain curve (megajoules per cubic meter; MJ / m 3 ) Calculate the toughness of the specimen.

[0057] Figure 4 The qualitative stress-strain curves in (a) show that the mechanical property strengthening effect of the E-Col gel film after (NH4)2SO4 treatment is significantly dependent on the (NH4)2SO4 concentration. The strengthening effect of the same (NH4)2SO4 treatment on the S-Col gel film is much smaller. Figure 4 The effect on Young's modulus is summarized in (b): when both networks are strengthened by 4M(NH4)2SO4 treatment, the modulus of E-Col increases 50-fold, while the modulus of S-Col increases only 6-fold. Figure 4 In (c), 4M (NH4)2SO4 treatment increased the toughness of the E-Col membrane by 16 times, but this treatment had little effect on the toughness enhancement of the S-Col membrane. These results indicate that the toughness of the E-Col network after (NH4)2SO4 treatment is significantly improved compared to the S-Col network, indicating the difference in the mechanical responsiveness of the two collagen gel membranes with different assembly structures to the Hofmeister effect.

[0058] Figure 5 This is a diagram showing the quantitative characterization of the mechanical properties of the E-Col membrane based on the Hofmeister effect.

[0059] The gel film was immersed in sodium carbonate Na2CO3 solutions of different concentrations (1M, 2M, and 2.5M) at room temperature for 12 hours. The tensile properties of the hydrogel at room temperature were then studied using an Electro-Force 3200 biodynamic tester. The tensile rate was set at 10 mm / min, and the stress-strain curve of the collagen film was obtained. Figure 5 As shown in (a), the stress-strain curves of the E-Col gel film after treatment with different concentrations of Na2CO3 indicate that Na2CO3, as a Hofmeister salt with strong hydration ability, can also strengthen the E-Col network. And the effect increases with the increase of salt concentration. Figure 5 As shown in (b), the E-Col gel membrane exhibits a significant strengthening effect after being treated with 2M Na2CO3 for 24 hours. However, after being treated with SBF (simulated body fluid) for 24 hours, the strengthened E-Col gel membrane gradually softens back to its initial soft state, indicating that the strengthening of the E-Col network by Na2CO3 is a reversible process and that the membrane will return to its soft state as the Hofmeister salt ions leach out. This demonstrates that the mechanical strengthening effect of the E-Col membrane based on the Hofmeister effect is reversible.

[0060] Figure 6 A test chart of the mechanically reinforced E-Col membrane used for in vivo arterial constriction.

[0061] Two-month-old New Zealand white rabbits were used as experimental subjects. Their hearts and surgical sites were as follows Figure 6 As shown in (a). E-Col membrane reinforced with Na2CO3 was used as a surgical band to reduce the diameter of the pulmonary artery, as shown in Figure 6 As shown in (b). Figure 6 The cardiac color Doppler ultrasound image in center (c) shows that the surgical band reduced the pulmonary artery diameter from a preoperative diameter of Φ = 0.63 cm to a postoperative diameter of Φ = 0.43 cm, a reduction of approximately 68%. This confirms that E-Col, enhanced with Na2CO3, provides high mechanical strength to significantly constrict the pulmonary artery diameter. The pulmonary artery diameter returned to 75% of its normal diameter (Φ = 0.48 cm) on the first postoperative day and returned to its normal preoperative diameter three days later.

[0062] Figure 7 Yes Figure 6 The arteries of the New Zealand white rabbits involved were examined by Doppler ultrasound to examine blood flow velocity and pressure gradient maps.

[0063] The results showed that the blood flow velocity decreased from 119 cm / s before the operation to 93.1 cm / s after the operation, and the pressure gradient decreased significantly from 6 mmHg before the operation to 3 mmHg after the operation, indicating that the enhanced E-Col band implanted around the artery can significantly constrict the pulmonary artery to achieve the effect of short-term restriction of blood flow velocity and reduction of blood flow pressure. DETAILED DESCRIPTION

[0064] This invention provides a technology for preparing collagen membranes with high-strength and high-toughness mechanical properties, as well as the resulting collagen membranes and their applications. This technology consists of two stages: improved EDP technology for collagen assembly and Hofmeister ion immersion. The collagen membrane material is characterized by: a composition of short-range, ordered, and densely packed collagen microfibers composited with Hofmeister salts; enhanced non-covalent bonds connecting the collagen microfibers; and a characteristic in which the strength and toughness of the collagen membrane increase with increasing Hofmeister salt concentration. With the dissolution of the Hofmeister salts, the mechanical properties of the collagen membrane decrease, and it eventually decomposes within the body. Therefore, it can be used as an in vivo "arterial constriction" bandage, temporarily restricting blood flow.

