An electric field periodic regulation biomimetic collagen membrane material, a preparation method and application thereof

Abstract

The application provides a kind of electric field period regulation's biomimetic collagen film material and its preparation method and application, it is related to organic polymer compound processing field, also it is related to medical material technical field.Collagen solution is deposited on electrode in the form of electrolysis in this application, and collagen is formed into film, surfactant and ionic strength regulator are added in collagen solution, surfactant stabilizes collagen dispersion, and film is easy to peel off; Moderate ionic strength regulator is compatible with pH value, reduces film-electrode electrostatic adsorption, and enhances film strength; Further, high concentration collagen solution is concentrated by specific hollow fiber ultrafiltration membrane to electrolyte, collagen is formed into uniform film, and it is ensured that collagen film obtained in continuous film forming process is uniform; By periodically switching electrode polarity, negative electric collagen is deposited into film, and positive switching is used to effectively remove film by electrostatic repulsion; Interface regulator and physical auxiliary are combined to realize deposition-film removal continuity; And the obtained collagen film is dense and uniform, and has high strength.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of organic polymer compound processing and the field of medical material technology, in particular to a kind of biomimetic collagen membrane material of electric field periodic regulation and preparation method and application thereof. BACKGROUND

[0002] Collagen electrolytic film formation is a technology based on the directional migration of collagen molecules driven by electric field and the deposition of thin film on the electrode surface. Collagen molecules are positively charged in acidic solution and migrate towards the cathode under the action of direct current or pulse electric field. When the local pH rises, charge neutralization occurs, forming a fiber network structure. Compared with traditional casting method, electrolytic film formation has the advantages of fast speed (minute level), controllable fiber arrangement, strong film-substrate bonding force, and can better maintain the triple helix structure of collagen, making it have wide application potential in the fields of tissue engineering, drug release and wound repair.

[0003] Currently, there are still several key problems in collagen electrolytic film formation technology: (1) uneven electric field distribution leads to inconsistent film thickness, affecting mechanical properties; (2) as the electrolysis proceeds, the collagen concentration in the electrolyte decreases, leading to poor consistency of the collagen film; (3) after film formation, manual methods are used for demolding, which can easily cause damage to the collagen film and structure; (4) the deposition process lacks real-time control, and collagen is prone to denaturation due to excessive local pH or temperature fluctuations; (5) traditional chemical cross-linking (such as glutaraldehyde) may introduce cytotoxicity, while physical cross-linking (such as ultraviolet light) usually needs to be performed separately after film formation, leading to process complexity.

[0004] Chinese patent application 202311694018.1 discloses a preparation method of collagen-chitosan drainage tube. The patent adds chitosan to the collagen solution and uses hydrogen peroxide as the electrolyte for electrolysis. After electrolysis, glutaraldehyde chemical cross-linking is performed on the working electrode, and then the working electrode is extracted after multiple rehydration and drying to obtain the final material. In this preparation process, the electrode is separated from the drainage tube only in the final step, which cannot be continuously utilized. Moreover, the introduction of chemical cross-linking agent to enhance the strength of the material during the preparation process increases its cytotoxicity. Chinese patent application 202311545675.X discloses a preparation method of collagen membrane for guided tissue regeneration. The collagen membrane is prepared by electrolysis of collagen deposition solution under low temperature and high voltage, followed by cross-linking under ultraviolet light, and then manually peeling off to obtain the collagen membrane material. The collagen membrane obtained by this patent is asymmetric and non-uniform, and the ultraviolet cross-linking time is as long as 300 minutes, which is a complex process. Manual removal of the membrane material is difficult. Moreover, this process can only increase the area of the electrode sheet to improve productivity. The concentration of collagen deposition solution after electrolysis decreases, which cannot be electrolyzed again for preparation, thus making it difficult to expand production. SUMMARY

[0005] In order to overcome the problems of non-uniformity of collagen electrolytic deposition film, difficulty in demolding after film formation, non-uniformity of collagen film caused by the decrease of collagen concentration in electrolyte during film formation, and cross-linking after film formation to increase the strength of the film, the application provides a biomimetic collagen film material with periodic electric field regulation and a preparation method and application thereof.

[0006] In the first aspect, the application provides a preparation method of a biomimetic collagen film material with periodic electric field regulation, comprising the following preparation steps:

[0007] Preparation of electrolytic collagen solution: take a collagen solution with a pH value of 2.5-5, add a surfactant to the solution system to make the final concentration of the surfactant 0.01-0.1wt%, and add a 50-150 mM ion strength regulator, and then mix to obtain an electrolytic collagen solution, wherein the ion strength regulator is a hydrolyzable ionic halide;

[0008] Collagen film deposition: placing positive and negative electrodes with a coating on the surface in the electrolytic collagen solution for collagen film deposition, wherein the coating is a hydrophilic coating or a conductive lubricating layer;

[0009] Electrolytic demolding: then switching the positive and negative electrodes for demolding to obtain a collagen film;

[0010] Hollow fiber ultrafiltration: performing hollow fiber ultrafiltration concentration on the electrolyte;

[0011] Continuous film formation: after the ultrafiltration and electrolytic demolding are completed synchronously, supplementing the collagen solution, repeating the electrolytic film formation step, realizing continuous film formation, and obtaining the biomimetic collagen film material after cleaning and drying.

