CN115137882B

CN115137882B - Method for modularly assembling collagen membrane by utilizing LB technology

Info

Publication number: CN115137882B
Application number: CN202210925035.0A
Authority: CN
Inventors: 李国英; 刘思聪; 张晓霞
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-08-03
Filing date: 2022-08-03
Publication date: 2023-07-21
Anticipated expiration: 2042-08-03
Also published as: CN115137882A

Abstract

The invention provides a method for modularly assembling a collagen membrane by utilizing an LB technology, which mainly comprises the following process steps: s1, preparing a collagen solution for LB film preparation; s2, preparing a substrate LB film; s3, modularly assembling the collagen membrane. Specifically, by using a collagen LB film-forming technique, a collagen film having a 67nm D period and maintained bioactivity in an orderly and oriented arrangement was constructed by limiting the average particle size of collagen aggregates in a collagen solution for LB film-forming and limiting a subphase solution. The preparation method has mild conditions and does not damage the collagen structure, and the prepared collagen film has the characteristics of 67nm D period, high orientation, good biocompatibility and no toxicity, and can be directly used as a biomedical material to be applied to the biomedical field.

Description

Method for modularly assembling collagen membrane by utilizing LB technology

Technical Field

The invention belongs to the technical field of biomedical material preparation, and relates to a method for modularly assembling a collagen film by utilizing an LB technology, in particular to a method for modularly assembling a 3D bionic anisotropic collagen film by utilizing the LB technology.

Background

Anisotropic microarchitecture is widely found in biological systems, such as muscle, skin, articular cartilage, wood, etc., to perform specific biological functions. The structure and the functional characteristics of the bionic natural material have great application prospects in the fields of sensors, actuators, cell culture, tissue engineering and the like, so that the bionic natural material is widely focused. Because of the complexity of the natural tissue structure, precise regulation of the anisotropic structure of the bionic natural material is always a challenging hot spot subject. Collagen is an important constituent structural protein for forming animal connective tissue, has low antigenicity, good biocompatibility and unique biological function, is an ideal important biomass material, and has been widely applied to the fields of tissue engineering, biological medicine, plastic repair and the like. In animals, collagen molecules self-assemble in a 1/4 staggered manner to form an anisotropic structure micro-architecture with a 67nmD periodic structure: for example, the collagen fibers in the tendon are parallel to a single direction, each layer of collagen fibers in the cornea is orthogonally arranged, and the collagen fibers in the bone have a layered structure.

In order to obtain collagen fibers with better directionality, many control modes such as electrospinning, fluid shearing, magnetic field assistance, spin coating techniques, etc. have been proposed successively. Electrospinning and fluid shearing are the most commonly used methods for inducing collagen to form a certain directionality by mechanical deformation, and a high-concentration collagen solution with sufficient mobility is used as a processing material, so that the formed "collagen fibers" are essentially fibrous aggregates of collagen molecules, and 67 nm-characteristic D periodic structures are not present (Y.Wakuda, S.Nishimoto, S.Suye, et al, native collagen hydrogel nanofibres with anisotropic structure using core-shell electrospinning [ J Scientific Reports,2018,8 (1): 6248-6248;S.Kwak,A.Haider,K.C.Gupta,et al.Nano Multilayered Scaffolds of PLGA and Collagen by Alternately Electrospinning for Bone Tissue Engineering[J ]. Nanoscale Research Letters,2016,11 (1): 323-323.). The magnetic field is assisted by taking the collagen composite solution added with the magnetic nano particles as a processing material, and collagen molecules in the solution form 2D (two-dimensional) uniaxially arranged aggregation fibers under the action of an external magnetic field. The spin coating technique can achieve 3D lamination of different directions between 2D collagen fiber layers, but the fiber growth direction on the 2D collagen fiber layer is easily reversed or generates a "hook" shape (M.Antmanpassig, O.Shefi.Remote magnetic orientation of 3D collagen hydrogels fbr directed neuronal regeneration[J ]. Nano Letters,2016,16 (4): 2567-2573.; C.Guo, L.J.Kaufrnan.Flow and magnetic field induced collagen alignment [ J ]. Biomaterials,2007,28 (6): 1105-1114.). However, the "collagen fibers" formed by the current regulatory technology are substantially fibrous aggregates formed by collagen molecules, and 67nm characteristic D periodic structures do not exist.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for modularly assembling a collagen film by utilizing an LB technology, which has mild conditions, and builds the collagen film which has 67nm D period and is orderly and directionally arranged and keeps the bioactivity by controlling the cooperative action of a collagen solution aggregation state structure, a subphase solvent system, a sliding barrier moving speed and surface pressure under the condition that the collagen denaturation temperature is not exceeded; the preparation method has mild conditions and does not damage the collagen structure, and the prepared collagen film has the characteristics of 67nm D period, high orientation, good biocompatibility and no toxicity, and can be directly used as a biomedical material in the biomedical field.