[0065] The present invention will be further described below with reference to the examples.

[0066] Example 1: Preparation of collagen material I

[0067] (1) Preparation of collagen solution: Accurately weigh 400 mg of type I collagen in 40 mL of ultrapure water, add glacial acetic acid dropwise and stir thoroughly to dissolve the collagen completely. Adjust the pH value of the final solution to 3.5. Place it in a dialysis bag (M Wcut off =7.0 kDa) and placed in a beaker containing 1000 ml of water and 15 ml of glacial acetic acid, and dialyzed at 4°C for 72 hours to remove small molecular impurities. After dialysis, 10 mg / ml of collagen viscous liquid was obtained.

[0068] (2) Add 80 μl / ml of hydrogen peroxide to the collagen solution described in step (1), stir evenly, and centrifuge at 8000 rpm / min at 4°C to remove bubbles. The collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of hydrogen peroxide.

[0069] (3) Select a titanium sheet (cathode) as the working electrode (electrode size 2cm x 3cm) and a platinum wire or platinum sheet (anode) as the counter electrode. Place the two electrodes horizontally and parallel in the electrolytic cell (e.g. Figure 1 In (b), the distance between the electrodes is controlled at 1.5 cm. The collagen solution (concentration of 10 mg / ml) prepared in step (2) is carefully added to the electrolytic cell slowly to prevent bubbles from forming due to the high viscosity of the solution.

[0070] (4) The electrode was then connected to an electrochemical workstation CHI660E, a cathode voltage was applied, and constant current deposition was performed with a current density of 6.67 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , deposition time 800 seconds, the electrode half-reaction that occurred is shown below.

[0071] Anode: 2H2O-4e - →4H + +O2

[0072] Cathode: 4H2O+4e - →4OH - +2H2.

[0073] After the experiment, a layer of collagen gel film appeared on the cathode, such as Figure 1 As shown in (a). The working electrode with the collagen hydrogel membrane was washed several times with ultrapure water, and then the collagen material E-Col was peeled off from the electrode. Both horizontal and vertical electrodes can be used to prepare collagen materials, but it was found that the materials prepared with vertical electrodes tended to be thinner at the top and thicker at the bottom due to gravity. Using horizontal electrodes can avoid this. The E-Col collagen material has a very uniform appearance and is highly transparent in both dry and wet states. Figure 1 As shown in (b).

[0074] Example 2: Preparation of collagen material II

[0075] (1) Preparation of collagen solution: Accurately weigh 800 mg of type I collagen in 40 mL of ultrapure water, add glacial acetic acid dropwise and stir thoroughly to dissolve the collagen completely. Adjust the pH value of the final solution to 3.5. Place it in a dialysis bag (M Wcut off =7.0 kDa) and placed in a beaker containing 1000 ml of water and 15 ml of glacial acetic acid, and dialyzed at 4°C for 72 hours to remove small molecular impurities. After dialysis, 20 mg / ml of collagen viscous liquid was obtained.

[0076] (2) Add 160 μl / ml of hydrogen peroxide to the collagen solution described in step (1), stir evenly, and centrifuge at 8000 rpm / min at 4°C to remove bubbles. The collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of hydrogen peroxide.

[0077] (3) Select a titanium sheet (cathode) as the cathode and a platinum wire or sheet (anode) as the anode. Place the two electrodes horizontally and parallel in the electrolytic cell (e.g. Figure 1In (b), the distance between the electrodes is controlled at 1.5 cm. The collagen solution (concentration of 20 mg / ml) prepared in step (2) is carefully added to the electrolytic cell slowly to prevent bubbles from forming due to the high viscosity of the solution.