[0012] The preparation method of the application realizes the directional migration and controllable film formation of collagen molecules by using a hollow fiber ultrafiltration membrane to regulate the concentration of the electrolyte in real time, combining the periodic electrode polarity switching technology (negative pressure deposition / positive pressure demolding), modifying the electrode-collagen interface by composite surfactants, and synergistically acting with metal ions to reduce the binding energy of the collagen film-electrode while improving the cohesion of the film; the electrode gradient rotation system (low-speed rotation in the deposition stage / high-speed shearing in the demolding stage) is innovatively designed to realize efficient demolding of the negative electrode; the obtained collagen film has the characteristics of narrow pore size distribution and high tensile strength, and is suitable for medical scenarios such as wound repair, tissue hemostasis, and soft tissue regeneration; the technology breaks through the limitations of traditional intermittent production, realizes continuous film formation at a minute level, and provides a new strategy for the industrialized production of biomedical materials.

[0013] The ionic strength regulator promotes collagen aggregation by charge shielding (weakening electrostatic repulsion), but can cause excessive aggregation due to salt effect; the surfactant inhibits aggregation by steric hindrance, and the surfactant and the ionic strength regulator cooperatively balance the solubility-aggregation state of collagen, the use of the ionic strength regulator alone is easy to precipitate, the use of the surfactant alone is easy to over-disperse, and both will make the collagen layer deposition effect poor.

[0014] Further, the pH value of the collagen solution is 2.5-3.5, and the surfactant is a non-ionic surfactant.

[0015] Optionally, the surfactant includes but is not limited to Tween 20, Tween 80, Triton X-100.

[0016] Optionally, the ionic strength regulator includes but is not limited to NaCl, KCl, CaCl 2。

[0017] Further, the concentration of the collagen solution is 6-8 mg / mL.

[0018] Further, the material of the positive and negative electrodes is platinum, and the electrode spacing is 5-6 cm.

[0019] Further, the hydrophilic coating includes a polyethylene glycol coating, and the conductive lubricating layer includes a graphene coating.

[0020] Further, in the collagen membrane deposition step, the rotation speed of the positive and negative electrodes is adjusted to 5-15 rpm.

[0021] Preferably, in the collagen membrane deposition step, the rotation speed of the positive and negative electrodes is adjusted to 5-10 rpm.

[0022] Further, in the collagen membrane deposition step, the set voltage in the constant current mode is 2-3 V.

[0023] Further, in the electrolytic membrane stripping step, the electrode rotation speed is adjusted to 60-80 rpm.

[0024] Further, in the electrolytic membrane stripping step, the set voltage in the constant current mode is 2-3 V, and the electrolytic membrane stripping is performed for 18-40 s.

[0025] Further, in the hollow fiber ultrafiltration step, the molecular weight cut-off of the hollow fiber is 5-30 kDa.

[0026] Further, in the hollow fiber ultrafiltration step, the molecular weight cut-off of the hollow fiber is 5-10 kDa, the effluent pressure is set to 0.05-0.15 bar, and the ultrafiltration is performed for 18-40 s.

[0027] In a second aspect, the application provides a biomimetic collagen membrane material prepared by the method described in the application.

[0028] In a third aspect, the application provides use of the biomimetic collagen membrane material in preparation of a medical wound repair, tissue hemostasis and soft tissue regeneration material.

[0029] Advantages:

[0030] 1. In the application, collagen solution is deposited on the electrode in the form of collagen film by electrolysis, a surfactant and an ionic strength regulator are added to the collagen solution, the surfactant stabilizes the collagen dispersion and enhances the hydrophilicity to introduce charge repulsion, so that the film is easy to peel off; the ionic strength regulator adjusts the pH value of the collagen solution, the moderate ionic strength of which shields the collagen charge, reduces the electrostatic adsorption of the film-electrode, and ensures that the collagen is driven at a relatively uniform speed under the action of the electric field, thereby enhancing the film strength; further, the high-concentration collagen solution is concentrated by ultrafiltration of the electrolyte through a specific hollow fiber ultrafiltration membrane, so that the collagen concentration in the electrolyte remains unchanged during the electrolysis process, the collagen film is uniform, and the collagen film obtained by the continuous film forming process is uniform; by periodically switching the electrode polarity, negative collagen is deposited to form a film, and the film is effectively removed by electrostatic repulsion after switching the positive electrode; the deposition-film removal process is continuous by combining the interface regulator with physical assistance; the obtained collagen film is dense and uniform, the collagen film strength is improved, and the collagen film pore structure is uniform and controllable, and the specific surface area is high.

