Method for modular and efficient assembly of bionic collagen membrane with D-period characteristics based on LB technology

Abstract

The invention provides a method for modular efficient assembly of a bionic collagen membrane with D period characteristics based on an LB technology. The method comprises the following main process steps: S1, preparing a collagen solution for LB membrane preparation; s2, preparing a substrate LB film; s3, standing and micro-amplitude pressure circulation are carried out; and S4, modularly assembling the collagen membrane. According to the method, on the premise of not changing a collagen formula and mild environmental conditions, two key process steps of continuously standing after balanced mould pressing and forming a'compression-relaxation 'effect with micro-amplitude pressure circulation are introduced, so that the spreading and rearrangement sufficiency of interface molecules is improved, residual interface stress is released, and the collagen quality is improved. Therefore, the structural integrity and the preparation stability of the membrane layer are remarkably improved, the collagen membrane which has higher membrane forming yield and D-period success rate and keeps biological activity is obtained, the stability of the collagen membrane with the D-period characteristic assembled on the basis of an LB technical path is promoted to step from a sample level to a process level, and a reliable manufacturing basis is provided for subsequent application and amplification.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medical material preparation and collagen membrane, and relates to a method for modular high-efficiency assembly of D-period characteristic biomimetic collagen membranes based on LB technology. BACKGROUND

[0002] Collagen is the most abundant structural protein in animal body, widely existing in skin, bone, tendon and blood vessel, etc. Among them, type I collagen is the most common. Collagen polypeptide chain is characterized by repeating sequence of Gly-X-Y (X and Y positions are often proline / hydroxyproline), and three left-handed helical polypeptide chains are wound to form a right-handed triple helix tropocollagen molecule. At a higher level, the tropocollagen molecules are further assembled into a hierarchical structure of microfibril-fibril-fiber through end peptide segment interaction, side chain interaction and enzymatic / non-enzymatic crosslinking; the fibrils are periodically staggered along the axial direction, thereby forming a D-periodic banding of about 67 nm, and the structure is often regarded as an important symbol of the hierarchical architecture and mechanical / functional attributes of natural collagen.

[0003] At the tissue scale, collagen bears basic mechanical support and tensile strength, and its orientation, crosslinking degree and hierarchical arrangement jointly determine the stiffness, elasticity and fatigue behavior of the tissue. The spatial distribution of collagen in different tissues is different: in the tendon, the collagen bundles are highly oriented along the stress direction; in the bone tissue, collagen and mineralization are combined to form a lamellar structure; in the skin and blood vessel wall, collagen and elastin, proteoglycan, etc. together constitute the extracellular microenvironment. Extracellular matrix (ECM) is composed of collagen, elastin, laminin, fibronectin and proteoglycan / glycosaminoglycan, etc., which not only provides a three-dimensional structural scaffold, but also transmits biochemical and biomechanical signals through interaction with cell receptors (such as the integrin family), to regulate adhesion, differentiation, migration and tissue remodeling processes.

[0004] When the vascular endothelium is damaged, the extracellular matrix is exposed to the blood flow. Collagen fibers, as the main structural protein exposed, can specifically recognize platelet membrane receptors such as GPVI and α2β1; at the same time, von Willebrand factor (vWF) mediates the rapid adhesion of GPIb-IX-V under high shear flow field, followed by stable binding of GPVI and α2β1 into the adhesion-activation-aggregation cascade. The natural subendothelial collagen is usually parallel and orderly oriented along the direction of blood flow, maintaining a D periodic band of about 67 nm; such nanotopology helps to increase the probability of receptor recognition and effective contact time, optimizing platelet adhesion kinetics under high shear conditions. Therefore, the controllable replication of the collagen topology of "directional arrangement + D periodicity" in material engineering is considered to be closely related to key performance such as biomimetic hemostasis, blood compatibility and cell compatibility.

[0005] In order to achieve the above-mentioned objectives, various preparation methods have been proposed. The bulk processing method (such as freeze-drying, solution casting, electrospinning, fluid shear, external field induction, spin coating, etc.) has a relatively simple process and is suitable for macro-molding, but the obtained structure is mostly macro-orientation of fiber bundles or aggregates, which is difficult to simultaneously reconstruct the ordered stacking of triple helix and 67 nm D periodic band at the nanoscale; at the same time, the participation of high temperature, strong acid and alkali or part of organic solvent may cause partial denaturation of collagen molecules, reducing the replicability and stability of "band + orientation".