[0078] (4) The electrode was then connected to an electrochemical workstation CHI660E, a cathode voltage was applied, and constant current deposition was performed with a current density of 6.67 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , the deposition time was 800 seconds, and a layer of collagen gel film was successfully prepared on the cathode after the experiment.

[0079] Example 3: Preparation of collagen material III

[0080] (1) Preparation of collagen solution: Accurately weigh 40 mg of type I collagen in 40 mL of ultrapure water, add glacial acetic acid dropwise and stir thoroughly to dissolve the collagen completely. Adjust the pH value of the final solution to 3.5. Place it in a dialysis bag (M Wcut off =7.0 kDa) and placed in a beaker containing 1000 ml of water and 15 ml of glacial acetic acid, and dialyzed at 4°C for 72 hours to remove small molecular impurities. After dialysis, 1 mg / ml of collagen liquid was obtained.

[0081] (2) Add 50 μl / ml of hydrogen peroxide to the collagen solution described in step (1), stir evenly, and centrifuge at 8000 rpm / min at 4°C to remove bubbles. The collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of hydrogen peroxide.

[0082] (3) Select a titanium sheet (cathode) as the cathode and a platinum wire or sheet (anode) as the anode. Place the two electrodes horizontally and parallel in the electrolytic cell (e.g. Figure 1 In (b), the distance between the electrodes is controlled at 1.5 cm. The collagen solution (concentration of 1 mg / ml) prepared in step (2) is carefully added to the electrolytic cell slowly to prevent bubbles from forming due to the high viscosity of the solution.

[0083] (4) The electrode was then connected to an electrochemical workstation CHI660E, a cathode voltage was applied, and constant current deposition was performed with a current density of 6.67 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , the deposition time was 800 seconds, and a layer of collagen gel film was successfully prepared on the cathode after the experiment.

[0084] Example 4: Preparation of collagen material IV

[0085] (1) Preparation of collagen solution: Accurately weigh 400 mg of type I collagen in 40 mL of ultrapure water, add glacial acetic acid dropwise and stir thoroughly to dissolve the collagen completely. Adjust the pH value of the final solution to 2.0. Place it in a dialysis bag (M Wcut off =7.0 kDa) and placed in a beaker containing 1000 ml of water and 200 ml of glacial acetic acid, and dialyzed at 4°C for 72 hours to remove small molecular impurities. After dialysis, 10 mg / ml of collagen liquid was obtained.

[0086] (2) Add 80 μl / ml of hydrogen peroxide to the collagen solution described in step (1), stir evenly, and centrifuge at 8000 rpm / min at 4°C to remove bubbles. The collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of hydrogen peroxide.

[0087] (3) Select a titanium sheet (cathode) as the cathode and a platinum wire or sheet (anode) as the anode. Place the two electrodes horizontally and parallel in the electrolytic cell (e.g. Figure 1 In (b), the distance between the electrodes is controlled at 1.5 cm. The collagen solution (concentration of 10 mg / ml) prepared in step (2) is carefully added to the electrolytic cell slowly to prevent bubbles from forming due to the high viscosity of the solution.

[0088] (4) The electrode was then connected to an electrochemical workstation CHI660E, a cathode voltage was applied, and constant current deposition was performed with a current density of 6.67 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , the deposition time was 800 seconds, and a layer of collagen gel film was successfully prepared on the cathode after the experiment.

[0089] Example 5: Preparation of collagen material V

[0090] (1) Preparation of collagen solution: Accurately weigh 400 mg of type I collagen in 40 mL of ultrapure water, add glacial acetic acid dropwise and stir thoroughly to dissolve the collagen completely. Adjust the pH value of the final solution to 4.0. Place it in a dialysis bag (M Wcut off =7.0 kDa) and placed in a beaker containing 1000 ml of water and 20 μl of glacial acetic acid, and dialyzed at 4°C for 72 hours to remove small molecular impurities. After dialysis, 10 mg / ml of collagen liquid was obtained.

[0091] (2) Add 80 μl / ml of hydrogen peroxide to the collagen solution described in step (1), stir evenly, and centrifuge at 8000 rpm / min at 4°C to remove bubbles. The collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of hydrogen peroxide.