[0031] 2. Further, the electrode polarity is specially modified to reduce the adsorption of the electrode to the film, and the charge repulsion of the surfactant in the film layer is compatible, so that the film is easy to peel off, continuous and rapid electrolysis film forming is realized, and the electrode is sheared and rotated to effectively remove the formed film.

[0032] 3. Further, the electrode polarity mode is switched in stages and the electrode is slowly rotated during the film forming process to make the film dense and uniform, improve the collagen film strength, and the slow rotation during the film forming process makes the film dense and uniform; the film is quickly removed by shearing force during the film removal process; the electrode is variable-speed rotated during the film forming process, which further improves the film density uniformity, uniformity, strength, realizes the continuous deposition-film removal process, and improves the film quality and the ease of demolding.

[0033] 4. The collagen film obtained by the application has the characteristics of narrow pore size distribution and high tensile strength, and is suitable for medical scenes such as wound repair, tissue hemostasis and soft tissue regeneration; the technology breaks through the limitations of traditional intermittent production, realizes continuous film forming in minutes, and provides a new strategy for the industrialized production of biomedical materials. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1is the electrolyte collagen concentration change chart after continuous ten times of film forming by the method of the present application / embodiments 1-3 and comparative examples 9-11;

[0035] Figure 2 is the tensile strength chart of the fifth collagen membrane obtained by the method of the present application / embodiments 1-3 and comparative examples 9-11. DETAILED DESCRIPTION

[0036] In order to make the technical scheme of the present application clearer, the present application will be further described in detail below in combination with the drawings and specific embodiments.

[0037] Embodiment 1: A biomimetic collagen membrane material periodically regulated by electric field and a preparation method thereof, comprising the following preparation steps:

[0038] Preparation of electrolytic collagen solution: take collagen solution (concentration 6 mg / ml, pH 3.5) 3 L, add Tween 20 to make the final concentration 0.01 wt%, and synchronously add 150 mM NaCl, and after mixing, obtain the electrolytic collagen solution.

[0039] Pre-coating layer of positive and negative electrodes: the positive and negative electrodes in the electrolytic cell are made of platinum, the electrode surface is pre-coated with a hydrophilic coating-polyethylene glycol, the electrode spacing is 5 cm, and the electrode can be adjusted to rotate; 3 L of electrolytic collagen solution is placed in a 3 L electrolytic cell.

[0040] Collagen membrane deposition: the rotation speed of the positive and negative electrodes is set to 5 rpm, the electrolysis is started, the voltage is set to 2.5 V in constant current mode, and the collagen membrane deposition is timed for 3 min.

[0041] Electrolytic membrane stripping: then switch the positive and negative electrodes, set the voltage to 2.5 V in constant current mode, and time the electrolytic membrane stripping for 30 s, while adjusting the electrode rotation speed to 70 rpm, and performing positive voltage stripping of the original negative electrode.

[0042] Hollow fiber ultrafiltration: after cutting the collagen membrane that falls and floats along the busbar, slowly transfer it to physiological saline for standing; at the beginning of electrolytic membrane stripping, simultaneously perform hollow fiber ultrafiltration concentration on the electrolyte in the electrolytic cell, select the molecular weight cut-off of the hollow fiber to be 5 kDa, set the outflow pressure to be 0.10 bar, and the ultrafiltration time to be 30 s.

[0043] Continuous membrane preparation: after the ultrafiltration and electrolytic membrane stripping are simultaneously completed, repeat the electrolytic membrane preparation steps after supplementing the original electrolytic collagen solution to 3 L, to realize continuous membrane preparation; after washing the collected collagen membrane with physiological saline for multiple times, dry to obtain the biomimetic collagen membrane material.

[0044] Embodiment 2: A biomimetic collagen membrane material periodically regulated by electric field and a preparation method thereof, comprising the following preparation steps:

[0045] Preparation of electrolytic collagen solution: Take collagen solution (concentration 8 mg / ml, pH 2.5) 3 L, add Tween 80 to make its final concentration 0.05 wt%, synchronously add 100 mM KCl, after mixing, the electrolytic collagen solution is obtained.

[0046] Pre-coating of positive and negative electrodes: The positive and negative electrodes with platinum as electrode material are placed in the electrolytic cell, the electrode surface is pre-coated with a hydrophilic coating-polyethylene glycol, the electrode spacing is 5 cm, and the electrode can be adjusted to rotate at a speed.