In order to achieve the above object, the present invention is realized by adopting the technical scheme comprising the following technical measures.

In one aspect, the invention provides a method for modularly assembling a collagen membrane by utilizing LB technology, which mainly comprises the following process steps:

s1, preparing collagen solution for LB film preparation:

preparing collagen into a collagen solution, regulating the pH value of the collagen solution, and adding an organic solvent to prepare a collagen solution for LB film making, wherein the average grain size of the collagen aggregate is not more than 2000 nm;

s2, preparing a substrate LB film:

regulating the temperature of the collagen solution for LB film preparation obtained in the step S1 to 24-30 ℃, uniformly dispersing 500-1500 mu L of the collagen solution for LB film preparation on the subphase surface of an LB film analyzer by using a syringe, standing until the film pressure approaches to equilibrium, pressing the film at a film moving speed of 1-10 cm/min, and finally, forming a film with a surface area of 10-20 cm ² The tent sheet stops moving, so that the surface pressure is stabilized at 10-30 mN/m;

wherein the subphase comprises a metal ion solution with the ionic strength of 0.2-0.5, the temperature is controlled at 24-30 ℃ and the pH value is 5-9;

s3, modularly assembling a collagen film:

transferring the collagen to the substrate under the condition of keeping the surface pressure stable at 10-30 mN/m by adopting a vertical pulling method to form an LB single-layer collagen film, cleaning and drying the LB single-layer collagen film on the substrate, then adopting the vertical pulling method again, repeating the steps, transferring the collagen to the substrate, and forming an LB stacked collagen film to obtain the collagen film.

The collagen described in step S1 is an animal-derived collagen, and the person skilled in the art can select the industrial/experimental animal-derived collagen type described in the prior art, and can be obtained commercially or by self-made preparation, preferably an animal organism tissue part rich in natural collagen, including but not limited to any one of cow hide, pig hide, fish hide, sheep hide, bullfrog hide, rat tail, bovine achilles tendon, pig achilles tendon and sheep achilles tendon.

In one technical scheme, the specific concentration of the collagen solution for LB film making in the step S1 can refer to the prior art documents related to the LB film making of the collagen in the field, and can be optionally selected within the mass concentration range of 0.1-0.7 mg/mL.

In one embodiment, the step S1 of adjusting the pH of the collagen solution, wherein the specific pH is selected based on the collagen self-assembly in the subject matter and technical object of the present invention, and the person skilled in the art can clearly know the conventional self-assembly applicable pH range of collagen, such as pH adjustment to 5-9, according to the existing literature in the art; it should also be apparent that conventional pH adjusting agents for collagen, such as acetic acid solutions, are known without disrupting the structure of the collagen.

In one embodiment, the organic solvent in step S1 is used to facilitate the uniform dispersion of the collagen solution on the sub-phase, and the specific selection thereof may be described in the prior art for preparing collagen LB films, and may be a small molecular alcohol solvent (carbon chain length is not longer than C4), including but not limited to methanol, ethanol, n-propanol, isopropanol, glycerol, and n-butanol.

In one embodiment, the adding in step S1 includes adding an organic solvent, which may be only adding an organic solvent, or adding an organic solvent and other auxiliary agents, where the other auxiliary agents include, but are not limited to, formic acid, trichloroacetic acid, amide auxiliary agents (e.g., N-methylacetamide), and the methods and technical effects of using the other auxiliary agents are described in the prior art documents in the field.

Note that the specific selection and concentration of the above-mentioned agents including pH adjusting agents and organic agents should satisfy the aggregation state index characterization range, that is, the average particle size of the collagen aggregate is not more than 2000nm.