[0006] In view of the defects and problems existing in the prior art, the inventors of the present application have previously granted a patent for "Method for modularly assembling collagen membranes using LB technology" (CN115137882B), which discloses a method for modularly assembling collagen membranes using LB technology, the main process steps of which are as follows: S1, preparation of collagen solution for LB membrane preparation; S2, preparation of substrate LB membrane; S3, modular assembly of collagen membrane. Specifically, by using collagen LB membrane preparation technology, the collagen aggregates in the collagen solution for LB membrane preparation are limited in average particle size, and the sub-phase solution is limited, thereby constructing a collagen membrane with 67 nm D periodicity and orderly directional arrangement, which maintains biological activity.

[0007] In this patent technology, the technical bottleneck that the "collagen fibers" assembled by the instruments based on Langmuir-Blodgett and Langmuir-Schäfer technology in the past are essentially fibrous aggregates formed by collagen molecules and do not have the characteristics of 67 nm D periodic band is broken through, providing a new technical strategy for precise regulation of biomimetic anisotropic natural collagen fibers.

[0008] But in the subsequent further application of the patent technology process, it is found that although the LB path can obtain the collagen membrane with D period characteristics in the sample level verification, there are common problems in process manufacturability and batch stability. First, the interface spreading-compression-equilibrium process is highly sensitive to temperature, ion strength, surface pressure, compression rate, etc. If the parameters deviate from the appropriate interval or the equilibrium judgment and the transfer timing do not match, defects such as molecular stacking and voids may occur, which may cause the strip to be interrupted and the orientation consistency to be reduced, thereby affecting the formation of the D period structure. Second, even within the commonly used window, the film formation yield and the D period success rate may still fluctuate within / between batches, which may be caused by factors such as but not limited to solution aging time and effective particle size distribution, sample volume and diffusion-merging behavior, compression path and equilibrium time determination caliber, etc. If these "hidden variables" are not included in the process procedures with a unified caliber, the phenomenon of "the same formula but different interface behaviors" may occur. Third, in the multi-layer construction, the predictability and consistency of the layer-to-layer transfer ratio (TR) are coupled with factors such as interface state and compression behavior. Once the previous layer has defects or the interface relaxation is insufficient, the subsequent layer may amplify the error, which may result in uneven thickness and orientation and damaged film formation yield.

[0009] In summary, based on the LB process technology, the collagen membrane with "directional arrangement + D periodicity" is replicated under mild conditions, which has methodological advantages. However, for the control of interface defects, the statistical stability of yield and success rate, the consistency of TR and the repeatability of multi-layer construction, further improvement of the process is still needed based on the existing technical solutions to promote the transition of the collagen membrane with D period characteristics from sample level feasibility to process level stability, and to provide a reliable manufacturing basis for subsequent application and scaling-up. SUMMARY

[0010] The purpose of the present application is to overcome the shortcomings of the prior art and provide a method for modularizing and efficiently assembling D period characteristic biomimetic collagen membrane based on LB technology. The method does not change the collagen formula and the mild environmental conditions, and introduces two key process steps of "compression-relaxation" effect formed by continuing to stand after balance molding and micro-pressure cycle to improve the spreading and rearrangement of interface molecules, release residual interface stress, thereby significantly improving the structural integrity and preparation stability of the film layer, obtaining collagen membrane with higher film formation yield, D period success rate and biological activity, promoting the transition of D period characteristic collagen membrane based on LB technology from sample level to process level stability, and providing a reliable manufacturing basis for subsequent application and scaling-up.

[0011] To achieve the above-mentioned purpose, the present application is realized by the following technical measures.

[0012] In an aspect, the present application provides a method for modularly assembling a D-periodic characteristic biomimetic collagen membrane based on LB technology, the main process steps of which are as follows:

[0013] S1, preparation of collagen solution for LB film preparation:

[0014] The collagen is prepared into a collagen solution, and then the pH value of the collagen solution is adjusted, and an additive including an organic solvent is added, to prepare the collagen solution for LB film preparation, wherein the average particle size of the collagen aggregate is not greater than 2000 nm;

[0015] S2, preparation of a substrate LB film:

[0016] The collagen solution for LB film preparation prepared in step S1 is temperature-adjusted to 24-26℃, 500-1500 μL of the collagen solution for LB film preparation is uniformly dispersed on the subphase surface of an LB film analyzer by using a syringe, and after the film pressure is close to equilibrium after standing, the film is pressed at a belt moving speed of 1-10 cm / min, and finally the belt is stopped moving when the surface area reaches 10-20 cm 2 , so that the surface pressure is stabilized at 16-20 mN / m;

[0017] The subphase includes a metal ion solution with an ionic strength of 0.2-0.3, and the temperature is controlled at 24-26℃ and the pH value is 5-9;

[0018] S3, standing and micro-amplitude pressure cycle:

[0019] After the surface pressure is stabilized at 16-20 mN / m, continue to stand for 5-15 min, and after the time arrives, the belt is moved slightly to make the surface pressure cycle between Π-ΔP and Π+ΔP for 2-4 times, and after the cycle is completed, the belt is returned to the position before the slight belt movement;