[0047] Collagen membrane deposition: The rotation speed of the positive and negative electrodes is set to 5 rpm, electrolysis is started, the voltage is set to 2 V in constant current mode, and collagen membrane deposition is timed for 3 min.

[0048] Electrolytic membrane stripping: Then switch the positive and negative electrodes, set the voltage to 2.5 V in constant current mode, and time the electrolytic membrane stripping for 30 s, while adjusting the electrode rotation speed to 60 rpm, and performing positive voltage stripping of the original negative electrode.

[0049] Hollow fiber ultrafiltration: The collagen membrane that falls off and floats is cut along the busbar and slowly transferred to physiological saline for standing; at the beginning of electrolytic membrane stripping, the electrolyte in the electrolytic cell is simultaneously subjected to hollow fiber ultrafiltration and concentration, the molecular weight cut-off of the hollow fiber is selected to be 10 kDa, the outflow pressure is set to 0.15 bar, and the ultrafiltration time is 30 s.

[0050] Continuous membrane preparation: After the ultrafiltration and electrolytic membrane stripping are completed synchronously, the initial collagen solution to be electrolyzed is supplemented to 3 L, and the electrolytic membrane preparation steps are repeated to realize continuous membrane preparation; the collected collagen membrane is washed with physiological saline for multiple times and dried to obtain a collagen membrane material.

[0051] Example 3: A biomimetic collagen membrane material periodically regulated by an electric field and a preparation method thereof, comprising the following preparation steps:

[0052] Preparation of electrolytic collagen solution: collagen solution (concentration 7 mg / ml, pH 3) 3 L, add Triton X-100 to make its final concentration 0.1 wt%, synchronously add 150 mM CaCl2, after mixing, the electrolytic collagen solution is obtained.

[0053] Pre-coating of positive and negative electrodes: 3 L of the above solution is placed in a 3 L electrolytic cell, the positive and negative electrodes with platinum as electrode material are placed in the electrolytic cell, the electrode surface is pre-coated with a conductive lubricating layer-graphene, and the electrode spacing is 5 cm.

[0054] Collagen membrane deposition: The rotation speed of the positive and negative electrodes is set to 10 rpm, electrolysis is started, the voltage is set to 3 V in constant current mode, and collagen membrane deposition is timed for 3 min.

[0055] Electrolytic membrane stripping: then switch the positive and negative electrodes, set the voltage to 2.5 V in constant current mode for 30 s, while adjusting the electrode speed to 80 rpm, and the positive voltage of the original negative electrode is stripped.

[0056] Hollow fiber ultrafiltration: the detached and floated collagen membrane is cut along the busbar and slowly transferred to physiological saline for standing. At the beginning of electrolytic membrane stripping, the electrolyte in the electrolytic tank is simultaneously subjected to hollow fiber ultrafiltration concentration, the molecular weight cut-off of the hollow fiber is selected to be 8 kDa, the effluent pressure is set to be 0.05 bar, and the ultrafiltration time is 30 s.

[0057] Continuous membrane preparation: after the ultrafiltration and electrolytic membrane stripping are simultaneously completed, the initial collagen solution to be electrolyzed is supplemented to 3 L, and then the electrolytic membrane preparation step is repeated to realize continuous membrane preparation; the collected collagen membrane is washed with physiological saline for multiple times and dried to obtain a collagen membrane material.

[0058] Comparative Example 1: a kind of biomimetic collagen membrane material and its preparation method controlled by periodic electric field, compared with Example 1, the difference lies in that in the preparation step of the electrolytic collagen solution, the pH of the collagen solution is 6.5.

[0059] Comparative Example 2: a kind of biomimetic collagen membrane material and its preparation method controlled by periodic electric field, compared with Example 1, the difference lies in that in the preparation step of the electrolytic collagen solution, no surfactant is added.

[0060] Comparative Example 3: a kind of biomimetic collagen membrane material and its preparation method controlled by periodic electric field, compared with Example 1, the difference lies in that in the preparation step of the electrolytic collagen solution, no ionic strength regulator is added.

[0061] Comparative Example 4: a kind of biomimetic collagen membrane material and its preparation method controlled by periodic electric field, compared with Example 1, the difference lies in that the positive and negative electrodes are not pre-coated with a coating.

[0062] Comparative Example 5: a kind of biomimetic collagen membrane material and its preparation method controlled by periodic electric field, compared with Example 1, the difference lies in that in the collagen membrane deposition step, the electrode speed is set to 20 rpm.

[0063] Comparative Example 6: a kind of biomimetic collagen membrane material and its preparation method controlled by periodic electric field, compared with Example 1, the difference lies in that in the collagen membrane deposition step, the collagen membrane deposition voltage is 5 V.