In one technical scheme, the subphase in the step S2 comprises a metal ion solution with the ionic strength of 0.2-0.5, wherein the metal ion solution can be used for forming the subphase independently, or the metal ion solution and other subphase additives can be used for forming the subphase together. The metal ion solution is based on collagen self-assembly in the subject matter and technical purpose, and the metal ion can be selected from transition metal ions and non-transition metal ions, preferably non-transition metal ions, such as sodium ions, potassium ions, calcium ions and magnesium ions. The other sub-phase auxiliary agents are sub-phase auxiliary agents which are conventionally used in LB film making technology, such as EDC, NHS, succinic anhydride and lauroyl chloride, and the use method and the technical effect of the other sub-phase auxiliary agents are recorded in the prior art documents.

In this context, the LB film analyzer should be one which uses Langmuir-Blodgett and Langmuir-Technical instruments.

In one embodiment, the LB film analyzer described herein has a substrate that is held vertically according to standard operating specifications of the vertical pull method. In the process of repeating the vertical pulling method and forming the LB stacked collagen film, the clamping position of the substrate can be kept all the time, and the LB stacked collagen film with consistent orientation degree is formed; the clamping orientation of the substrate can also be selectively adjusted, namely, the next layer of collagen film is transferred after the substrate is rolled (namely, the clamping orientation of the substrate is adjusted before each time of collagen transfer onto the substrate), so that the effect of constructing the collagen fiber bracket with the 3D structure is achieved.

In one embodiment, the substrate of the LB film analyzer described herein may be an inorganic material substrate, including but not limited to any one of a silicon wafer, a mica sheet, a glass sheet, a cell climbing sheet, an aluminum sheet, and a titanium sheet; the substrate may also be an organic material substrate including, but not limited to, any of collagen membrane sheet, polyvinyl alcohol (PVA) membrane sheet, gelatin sponge sheet.

In one embodiment, the substrate of the LB film analyzer described herein may be any shape, such as rectangular, square, trapezoidal, circular, oval, spherical.

It should be noted that, based on the general principle of LB film formation, the surface area of one side of the substrate should be smaller than or equal to the surface area provided by film pressing.

In this context, the vertical pulling method is a conventional operation of the LB film analyzer, and specifically may refer to an operation description of the LB film analyzer, and may refer to an existing literature description of the art of collagen LB film production.

In one technical scheme, the vertical pulling method is adopted in the step S3, and the specific pulling speed can be described by referring to the prior literature of collagen LB film making in the field, or can be arbitrarily selected within the pulling speed range of 1-10 mm/min.

In one embodiment, after the LB monolayer collagen film on the substrate is washed and dried in step S3, a specific washing and drying manner thereof may be selected from collagen drying manners or methods that are conventional in the art, such as low-temperature drying and vacuum drying; the cleaning mode is the same as that of deionized water.

In a preferred technical scheme, the vertical pulling method in step S3 keeps the clamping orientation of the substrate, and the LB-superimposed collagen film with 3-5 layers is repeatedly formed, so that the method can be applied to the biomedical field, for example, the method can be used as skin auxiliary materials.

In a preferred technical scheme, the vertical pulling method in step S3 keeps the clamping orientation of the substrate, and the LB-superimposed collagen film with 10-15 layers is repeatedly formed, so that the method can be applied to the biomedical field, for example, the method can be used as artificial cornea tissue.

In a preferred technical scheme, the vertical pulling method in step S3 defines that the clamping orientation of the substrate is 0 ° when the first collagen film is transferred, and the substrate is rolled by adjusting the clamping orientation of the substrate by 90 ° when the second collagen film is transferred, and the LB-superimposed collagen film with 15-20 layers is repeatedly formed, so that the vertical pulling method can be applied to the biomedical field, for example, used as artificial tendon-like tissue.

In one embodiment, after forming the LB-laminated collagen film, a sterilization treatment for forming the LB-laminated collagen film is further included in consideration of the repeated operations in step S3, and the sterilization treatment may be a sterilization treatment method conventional in the field of biological materials, such as irradiation sterilization.

The principle of the invention is that the interface horizontal thrust of LB technology is utilized to couple the action of the collagen assembling rearrangement driving force, the high fitting of each key factor is achieved from the regulation and control of the collagen aggregation state structure, the system constructs the 2D bionic collagen fiber structure module with 67nm D period and orderly and directionally arranged, and then the collagen membrane is assembled in a modularized way, in particular to the 3D bionic anisotropic natural collagen fiber micro-architecture.