[0092] (3) Select a titanium sheet (cathode) as the cathode (electrode size 2cm x 3cm) and a platinum wire or platinum sheet (anode) as the anode. Place the two electrodes horizontally and parallel in the electrolytic cell (e.g. Figure 1 In (b), the distance between the electrodes is controlled at 1.5 cm. The collagen solution (concentration of 10 mg / ml) prepared in step (2) is carefully added to the electrolytic cell slowly to prevent bubbles from forming due to the high viscosity of the solution.

[0093] (4) The electrode was then connected to an electrochemical workstation CHI660E, a cathode voltage was applied, and constant current deposition was performed with a current density of 6.67 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , the deposition time was 800 seconds, and a layer of collagen gel film was successfully prepared on the cathode after the experiment.

[0094] Example 6: Preparation of collagen material VI

[0095] (1) Preparation of collagen solution: Accurately weigh 400 mg of type I collagen in 40 mL of ultrapure water, add glacial acetic acid dropwise and stir thoroughly to dissolve the collagen completely. Adjust the pH value of the final solution to 3.5. Place it in a dialysis bag (M Wcut off =7.0 kDa) and placed in a beaker containing 1000 ml of water and 15 ml of glacial acetic acid, and dialyzed at 4°C for 72 hours to remove small molecular impurities. After dialysis, 10 mg / ml of collagen liquid was obtained.

[0096] (2) Add 50 μl / ml of hydrogen peroxide to the collagen solution described in step (1), stir evenly, and centrifuge at 8000 rpm / min at 4°C to remove bubbles. The collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of hydrogen peroxide.

[0097] (3) Select a titanium sheet (cathode) as the cathode and a platinum wire or sheet (anode) as the anode. Place the two electrodes horizontally and parallel in the electrolytic cell (e.g. Figure 1 In (b), the distance between the electrodes is controlled at 1.5 cm. The collagen solution (concentration of 10 mg / ml) prepared in step (2) is carefully added to the electrolytic cell slowly to prevent bubbles from forming due to the high viscosity of the solution.

[0098] (4) The electrode was then connected to an electrochemical workstation CHI660E, a cathode voltage was applied, and constant current deposition was performed with a current density of 6.67 mA / cm 2 , the voltage range is 1-1.5V / cm 2, the deposition time was 800 seconds, and a layer of collagen gel film was successfully prepared on the cathode after the experiment.

[0099] Example 7: Preparation of collagen material VII

[0100] (1) Preparation of collagen solution: Accurately weigh 400 mg of type I collagen in 40 mL of ultrapure water, add glacial acetic acid dropwise and stir thoroughly to dissolve the collagen completely. Adjust the pH value of the final solution to 3.5. Place it in a dialysis bag (M Wcut off =7.0 kDa) and placed in a beaker containing 1000 ml of water and 15 ml of glacial acetic acid, and dialyzed at 4°C for 72 hours to remove small molecular impurities. After dialysis, 10 mg / ml of collagen liquid was obtained.

[0101] (2) Add 200 μl / ml of hydrogen peroxide to the collagen solution described in step (1), stir evenly, and centrifuge at 8000 rpm / min at 4°C to remove bubbles. The collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of hydrogen peroxide.

[0102] (3) Select a Pt sheet (cathode) as the cathode (electrode size 2cm x 3cm) and a platinum wire or platinum sheet (anode) as the anode. Place the two electrodes horizontally and parallel in the electrolytic cell (e.g. Figure 1 (b) The distance between the electrodes was controlled at 3.0 cm. The collagen solution (concentration 10 mg / ml) prepared in step (2) was carefully added to the electrolytic cell slowly to prevent bubbles from forming due to the high viscosity of the solution.

[0103] (4) The electrode was then connected to an electrochemical workstation CHI660E, a cathode voltage was applied, and constant current deposition was performed with a current density of 6.67 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , deposition time 500 seconds, and a layer of collagen gel film was successfully prepared on the cathode after the experiment.