[0064] Comparative Example 7: a kind of biomimetic collagen membrane material and its preparation method controlled by periodic electric field, compared with Example 1, the difference lies in that in the electrolytic membrane stripping step, the electrolytic membrane stripping time is set to 15 s.

[0065] Comparative Example 8: A biomimetic collagen membrane material periodically regulated by electric field and a preparation method thereof, which is different from Example 1 in that the rotating speed is set to 40 rpm in the electrolytic membrane separation step.

[0066] Comparative Example 9: A biomimetic collagen membrane material periodically regulated by electric field and a preparation method thereof, which is different from Example 2 in that the molecular weight cut-off of the hollow fiber is selected to be 50 kda in the hollow fiber ultrafiltration.

[0067] Comparative Example 10: A biomimetic collagen membrane material periodically regulated by electric field and a preparation method thereof, which is different from Example 2 in that the outflow pressure of the hollow fiber ultrafiltration is set to 0.5 bar in the hollow fiber ultrafiltration.

[0068] Comparative Example 11: A biomimetic collagen membrane material periodically regulated by electric field and a preparation method thereof, which is different from Example 2 in that the time of the hollow fiber ultrafiltration is shortened to 15 s in the hollow fiber ultrafiltration.

[0069] Performance detection:

[0070] 1. Ultrafiltration concentration parameter determination

[0071] Examples 1, 2, 3 and Comparative Examples 9, 10 and 11: The changes of the concentration of collagen in the electrolyte after each preparation and after the ultrafiltration concentration replenishment in the 10 consecutive preparation processes were determined to judge the influence of the selected hollow fiber ultrafiltration process on the change of the electrolyte concentration in the continuous preparation process and to exclude the influence of the electrolyte concentration fluctuation on the film forming performance. The changes of the collagen concentration in the electrolyte after each film forming of the samples in each group were determined by the hydroxyproline determination method (see Appendix); the changes of the collagen concentration in the electrolyte after the continuous ten times of film forming are shown in Table 1; the test method of the collagen concentration in the electrolyte is as follows: Figure 1

[0072] Principle: The sample is hydrolyzed in a 6M hydrochloric acid solution at 105°C to hydrolyze the hydroxyproline. The hydroxyproline is oxidized by chloramine T to generate an oxide containing a pyrrole ring. The excess chloramine T is destroyed by perchloric acid. The hydroxyproline oxide reacts with p-dimethylaminobenzaldehyde to generate a red compound, which is determined by colorimetry at a wavelength of 560 nm.

[0073] Instrument equipment: air drying oven, balance, ultraviolet visible spectrophotometer

[0074] Reagents: sodium acetate trihydrate, sodium citrate, citric acid monohydrate, sodium hydroxide, isopropyl alcohol, chloramine T, p-dimethylaminobenzaldehyde, perchloric acid (60% or more), L-hydroxyproline standard; all reagents are analytical pure except otherwise specified.

[0075] Preparation of reagents:

[0076] ​1) Hydrochloric acid solution, c(HCl) = 6 mol / L: mix equal volume of extra pure hydrochloric acid and water.

[0077] 2) pH = 6.0 buffer solution: weigh 57 g of sodium acetate trihydrate, 37.5 g of trisodium citrate, 5.5 g of citric acid monohydrate, 385 mL of isopropyl alcohol, add water to 500 mL, adjust pH to 6.0 with citric acid monohydrate, and dilute to 1000 mL with water.

[0078] 3) Chloramine T solution: weigh 3.5 g of chloramine T, dilute to 50 mL with water, and prepare immediately before use.

[0079] 4) Oxidant solution: mix chloramine T solution and pH = 6.0 buffer solution at a ratio of 1:4.

[0080] 5) 60% perchloric acid solution: measure 43 mL of perchloric acid, dilute to 50 mL with water.

[0081] 6) p-Dimethylaminobenzaldehyde solution: weigh 10 g of p-dimethylaminobenzaldehyde, dissolve in 15 mL of 60% perchloric acid solution.

[0082] 7) Color developing agent: measure 15 mL of p-dimethylaminobenzaldehyde solution, dissolve in 65 mL of isopropyl alcohol.

[0083] 8) Sodium hydroxide solution, c(NaOH) = 6 mol / L: weigh 24 g of sodium hydroxide, dilute to 100 mL with water.

[0084] C.4.2 Preparation of L-hydroxyproline control solution

[0085] 9) L-hydroxyproline control stock solution: accurately weigh L-hydroxyproline control, dissolve in water and dilute to a concentration of 20 μg per 1 mL.

[0086] 10) L-hydroxyproline control series solution: accurately measure 2.5 mL, 3.75 mL, 5.0 mL, 7.5 mL, and 10.0 mL of L-hydroxyproline control stock solution, respectively, into 10 mL volumetric flasks, and dilute to volume with water to prepare L-hydroxyproline control series solution.