Wherein, the rod-shaped collagen molecules floating on the interface are subjected to molecular rearrangement by subphase self-assembly driving force and tend to be parallel to the membrane barrier due to horizontal opposite force generated by movement of the membrane barrier, so that ordered directional growth is realized.

In another aspect, the present invention provides a collagen membrane prepared by the above method.

The invention has the following beneficial effects:

1. the invention has mild condition and is prepared at the low temperature of 24-30 ℃, and the obtained collagen film keeps the special triple helix structure of undenatured collagen and ensures the bioactivity of the collagen. After sterilization treatment, the safety is high, the heavy metal content is less than or equal to 10mg/kg, and the method can be widely applied to the biomedical field.

2. The collagen membrane prepared by the invention has anisotropy, the natural collagen fiber micro-architecture of the collagen membrane not only maintains the biology and structural integrity of tissues, but also has 67nmD periodic and orderly directional arranged dual structural characteristics of the structural module. The precise regulation and control of the bionic anisotropic collagen fiber micro-architecture has important guiding significance for the application of the bionic anisotropic collagen fiber micro-architecture in the fields of cell culture, tissue engineering and the like.

3. According to the invention, the macroscopic horizontal thrust of an interface is input by utilizing an LB technology, the self-assembly driving force action of collagen is coupled, the collagen is discharged from a structure for regulating and controlling the aggregation state of the collagen, and a regulating and controlling mechanism of a 2D bionic collagen fiber structure module which has 67nmD cycles and is orderly and directionally arranged is established by a system; by designing the lamination direction, the 3D bionic anisotropic collagen fiber micro-architecture is assembled in a modularized mode, and then the guiding effect of the 3D bionic anisotropic collagen fiber micro-architecture on cell behaviors is explored. The invention provides a new technical strategy for accurate regulation and control of bionic anisotropic natural collagen fibers.

Drawings

FIG. 1 is a photograph of a sample of the collagen membrane prepared in example 2 of the present invention. The collagen membrane has a small number of layers and a thickness of nanometer level, and is almost completely transparent and can not be observed by naked eyes.

FIG. 2 is an AFM photograph of a collagen film prepared in example 5 of the present invention.

FIG. 3 is an AFM photograph of a collagen film prepared in example 5 of the present invention. By further enlargement of fig. 2, the presence of a nodular structure on the fiber is visible from the figure, demonstrating self-assembly to form a 67nm feature D periodic structure.

FIG. 4 is an AFM photograph of a collagen film prepared in example 5 of the present invention. By further enlargement of fig. 3, the nodular structure is clearly visible from the figure, demonstrating self-assembly to form a 67nm feature D periodic structure.

FIG. 5 is an AFM photograph of LB-superimposed collagen film prepared in comparative example 5 of the present invention. A complete confusion of the fiber orientation was observed.

FIG. 6 is an AFM photograph of LB-superimposed collagen film prepared in comparative example 5 of the present invention. By further enlargement of fig. 5, it can be seen from the figure that no nodular structure is found on the fiber, demonstrating that it does not form a self-assembled 67nm feature D periodic structure.

Fig. 7 is a photograph of an embodiment of the present invention during the manufacturing process. In the figure, LB film analyzer is shown.

Detailed Description

For a further understanding of the present invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are merely intended to illustrate further features and advantages of the invention and are not limiting of the invention claims. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention. While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to aid in the description of the presently disclosed subject matter.

As used herein, the term "comprising" is synonymous with "including mainly" and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. "comprising" is a technical term used in claim language to mean that the element is present, but other elements may be added and still form an element or method within the scope of the claim.

The invention provides a method for modularly assembling a collagen membrane by utilizing an LB technology, which mainly comprises the following process steps:

s1, preparing collagen solution for LB film preparation:

s2, preparing a substrate LB film:

s3, modularly assembling a collagen film:

In one embodiment, the collagen solution for LB film formation in step S1 may be any collagen concentration in the range of 0.1 to 0.7mg/mL, for example, 0.15mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.65mg/mL, or any range or point value therebetween, with reference to the prior art documents related to LB film formation by collagen in the art.