[0104] Example 8: Preparation Example VIII of Collagen Material

[0105] (1) Preparation of collagen solution: Accurately weigh 400 mg of type I collagen in 40 mL of ultrapure water, add glacial acetic acid dropwise and stir thoroughly to dissolve the collagen completely. Adjust the pH value of the final solution to 3.5. Place it in a dialysis bag (M Wcut off =7.0 kDa) and placed in a beaker containing 1000 ml of water and 15 ml of glacial acetic acid, and dialyzed at 4°C for 72 hours to remove small molecular impurities. After dialysis, 10 mg / ml of collagen liquid was obtained.

[0106] (2) Add 200 μl / ml of hydrogen peroxide to the collagen solution described in step (1), stir evenly, and centrifuge at 8000 rpm / min at 4°C to remove bubbles. The collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of hydrogen peroxide.

[0107] (3) Select a Pt sheet (cathode) as the cathode (electrode size 2cm x 3cm) and a platinum wire or platinum sheet (anode) as the anode. Place the two electrodes horizontally and parallel in the electrolytic cell (e.g. Figure 1 (b) The distance between the electrodes was controlled at 3.0 cm. The collagen solution (concentration 10 mg / ml) prepared in step (2) was carefully added to the electrolytic cell slowly to prevent bubbles from forming due to the high viscosity of the solution.

[0108] (4) The electrode was then connected to an electrochemical workstation CHI660E, a cathode voltage was applied, and constant current deposition was performed with a current density of 6.67 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , the deposition time was 3000 seconds, and a layer of collagen gel film was successfully prepared on the cathode after the experiment.

[0109] Example 9: Mechanical enhancement of E-Col membrane based on Hofmeister effect

[0110] E-Col was prepared using the same method as in Example 1. By controlling the current intensity and application time, a gel-like E-Col with a thickness of approximately 500 μm was obtained. For comparison, the dialyzed collagen solution was simultaneously prepared into a collagen membrane, S-Col, using a solution method. The acidic collagen solution (5 mg / mL; pH = 3.5) was adjusted to a neutral pH = 7.2 with 0.5 M NaOH and then cast into a circular film culture dish (the collagen content per unit area was the same as the collagen mass per unit area of ​​the EDP assembly). The membrane was incubated at 37°C for 12 hours to achieve complete gelation. The gel was then dehydrated at room temperature for 48 hours, forming a milky white, translucent gel membrane, S-Col, with a thickness of approximately 500 μm.

[0111] The E-Col and S-Col films were cut into rectangular strips with a length of 30 mm and a width of 10 mm, and then immersed in a (NH4)2SO4 (2 mol / L) solution for 24 h (as shown in Figure 2). Figure 2 As shown in (a) in the figure, the changes in the mechanical properties of the gel film were observed. Figure 2 As shown in (b), the network of the E-Col collagen membrane can show obvious hardening phenomenon under the stimulation of the classic Hofmeister salt - ammonium sulfate, and the mechanical properties of the gel membrane are significantly enhanced.

[0112] After studying the mechanical strengthening effect of (NH4)2SO4 on E-Col gel film, the solution-assembled S-Col gel film was selected as a control to explore the differences in the Hofmeister mechanical strengthening induced by (NH4)2SO4 with different assembly structures. Figure 2 As shown in (c-d), after treating the E-Col network with (NH4)2SO4 (2M, 24h), the transparent E-Col gel film can withstand a load of 1 kg while maintaining the network's flexibility. The film can be knotted without breaking. In contrast, S-Col treated with the same method showed less strengthening effect. The treated film could not withstand a load of 500 g and suffered brittle fracture.

[0113] In order to study the effect of different Hofmann ions on the mechanical properties of E-Col, five different salts were selected to soak E-Col: CO3 2- 、SO4 2- 、Cl - 、SCN - , I - .like Figure 2 As shown in (e), immersed in CO3 2- or SO4 2- The mechanical strength of the membrane is significantly enhanced, while the gel membrane will swell when soaked in NaI solution; NaSCN solution will directly dissolve the gel membrane.

[0114] Example 10: Changes in water content after treatment of E-Col membrane with different Hofmeister salt concentrations (dehydration effect)

[0115] The collagen membranes (E-Col membrane and S-Col membrane) prepared in Example 9 were then immersed in a series of (NH4)2SO4 solutions with different concentrations (concentration gradient: 0M, 1M, 2M, 2.5M, 4M) at room temperature for 24 h, and the water content of the gel after immersion was then tested.