[0087] Preparation of test solution:

[0088] Take sample 0.5 g, accurately weighed, add 6 mol / L HCl appropriate amount, melt seal. 105 ℃ hydrolysis 22 h~24 h, cooling, the hydrolysis product is transferred to a certain volume flask, add water to wash hydrolysis tube, the washing liquid is combined and transferred to the volumetric flask, add 2 drops of phenolphthalein indicator, add 6 mol / L NaOH to the solution pink color; with water to constant volume to 250 mL, shake well, prepared test solution. (L-hydroxyproline concentration in the test solution should be in the range of the control product series solution concentration).

[0089] C.5 Determination

[0090] Respectively, 0.5 mL water (blank), 0.5 mL hydroxyproline control product series solution and 0.5 mL test solution in the stoppered colorimetric tube, respectively, add 1 mL isopropyl alcohol, add 0.5 mL oxidant solution, shake well, room temperature for 4 min, then add 6.5 mL color developing agent, stopper shake well, put into 60 ℃ water bath heating color development 15 min, room temperature cooling. With blank control, measured at 560 nm absorbance.

[0091] Calculation:

[0092] According to the data obtained, a set of standard fitting curve equation with absorbance as the ordinate, L-hydroxyproline concentration as the abscissa.

[0093] ;

[0094] In the formula: C - collagen content, units of milligrams per gram (mg / mL);

[0095] A - calculated from the standard curve sample hydroxyproline content, units of micrograms per milliliter (μg / mL);

[0096] M - the mass of the sample, units of grams (g);

[0097] V - sample solution constant volume, units of milliliters (mL);

[0098] ρ - the density of the sample (ρ = 1.01), units of grams per milliliter (g / mL);

[0099] 7.59 - is the conversion factor between hydroxyproline and collagen.

[0100] From the above Figure 1It can be seen that after preparing ten membranes according to examples 1, 2 and 3, and supplementing the liquid after ultrafiltration according to the respective processes, the collagen concentration in the electrolyte is stabilized at 6.0 mg / ml, 8.2 mg / ml and 7.1 mg / ml, respectively. This shows that the molecular weight cut-off of the hollow fiber selected in the example process is suitable for the concentration of collagen in the electrolyte. During the ultrafiltration process, the solvent in the electrolyte is filtered out through the hollow fiber membrane, while the collagen inside the hollow fiber membrane cannot flow out due to the cut-off molecular weight, so it is concentrated and returned to the electrolyte tank, and the concentration of the electrolyte is stabilized after supplementing the liquid, ensuring the stability of the collagen concentration during continuous membrane preparation.

[0101] In comparative example 9, a hollow fiber membrane with a molecular weight cut-off of 50 kDA was selected for continuous membrane preparation. Since the molecular weight of 50 kda is greater than the molecular weight of collagen 30 kda in the solution. Therefore, during the ultrafiltration process, part of the collagen permeates the hollow fiber membrane and is filtered out, resulting in a continuous decrease in the collagen concentration in the electrolyte, affecting the stability of subsequent membrane preparation.

[0102] In comparative example 10, the effluent pressure was set to 0.5 bar. At a higher pressure, the collagen molecules flowing inside the hollow fiber may permeate the fiber membrane and flow out, while the solution flow rate decreases, reducing the efficiency of ultrafiltration concentration. Therefore, the collagen concentration in the electrolyte continues to decrease, affecting the stability of subsequent membrane preparation.

[0103] In comparative example 11, the ultrafiltration time was shortened to 15 s. According to the experimental results of the concentration determination, the collagen concentration in the electrolyte continuously decreased with the continuous preparation of the membrane, indicating that the ultrafiltration time set to 15 s was not sufficient to concentrate the collagen concentration in the electrolyte to the initial level.

[0104] 2. Membrane uniformity determination

[0105] According to different processes, 8 membranes were continuously prepared, and the second and eighth membranes prepared were taken. The thickness of each membrane was determined at 5 random points using a micrometer, and the standard deviation was calculated.

[0106] Table 1 Thickness of the second membrane

[0107]

[0108] Table 2 Thickness of the eighth membrane

[0109]

[0110] From the data of Table 1-2, it can be seen that the collagen membranes prepared in Examples 1, 2 and 3 have slight differences depending on the collagen concentration in the initial electrolyte. The thickness of 5 randomly selected points of the membranes prepared in Examples 1, 2 and 3 is uniform, and the standard deviation of the thickness of each membrane is below 0.7, indicating that the collagen membranes prepared have high uniformity. The thickness of the second and eighth membranes prepared in Examples 1, 2 and 3 is uniform, indicating that Examples 1, 2 and 3 can realize uniform and continuous preparation.