In one embodiment, the adjustment of the pH of the collagen solution described in step S1, the specific pH of which is selected based on collagen self-assembly in the subject and technical objects of the present invention, the person skilled in the art will be aware of the conventional self-assembly applicable pH range of collagen, e.g. the pH adjustment to a pH of 5-9, even more specific e.g. 5.5, 6, 7, 8, 8.5 or any range or point in between; it should also be apparent that conventional pH adjusting agents for collagen, such as acetic acid solutions, are known without disrupting the structure of the collagen.

In one embodiment, the organic solvent in step S1 is used to facilitate the uniform dispersion of the collagen solution on the sub-phase, and the specific choice thereof may be described in the prior art for preparing collagen LB films, and may be a small molecule alcohol solvent (carbon chain length is not longer than C4), such as any one or more of methanol, ethanol, n-propanol, isopropanol, glycerol, and n-butanol.

Note that the specific selection and concentration of the pH adjusting agent and the organic agent should satisfy the aggregation state index characterization range, that is, the average particle size of the collagen aggregate is not more than 2000nm.

In one embodiment, the adding in step S1 includes adding an organic solvent, which may be adding only an organic solvent, or adding an organic solvent and other auxiliary agents, preferably adding only an organic reagent; other adjuvants include, but are not limited to, formic acid, trichloroacetic acid, amide-based adjuvants (e.g., N-methylacetamide), methods of use and technical effects of other adjuvants are well documented in the art.

In one embodiment, the subphase in step S2 includes a metal ion solution having an ionic strength of 0.2 to 0.5, and the subphase may be formed by the metal ion solution alone or by the metal ion solution and other subphase additives. The metal ion solution is based on collagen self-assembly in the subject matter and technical purpose, and the metal ion can be selected from transition metal ions or non-transition metal ions, preferably non-transition metal ions, such as any one of sodium ions, potassium ions, calcium ions and magnesium ions. The other sub-phase auxiliary agents are sub-phase auxiliary agents which are conventionally used in LB film making technology, such as any one or more of EDC, NHS, succinic anhydride and lauroyl chloride, and the use method and the technical effect of the other sub-phase auxiliary agents are recorded in the prior art documents in the field.

In one embodiment, the LB film analyzer described herein has a substrate that is held vertically as specified by standard practice of vertical pull-up. In the process of repeating the vertical pulling method and forming the LB stacked collagen film, the clamping position of the substrate can be kept all the time, and the LB stacked collagen film with consistent orientation degree is formed; the clamping orientation of the substrate can also be selectively adjusted, namely, the next layer of collagen film is transferred after the substrate is rolled, so that the effect of constructing the collagen fiber support with the 3D bionic anisotropic structure is achieved, and the substrate can be rolled by an angle of 0-360 degrees, such as 5 degrees, 10 degrees, 15 degrees, 20 degrees, 30 degrees, 40 degrees, 45 degrees, 50 degrees, 60 degrees, 70 degrees, 75 degrees, 80 degrees, 90 degrees, 120 degrees, 150 degrees, 180 degrees, 210 degrees, 240 degrees, 270 degrees, 300 degrees, 330 degrees, 360 degrees or any point value between the two angles based on the difference of the clamping orientation.

In one embodiment, the LB film analyzer described herein, the substrate of which may be an inorganic material substrate, including but not limited to any one of a silicon wafer, a mica sheet, a glass sheet, a cell climbing sheet, an aluminum sheet, a titanium sheet; the substrate may also be an organic material substrate including, but not limited to, any of collagen membrane sheet, polyvinyl alcohol (PVA) membrane sheet, gelatin sponge sheet.

In one embodiment, the substrates of the LB film analyzer described herein may be any shape, such as rectangular, square, trapezoidal, circular, oval, spherical.

In one embodiment, the vertical pulling method is used in the step S3, and the specific pulling speed can be described in the prior art for preparing the collagen LB film, or can be arbitrarily selected in the pulling speed range of 1-10 mm/min, such as 1.5mm/min, 2mm/min, 3mm/min, 4mm/min, 5mm/min, 6mm/min, 7mm/min, 8mm/min, 9mm/min, 9.5mm/min or any range or point value between them.

In one embodiment, after the LB monolayer collagen film on the substrate is washed and dried in step S3, the specific washing and drying manner thereof may be selected from collagen drying manners or methods conventional in the art, such as low temperature drying and vacuum drying; the cleaning mode is the same as that of deionized water.