[0116] like Figure 3 As shown in the figure, the water content of both types of gels decreased to a certain extent after immersion, and decreased significantly with the increase of (NH4)2SO4 concentration. There was no significant difference in the water content of the two types of collagen membranes after treatment.

[0117] Example 11: Quantitative Characterization of Mechanical Properties of E-Col Membrane Based on the Hofmeister Effect

[0118] E-Col and S-Col collagen membranes were prepared using the same method as in Example 9. The collagen gel films (10 mm × 0.5 mm × 30 mm) were immersed in ammonium sulfate solutions of different concentrations (1 M, 2 M, 2.5 M, 4 M) at room temperature for 12 h to strengthen the hydrophobic and H-bond interactions. The mechanical properties of the collagen membranes at room temperature were studied using an Electro-Force 3200 biodynamic tester. The sample was stretched using a clamp at a strain rate of 10 mm / min. The Young's modulus (megapascals; MPa) of the sample was calculated using the slope of the initial linear region of the stress-strain curve. The integral area of ​​the tensile stress-strain curve (megajoules per cubic meter; MJ / m 3 ) Calculate the toughness of the specimen.

[0119] Figure 4 The qualitative stress-strain curves in (a) show that the mechanical property strengthening effect of the E-Col gel film after (NH4)2SO4 treatment is significantly dependent on the (NH4)2SO4 concentration. The strengthening effect of the same (NH4)2SO4 treatment on the S-Col gel film is much smaller. Figure 4 The effect on Young's modulus is summarized in (b): when both networks are strengthened by 4M(NH4)2SO4 treatment, the modulus of E-Col increases 50-fold, while the modulus of S-Col increases only 6-fold. Figure 4 The effect on toughness is summarized in (c): 4M (NH4)2SO4 treatment increased the toughness of the E-Col membrane by 16 times, but this treatment had little effect on the toughness of the S-Col membrane. These results indicate that the toughness of the E-Col network after (NH4)2SO4 treatment is significantly improved compared to the S-Col network, indicating the difference in the mechanical responsiveness of the two collagen gel membranes with different assembly structures to the Hofmeister effect.

[0120] Example 12: Quantitative Characterization of Mechanical Properties of E-Col Membrane Based on the Hofmeister Effect

[0121] E-Col films were prepared using the same method as in Example 9. Thin gel films (10 mm × 0.5 mm × 30 mm) were immersed in sodium carbonate NaCO solutions of varying concentrations (1 M, 2 M, and 2.5 M) at room temperature for 12 hours. The tensile properties of the hydrogels at room temperature were then studied using an Electro-Force 3200 biodynamic tester.

[0122] The stretching rate was set at 10 mm / min, and the stress-strain curve of the collagen membrane was obtained. Figure 5As shown in (a), the stress-strain curves of E-Col gel films after treatment with different concentrations of Na2CO3 indicate that Na2CO3, as a Hofmeister salt with strong hydration capacity, can also strengthen the E-Col network. The effect increases with increasing salt concentration.

[0123] The same method as in Example 2 was used to prepare the E-Col membrane. The gel film (10 mm × 0.5 mm × 30 mm) was immersed in a 2M Na2CO3 solution at room temperature for 12 h. Figure 5 As shown in (b), the E-Col gel film exhibits a significant strengthening effect after being treated with 2M Na2CO3 for 24 hours. However, after the strengthened E-Col gel film is treated with SBF (Guangzhou Yazhi Biotechnology Co., Ltd., PH1820) for 24 hours, it gradually softens and returns to its initial soft state, indicating that the strengthening of the E-Col network by Na2CO3 is a reversible process and will return to its soft state as the Hofmeister salt ions are leached out.

[0124] This indicates that the mechanical enhancement effect of E-Col membrane based on the Hofmeister effect is reversible.