[0020] The belt moving speed of the slight belt movement is 1-10 cm / min, and in the Π-ΔP and Π+ΔP, Π is the surface pressure before the slight belt movement (16-20 mN / m), and ΔP is 1-4 mN / m;

[0021] S4, modularly assembling a collagen membrane:

[0022] The vertical pulling method is adopted to transfer the collagen to the substrate under the condition of keeping the surface pressure stable, to form an LB monolayer collagen membrane, and after the LB monolayer collagen membrane on the substrate is washed and dried, the vertical pulling method is adopted again to repeat the above steps, to transfer the collagen to the substrate to form an LB stacked collagen membrane, i.e., a collagen membrane.

[0023] In the present application, the collagen in step S1 is animal-derived collagen. Those skilled in the art can select the animal-derived collagen species for industrial or experimental use as described in the prior art, which can be purchased or prepared by self-preparation. Preferably, the animal-derived collagen is a part of an animal tissue rich in natural collagen, including but not limited to any one of the following: cowhide, pigskin, fishskin, sheepskin, bullfrog skin, mouse tail, bovine Achilles tendon, porcine Achilles tendon, and sheep Achilles tendon.

[0024] In one of the embodiments, the collagen solution for LB film preparation in step S1 has a specific concentration, which can be selected according to the prior art related to LB film preparation of collagen, or can be selected within the range of 0.1-0.7 mg / mL.

[0025] In one of the embodiments, the pH of the collagen solution in step S1 is adjusted. The specific pH value is selected based on the self-assembly of collagen in the present application and the technical purpose. Those skilled in the art can clearly understand the applicable pH range for the conventional self-assembly of collagen according to the prior art, for example, the pH value is adjusted to 5-9. Those skilled in the art should also clearly understand the conventional pH adjustment reagent for collagen without destroying the structure of collagen, for example, acetic acid solution. Further, the step of preparing collagen solution and adjusting the pH of collagen solution can be combined, that is, a certain amount of pH adjustment reagent for collagen is added, for example, collagen is directly added to an aqueous acetic acid solution for dissolution and preparation.

[0026] In one of the embodiments, the additive including an organic solvent in step S1 is an additive that helps the uniform dispersion of the collagen solution in the subphase. The specific selection can be referred to the prior art related to LB film preparation of collagen, which can be a small molecular alcohol solvent (carbon chain length not higher than C4), including but not limited to methanol, ethanol, n-propanol, isopropanol, glycerol, n-butanol. Further, the additive including an organic solvent in step S1 can be only an organic solvent, or an organic solvent and other additives, which include but are not limited to formic acid, trichloroacetic acid, and amide additives (such as N-methyl acetamide). The use method and technical effect of other additives are described in the prior art.

[0027] Please note that the above specific selection and addition amount of collagen, pH adjustment reagent for collagen, organic solvent, and other additives should meet the characterization range of the aggregate state index, that is, the average particle size of the collagen aggregate is not greater than 2000 nm. Those skilled in the art can select appropriate collagen, pH adjustment reagent for collagen, organic solvent, and other additives and their addition amounts based on the technical purpose. On the basis of the specific index given above, the above specific selection and addition amount are all common knowledge in the art.

[0028] In one of the technical solutions, the sub-phase in step S2 includes a metal ion solution with an ionic strength of 0.2-0.3, which can be composed of the metal ion solution alone or together with other sub-phase assistants. Based on the collagen self-assembly in the subject and technical purpose of the present application, the metal ion is selected from transition metal ions / non-transition metal ions that can act on collagen self-assembly, preferably non-transition metal ions such as sodium ions, potassium ions, calcium ions, and magnesium ions. The other sub-phase assistants are sub-phase assistants commonly used in the LB film preparation process, such as PBS buffer, HEPES buffer, etc. The use method and technical effects of other sub-phase assistants are subject to the existing technical documents in the field.

[0029] In this paper, the LB film analyzer is an instrument using Langmuir-Blodgett and Langmuir-Schäfer technology.

[0030] In one of the technical solutions, the LB film analyzer in this paper vertically clamps the substrate according to the standard operation of the vertical pulling method. During the repeated vertical pulling method and the formation of the LB stacked collagen film, the clamping orientation of the substrate can be kept unchanged to form an LB stacked collagen film with uniform orientation; or the clamping orientation of the substrate can be adjusted, i.e. the substrate is rolled and then the next layer of collagen film is transferred, i.e. the clamping orientation of the substrate is adjusted before each collagen is transferred to the substrate, so as to achieve the effect of constructing a 3D structure collagen fiber scaffold.