In a preferred embodiment, the substrate in step S3 is disposed horizontally, and the laminated LB collagen film with 3-5 layers is formed repeatedly, so that the laminated LB collagen film can be applied to the biomedical field, for example, used as skin auxiliary materials.

In a preferred embodiment, the substrate in step S3 is horizontally disposed, and the LB-laminated collagen film with 10-15 layers is repeatedly formed, so that the method can be applied to the biomedical field, for example, used as artificial cornea tissue.

In a preferred embodiment, the vertical pulling method in step S3 defines that the holding direction of the substrate is 0 ° when transferring the first number of collagen films, and the substrate is rolled by adjusting the holding direction of the substrate by 90 ° when transferring the second number of collagen films, and the LB-superimposed collagen films with 15-20 layers are repeatedly formed, so that the vertical pulling method can be applied to the biomedical field, for example, used as artificial tendon-like tissues.

In one embodiment, after forming the LB-laminated collagen film, a sterilization treatment for forming the LB-laminated collagen film, which may be a sterilization treatment manner conventional in the field of biological materials, such as irradiation sterilization, is further included in consideration of the repeated operations in step S3.

The present application will be explained in further detail with reference to examples. However, those skilled in the art will appreciate that these examples are provided for illustrative purposes only and are not intended to limit the present application.

Examples

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustration of the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. The present application should not be construed as limited to the particular embodiments described.

1. Preparation method

S1, preparing collagen solution for LB film preparation:

1 part by weight of collagen derived from cow leather (theoretical weight can also be regarded as dry weight) is mixed with 1800 parts of acetic acid solution with 0.1mol/L molar concentration and 200 parts of isopropanol, and stirred at a temperature of 4 ℃ for 8 hours to prepare a collagen solution for LB film preparation, wherein the average particle size of collagen aggregate is not more than 2000nm (when the collagen is 1 part, the average particle size is 1586.2 nm);

s2, preparing a substrate LB film:

wherein the subphase is sodium ion solution with the ionic strength of 0.2-0.5, the temperature is controlled at 24-30 ℃ and the pH value is 5-9;

s3, modularly assembling a collagen film:

2. Test method

The surface pressure, orientation degree, transfer ratio, number of layers and thickness of the collagen films prepared in the following examples and comparative examples were measured by the following devices and by the conventional techniques.

The surface pressure and transfer ratio of the collagen film were measured using an LB film analyzer (JML 04).

The degree of orientation of the fibers in the collagen membrane was calculated using an insert orientation j analysis in ImageJ.

The thickness of the collagen film was measured using an ellipsometer.

The number of layers of the collagen film was measured using an ultraviolet absorption spectrometer.

And measuring the heavy metal content in the collagen membrane by using a heavy metal detector.

Examples 1 to 4 and comparative examples 1 to 2

Examples 1 to 4 and comparative examples 1 to 2 examined the effect of the subphase temperature conditions as variables on whether the prepared collagen film self-assembles to form a 67nm characteristic D periodic structure and the degree of alignment of fibers in the same direction, as shown in the following table:

table 1: with sub-phase temperature conditions as variables

The results of the table show that collagen cannot self-assemble to form a D period when the temperature is too low; self-assembly occurs when the temperature is in a proper range, and collagen fibers are arranged in an oriented manner (high orientation degree); however, when the temperature condition is continuously increased, the LB film pressing time and the self-assembly time cannot be matched due to the fact that the self-assembly is too fast, a molecular lamination phenomenon and a cavity area occur, and the orientation degree is reduced.

Examples 5 to 8 and comparative examples 3 to 4

Examples 5 to 8 and comparative examples 3 to 4 examined the effect of the subphase ionic strength conditions as variables on whether the prepared collagen film self-assembles to form a 67nm characteristic D periodic structure and the degree of alignment of fibers in the same direction, as shown in the following table:

table 2: takes the condition of subphase ion intensity as a variable (subphase temperature is unified to be 25 ℃)

As can be seen from the results of the above table, self-assembly is difficult to occur when the ion strength is too low; when the ionic strength is in a proper range, the self-assembly of collagen is promoted to form a D period, and the fiber arrangement directivity is good; when the ion strength of the subphase is continuously increased, although self-assembly can occur, a molecular lamination phenomenon and a cavity area appear, and the fiber arrangement order is reduced; too high an ionic strength inhibits the occurrence of self-assembly and does not form the D period.