[0125] Example 13: In vivo arterial ringing application of mechanically reinforced E-Col membrane

[0126] (1) Construction of pulmonary artery ring contraction experimental model

[0127] Two-month-old New Zealand white rabbits were treated with pentobarbital 40-50 mg kg -1 Anesthesia was performed by ear vein injection. Before the pulmonary artery decompression surgery, the pulmonary artery was observed with color Doppler ultrasound and the pulmonary artery diameter was recorded. Then, the blood velocity (VEL) and pressure gradient (PG) of each rabbit were detected. Next, the preparatory work was completed in advance, such as endotracheal intubation, respiratory support, inhalation anesthesia, and nutrient solution supply. The left ventricle (LV) was exposed after thoracotomy. First, the pulmonary artery was exposed, the ring contraction position was determined, and the electro-assembled collagen microfiber hydrogel strip (2M Na2CO3 enhanced treatment) was passed around the pulmonary artery. A sliding hydrogel knot was tied, and then the degree of ring contraction was adjusted, the gel knot was tightened, and finally the excess gel material was removed. After the operation, the rabbit chest was closed according to routine clinical procedures. The postoperative pulmonary artery constriction diameter, VEL, and PG values ​​were observed to determine the decompression effect. Color Doppler ultrasound was used to observe the recovery of the pulmonary artery diameter 1d and 3d after surgery. Figure 6 shown.

[0128] (2) Postoperative color Doppler ultrasound image results

[0129] The experiment used New Zealand rabbits as an animal model and used E-Col membrane enhanced by Na2CO3 as a surgical band to reduce the diameter of the pulmonary artery. Figure 6 As shown in (b). Figure 6 The cardiac color Doppler ultrasound image in (c) shows that the surgical band reduced the pulmonary artery diameter from a preoperative diameter of φ = 0.63 cm to a postoperative diameter of φ0 = 0.43 cm, a decrease of approximately 68%. This confirms that E-Col reinforced with Na2CO3 can provide high mechanical strength to significantly shrink the pulmonary artery diameter. The pulmonary artery diameter recovered to 75% of its normal diameter (φ1 = 0.48 cm) on the first day after surgery and returned to its normal preoperative diameter three days later. In addition, Doppler ultrasound examination of the arterial vessels showed blood flow velocity and pressure gradient, as shown in Figure 2. Figure 7 The results showed that the blood flow velocity decreased from 119 cm / s before the operation to 93.1 cm / s after the operation, and the pressure gradient decreased significantly from 6 mmHg before the operation to 3 mmHg after the operation, indicating that the enhanced E-Col band implanted around the artery can significantly constrict the pulmonary artery to achieve the effect of short-term blood flow restriction and blood pressure reduction.

[0130] The above descriptions and embodiments are provided to facilitate understanding and application of the present invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these contents and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above descriptions and embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should fall within the scope of protection of the present invention.

Claims

1. A bandage, characterized in that: The bandage is composed of a high-strength and high-toughness short-range oriented collagen gel film, which can restore its soft state and dynamically relax; The high strength mentioned above means a breaking strength of not less than 2.0 MPa or a Young's modulus of not less than 9.0 MPa; The high toughness mentioned above means that the toughness value is not less than 0.5MJ / M 3 ; The preparation method of the bandage comprises the following steps: S1, preparation of collagen solution: adding acetic acid to the collagen solution to completely dissolve the collagen, adjusting the pH value of the final solution to 1.5-4.0, removing impurities and concentrating to obtain a collagen solution with a concentration in the range of 1-20 mg / ml; S2, adding a standard hydrogen peroxide solution to the collagen solution obtained in step S1 to a final volume percentage of 5%-17% in the solution, stirring, removing bubbles, and placing at 0-10°C for later use; S3, placing the anode and cathode parallel to each other in an electrolytic cell with the distance between the electrodes controlled to be 0.5-3.0 cm, and slowly adding the collagen solution prepared in step S2 into the electrolytic cell; S4, performing electrochemical deposition for 8-60 minutes to obtain a collagen gel film that can be directly peeled off from the cathode; S5, soaking the collagen gel membrane prepared in step S4 in the first salt solution for 8-60 hours; The first salt solution is selected from a salt solution containing CO3 2- or SO4 2- of soluble salts.

2. The bandage according to claim 1, wherein The thickness of the bandage is 50-1000 μm; or, the bandage is composed of a short-range oriented collagen gel film with composite Hofmeister ions.