[0111] The pH of the collagen electrolyte used in Comparative Example 1 is 6.5, and in the neutral state, the solubility of collagen molecules decreases, and the efficiency of collagen deposition on the negative electrode during electrolysis is low, resulting in uneven and thin film formation. The thickness of different parts of the second membrane is between 10-21 microns, with a standard deviation of 4.39, and the film formation is uneven; the thickness of the eighth membrane after continuous film formation in Comparative Example 1 gradually increases, which is due to the thin film formation during continuous film formation, and the collagen content in the consumed electrolyte is lower than the increased collagen content, resulting in an increase in the collagen concentration of the electrolyte.

[0112] In Comparative Examples 2 and 3, the electrolyte does not contain a surfactant and an ionic strength regulator, respectively, resulting in a decrease in the efficiency of negative electrode film deposition during electrolysis, and thus the film is thin and uneven. The thickness of the eighth membrane after continuous film formation increases due to the increase in the collagen concentration of the electrolyte, but the film thickness is still uneven, with standard deviations of 2.40 and 3.61, respectively.

[0113] Comparative Example 4 only increases the coating of the electrode, which only affects the ease of film removal and has no significant effect on the thickness and uniformity of the film, so the collagen membrane obtained in Comparative Example 4 has no significant difference from that of Example 1.

[0114] Comparative Example 5 increases the rotation speed of the electrode during film deposition, and the adhesion of collagen deposition to the film decreases during high-speed rotation of the electrode, resulting in poor uniformity of the film, with the thickness of different parts of the single film fluctuating between 20-25 microns.

[0115] Comparative Example 6 increases the voltage between the electrodes during electrolysis, resulting in a change in the speed of negative electrode collagen deposition, causing the film to become thicker but less uniform, with the film thickness fluctuating between 30-50 microns. The collagen in the electrolyte is consumed too quickly, resulting in a rapid decrease in the thickness of the eighth membrane.

[0116] Comparative Examples 7 and 8 change the film removal time and the rotation speed of the electrode during film removal, respectively. Since this experiment ensures that 8 membranes are prepared, the membranes obtained in these two examples have no significant difference from those of Example 1.

[0117] The ultrafiltration concentration process of Comparative Examples 9, 10 and 11 is different from that of Example 2. According to the experimental results of determining the ultrafiltration concentration parameters, the collagen concentration of the electrolyte continuously decreases in the continuous preparation process of the three comparative examples, so there is no obvious difference in the film thickness and uniformity of the second film and Example 2, but the film thickness and uniformity of the eighth film decrease.

[0118] 3. Difficulty of film detachment in continuous film process

[0119] Five films were continuously prepared according to different processes, and the difficulty of automatic film detachment of each film was evaluated. The values of 1-3 represent the difficulty of film detachment, 1 represents easy film detachment, 2 represents film partial adhesion to the electrode during the film detachment process, and manual peeling is required, and 3 represents that the film cannot be automatically detached. The maximum value of the total score is taken from the scores of the five films.

[0120] Table 3 Difficulty of film detachment during the preparation of five films

[0121]

[0122] According to the experimental results in Table 3, the film detachment of Examples 1, 2 and 3 is easy, which can meet the requirements of continuous film preparation in the process. Comparative Example 1 and Comparative Example 3 cause partial adhesion of film detachment due to uneven film formation caused by electrolyte pH and no addition of ionic strength regulator. Comparative Example 2 cannot automatically detach the film due to the strong adsorption between the film and the electrode caused by the absence of surfactant. Comparative Example 4 cannot be detached during the preparation process because the electrode has no coating, so it cannot be continuously prepared. Comparative Examples 5 and 6 only change the voltage and speed of electrodeposition, which has no effect on film detachment, so continuous film detachment can be achieved. Comparative Examples 7 and 8 cannot successfully detach the film during the continuous preparation process due to the reasons of shortening the film detachment time and slowing down the electrode speed during film detachment. In addition, the processes of Comparative Examples 9, 10 and 11 change the collagen concentration in the electrolyte due to the difference in ultrafiltration parameters, so there is no direct relationship between the difficulty of film detachment, so continuous film detachment can be achieved.

[0123] 4. Film strength experiment

[0124] The fifth collagen film material continuously prepared in each example and comparative example was cut into a cuboid with a length of 15 mm and a width of 40 mm, and the tensile strength of the sample was measured by a universal testing machine. The change of force value of the sample during tensile deformation was recorded, as shown in Figure 2 .

[0125] According to the above Figure 2 experimental results, the arrangement order of the breaking tension of each example and comparative example is as follows:

[0126] Example 2 > Example 3 > Example 1 > Comparative Example 6 > Comparative Example 7 > Comparative Example 8 > Comparative Example 4 > Comparative Example 2 > Comparative Example 3 > Comparative Example 5 > Comparative Example 1 > Comparative Example 10 > Comparative Example 11 > Comparative Example 9.