Comparative example 5

The comparative example uses ultrapure water as the sub-phase, and the other conditions are the same as in example 2.

The prepared collagen film was visually observed to be consistent with example 2, but it was confirmed from the AFM topography that it did not form a self-assembled 67nm feature D periodic structure, and that it was observed that the fiber orientation was completely disordered.

Comparative example 6

In this comparative example, 2 parts of a collagen solution (corresponding to a collagen mass concentration of 1 mg/mL) for LB film production was used, and the average particle size of collagen aggregates was larger than 2000nm, and the other conditions were the same as in example 2.

The prepared collagen film was visually observed to be consistent with example 2, but it was found from the AFM topography that the fiber orientation was completely disordered although the self-assembled 67nm feature D periodic structure was formed, consistent with comparative example 5.

The foregoing examples are illustrative of the present invention and are not intended to be limiting, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent and are within the scope of the present invention.

Claims

1. A method for modularly assembling a collagen membrane by utilizing LB technology is characterized by comprising the following main process steps:

s1, preparing collagen solution for LB film preparation:

s2, preparing a substrate LB film:

regulating the temperature of the collagen solution for LB film preparation obtained in the step S1 to 24-30 ℃, and uniformly dispersing 500-1500 mu L of the collagen solution for LB film preparation by using a syringeStanding the subphase surface of the LB film analyzer until the film pressure approaches equilibrium, pressing the film at a film moving speed of 1-10 cm/min, and finally, forming a film with a surface area of 10-20 cm ² Stopping the movement of the tent sheet to ensure that the surface pressure is stabilized at 10-30 mN/m;

wherein the subphase comprises a metal ion solution with the ionic strength of 0.2-0.5, the temperature is controlled at 24-30 ℃, and the pH value is 5-9;

s3, modularly assembling a collagen film:

transferring the collagen to the substrate by adopting a vertical pulling method under the condition of keeping the surface pressure stable at 10-30 mN/m to form an LB single-layer collagen film, cleaning and drying the LB single-layer collagen film on the substrate, and then repeating the steps by adopting the vertical pulling method to transfer the collagen to the substrate to form an LB stacked collagen film, so that the collagen film is obtained, and the structural module of the collagen film has the dual structural characteristics of 67nmD periodic and ordered directional arrangement.

2. The method according to claim 1, wherein: in the step S1, the collagen mass concentration of the collagen solution for LB film production is 0.1-0.7 mg/mL.

3. The method according to claim 1, wherein: the organic solvent in the step S1 is a small molecular alcohol solvent with the carbon chain length not longer than C4.

4. The method according to claim 1, wherein: the subphase in the step S2 comprises a metal ion solution with the ionic strength of 0.2-0.5, and the metal ion is selected from any one of sodium ion, potassium ion, calcium ion and magnesium ion.

5. The method according to claim 1, wherein: in the step S3, the LB-superimposed collagen film is formed, and the clamping orientation of the substrate may be selectively adjusted before each transfer of collagen onto the substrate.

6. The method according to claim 1, wherein:

step S3, maintaining the clamping direction of the substrate by the vertical pulling method, and repeatedly forming LB (LB) stacked collagen films with 3-5 layers;

or is the one or more than one of the following,

step S3, maintaining the clamping direction of the substrate by the vertical pulling method, and repeatedly forming LB (LB) stacked collagen films with the number of layers of 10-15 layers;

or is the one or more than one of the following,

and S3, defining the clamping direction of the substrate to be 0 degree when the first number of collagen films are transferred, rolling the substrate to be 90 degrees by adjusting the clamping direction of the substrate when the second number of collagen films are transferred, and repeatedly forming the LB stacked collagen films with 15-20 layers.

7. The method according to claim 1, wherein: and in the step S2, the subphase temperature is controlled to be 24-26 ℃.

8. The method according to claim 1, wherein: the subphase in the step S2 comprises a metal ion solution with the ionic strength of 0.2-0.3.

9. The method for modularly assembling a collagen membrane using LB technology of claim 1, wherein the collagen membrane is prepared.

10. Use of the collagen membrane of claim 9 as biomedical material.