3. The method for preparing the bandage according to claim 1, wherein: The following steps are involved: S1, preparation of collagen solution: adding acetic acid to the collagen solution to completely dissolve the collagen, adjusting the pH value of the final solution to 1.5-4.0, removing impurities and concentrating to obtain a collagen solution with a concentration in the range of 1-20 mg / ml; S2, adding a standard hydrogen peroxide solution to the collagen solution obtained in step S1 to a final volume percentage of 5%-17% in the solution, stirring, removing bubbles, and placing at 0-10°C for later use; S3, placing the anode and cathode parallel to each other in an electrolytic cell with the distance between the electrodes controlled to be 0.5-3.0 cm, and slowly adding the collagen solution prepared in step S2 into the electrolytic cell; S4, performing electrochemical deposition for 8-60 minutes to obtain a collagen gel film that can be directly peeled off from the cathode; S5, soaking the collagen gel membrane prepared in step S4 in the first salt solution for 8-60 hours; The first salt solution is selected from a salt solution containing CO3 2- or SO4 2- of soluble salts.

4. The preparation method according to claim 3, characterized in that In step S1, the mass of the added collagen raw material is adjusted so that the concentration of the final collagen solution is 5-10 mg / ml; Alternatively, the concentration of hydrogen peroxide added in step S2 is 20-150 μl / ml; Alternatively, the electrode distance in step S3 is 1.0-2.0 cm; Alternatively, in step S4, electrochemical deposition is performed in a constant current mode or a constant voltage mode; Alternatively, in step S5, the concentration of the first salt solution is 0.1-4M.

5. The preparation method according to claim 3, characterized in that In step S1, glacial acetic acid is added dropwise to the collagen solution to promote complete dissolution of the collagen; or, In step S2, the centrifugal speed is 6000-8000 rpm / min; or, In step S3, both electrodes are perpendicular or parallel to the bottom of the electrolytic cell; or, In step S4, the deposition time is 500-2000 seconds; In step S5, the concentration of the first salt solution is 0.5-2M; or the soaking time is 10-40 hours.

6. The preparation method according to claim 3, characterized in that The collagen solution in step S1 is prepared as follows: accurately weigh collagen and ultrapure water in a ratio of 400 mg type I collagen to 40 mL ultrapure water, dropwise add glacial acetic acid and stir thoroughly to completely dissolve the collagen, and adjust the pH of the final solution to 3.0-4.0; place the solution in a dialysis bag with a MW cutoff of 7.0 kDa and place it in an aqueous solution containing glacial acetic acid, and dialyze at 0-5°C for 3 days to remove small molecular impurities; after dialysis, a viscous collagen liquid is obtained; Alternatively, in step S2, 50-100 μl / ml of hydrogen peroxide is added to the collagen solution described in step S1, and the mixture is stirred evenly. The mixture is centrifuged at 5000-10000 rpm / min at 0-5°C to remove bubbles, and the collagen solution after centrifugation is stored in an ice-water mixed bath to prevent decomposition of the hydrogen peroxide. Alternatively, in step S3, a titanium sheet is selected as the cathode and a platinum wire or sheet is selected as the anode. The collagen solution prepared in step S2 is carefully added to the electrolytic cell slowly to prevent bubbles from being introduced due to the high viscosity of the solution. Alternatively, in step S4, the electrode is then connected to an electrochemical workstation, a cathode voltage is applied, and constant current deposition is performed with a current density of 5-10 mA / cm 2 , the voltage range is 1-1.5V / cm 2 , deposition time 1000-2000 seconds; Alternatively, in step S5, the collagen gel membrane peeled from the electrode is placed in 1-2 M Na2CO3 for treatment for 12-24 hours.

7. Use of the bandage according to claim 1, characterized in that: The bandage is used as an arterial cirriform bandage.

8. The use according to claim 7, characterized in that The method when the bandage is used as an artery ring bandage is as follows: Identify the location where the artery undergoes circumferential constriction; The bandage of claim 1 is passed around the location of the artery to be subjected to ring constriction, and a sliding hydrogel knot is tied; Adjust the ring contraction and remove any excess bandage.

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