[0127] Examples 1, 2, 3 show the regularity of breaking tension: Example 2 > Example 3 > Example 1 due to the difference of collagen concentration in electrolyte. Comparative Example 6 shows the initial film thickness is higher than Example 1 due to the increase of film-forming voltage. The strength of the fifth film is lower than Example 1 due to the consumption of collagen in electrolyte during the continuous film-forming process. Comparative Examples 7, 8, 4 and 2 show the increase of film-removing difficulty only, and the manual intervention during the film-removing process may lead to the local film strength decrease. Comparative Example 3 shows the decrease of film uniformity due to the addition of ionic strength adjuster. Comparative Example 5 shows the poor film uniformity due to the too high electrode rotation speed during electrodeposition, thus the breaking strength of the film is poor. Comparative Example 1 shows the low solubility of collagen due to the neutral pH of film-forming electrolyte, and the low breaking tension due to the poor film uniformity. Comparative Examples 9, 10, 11 show the difference of ultrafiltration and concentration process from Example 2. According to the experimental results of the determination of ultrafiltration and concentration parameters, the collagen concentration in electrolyte continuously decreases during the continuous preparation process of these three comparative examples, thus the thickness of the fifth film decreases, and the breaking strength of the film significantly decreases.

[0128] The above embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as the limitation of the scope of the present application. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A method for preparing an electric field periodically regulated biomimetic collagen membrane material, characterized in that, The preparation method comprises the following steps: Preparation of the electrolytic collagen solution: taking a collagen solution with a pH value of 2.5-5, adding a surfactant to the solution system to obtain a final concentration of 0.01-0.1 wt%, and adding a 50-150 mM ionic strength regulator, wherein the ionic strength regulator is a hydrolyzable ionic halide salt; and the surfactant is a non-ionic surfactant; Collagen membrane deposition: placing the positive and negative electrodes with a coating on the surface in the electrolytic collagen solution to perform collagen membrane deposition, wherein the coating is a hydrophilic coating or a conductive lubricating layer; Electrolytic membrane stripping: subsequently switching the positive and negative electrodes to perform membrane stripping to obtain a collagen membrane; Hollow fiber ultrafiltration: performing hollow fiber ultrafiltration concentration on the electrolyte; Continuous membrane preparation: after the ultrafiltration and electrolytic membrane stripping are simultaneously completed, supplementing the collagen solution, repeating the electrolytic membrane preparation step, and realizing continuous membrane preparation to obtain a biomimetic collagen membrane material after cleaning and drying.

2. The method according to claim 1, wherein, The pH value of the collagen solution is 2.5-3.

5.

3. The method of claim 1 or 2, wherein the method further comprises the step of: The concentration of the collagen solution is 6-8 mg / mL. ​ 4. The method of claim 1 or 2, wherein the method further comprises the step of applying an electric field to the collagen solution. The material of the positive and negative electrodes is platinum, and the electrode spacing is 5-6 cm.

5. The method for preparing a biomimetic collagen membrane material with periodically modulated electric field according to claim 4, characterized in that, The hydrophilic coating comprises a polyethylene glycol coating, and the conductive lubricating layer comprises a graphene coating.

6. The method of claim 1-2, 5, wherein, In the collagen membrane deposition step, the rotation speed of the positive and negative electrodes is adjusted to 5-15 rpm.

7. The method for preparing a biomimetic collagen membrane material with periodically modulated electric field according to claim 6, characterized in that, In the collagen membrane deposition step, the voltage is set to 2-3 V in the constant current mode.

8. The method of claim 1-2, 5, 7, wherein, In the electrolytic membrane stripping step, the electrode rotation speed is adjusted to 60-80 rpm.

9. The method for preparing a biomimetic collagen membrane material with periodically modulated electric field according to claim 8, characterized in that, In the electrolytic membrane stripping step, the voltage is set to 2-3 V in the constant current mode, and the electrolytic membrane stripping is performed for 18-40 s.

10. The method of claim 1-2, 5, 7, 9, wherein the method of preparing the electric field periodically regulated biomimetic collagen membrane material is characterized by, In the hollow fiber ultrafiltration step, the molecular weight cut-off of the hollow fiber is 5-30 kDa.

11. The method for preparing a biomimetic collagen membrane material with periodically modulated electric field according to claim 10, characterized in that, In the hollow fiber ultrafiltration step, the molecular weight cut-off of the hollow fiber is 5-10 kDa, the effluent pressure is set to 0.05-0.15 bar, and the ultrafiltration is performed for 18-40 s.

12. A biomimetic collagen membrane material obtained by the preparation method of any one of claims 1-11.

13. Use of the biomimetic collagen membrane material of claim 12 in the preparation of medical wound repair, tissue hemostasis and soft tissue regeneration materials.

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