[0031] In one of the technical solutions, the LB film analyzer in this paper can have 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; or an organic material substrate, including but not limited to any one of a collagen film sheet, a polyvinyl alcohol (PVA) film sheet, and a gelatin sponge sheet.

[0032] In one of the technical solutions, the LB film analyzer in this paper can have any shape, such as a rectangular shape, a square shape, a trapezoidal shape, a circular shape, an oval shape, and a spherical shape.

[0033] Please note that, based on the principle of LB film preparation, the surface area of the substrate should be less than or equal to the surface area set by the film pressing.

[0034] In this paper, the vertical pulling method is a conventional operation of the LB film analyzer, which can be referred to the operation instructions of the LB film analyzer or the existing literature on collagen LB film preparation in the field.

[0035] In one of the technical solutions, the vertical pulling method in step S4 can refer to the existing literature on collagen LB film forming in the field, or can be selected at random within the pulling speed range of 1-10 mm / min.

[0036] In one of the technical solutions, after the LB monolayer collagen film on the substrate is washed and dried, the specific washing and drying method can be the conventional collagen drying method in the field, such as low-temperature drying and vacuum drying. The washing method is the same, such as deionized water washing.

[0037] In one of the preferred technical solutions, the vertical pulling method in step S4 maintains the clamping orientation of the substrate, and forms an LB stacked collagen film with 3-5 layers through repeated formation. It can be applied in the biomedical field, such as used as a skin auxiliary material.

[0038] In one of the preferred technical solutions, the vertical pulling method in step S4 maintains the clamping orientation of the substrate, and forms an LB stacked collagen film with 10-15 layers through repeated formation. It can be applied in the biomedical field, such as used as an artificial keratoprosthesis tissue.

[0039] It should be noted that the number of layers of the LB stacked collagen film in the above preferred technical solutions is limited based on the application purpose, not the limit of the number of layers that can be stacked in the method.

[0040] In one of the preferred technical solutions, the vertical pulling method in step S4 defines the clamping orientation of the substrate as 0° when transferring the first odd-numbered collagen film, and adjusts the clamping orientation of the substrate to roll the substrate by 90° when transferring the first even-numbered collagen film. An LB stacked collagen film with 15-20 layers is formed through repeated formation. It can be applied in the biomedical field, such as used as an artificial tendon tissue.

[0041] In one of the technical solutions, considering the multiple repeated operations in step S4, after the LB stacked collagen film is formed, a sterilization treatment of the formed LB stacked collagen film is further included. The sterilization treatment can be a conventional sterilization treatment in the biological material field, such as irradiation sterilization.

[0042] Based on the technical solution described in the inventor's prior patent "Method for Modular Assembly of Collagen Membranes Using LB Technology" (CN115137882B), there is a clear need for process-level improvements to move from experimental feasibility to engineering manufacturability and large-scale production. Within a known parameter window, interval narrowing and coupling optimization are performed. Without altering the intrinsic properties of the material, the pairing relationship between temperature, ionic strength, and surface pressure path is clearly defined within executable intervals / levels to reduce the probability of interface defects and improve the statistical success rate of D-cycle formation. Based on satisfying the surface pressure balance criterion, two key process steps are introduced: continued static setting after balanced molding and micro-pressure cycling to create a "compression-relaxation" effect. This improves the sufficiency of interfacial molecule spreading and rearrangement, releases residual interfacial stress, and thus significantly improves the integrity of the membrane structure and the stability of the preparation. The statistical caliber and recording dimensions of the "successful membrane" criteria (coverage, defect threshold) and the D-cycle criteria (strip / FFT main peak identification) are unified to improve the comparability and traceability of control experiments.

[0043] By employing the above-described process of the present invention, the occurrence rate of interface defects such as molecular stacking and voids can be effectively reduced without changing the collagen system formulation, thereby improving film yield and D-cycle formation success rate, thus improving batch repeatability and meeting the needs of large-scale and standardized preparation.

[0044] The present invention has the following beneficial effects:

[0045] 1. This invention provides a method for modularly and efficiently assembling D-cycle characteristic biomimetic collagen membranes based on LB technology. This method introduces two key process steps, namely, balanced molding followed by static rest and micro-pressure cycling to form a "compression-relaxation" effect, without changing the collagen formula and mild environmental conditions. These steps enhance the sufficiency of interfacial molecule spreading and rearrangement, and release residual interfacial stress, thereby significantly improving the integrity of the membrane structure and the stability of the preparation. The result is a collagen membrane with higher film-forming yield, D-cycle success rate, and maintained bioactivity. This promotes the assembly of D-cycle characteristic collagen membranes based on LB technology from the sample level to the process level, providing a reliable manufacturing foundation for subsequent applications and scale-up.

[0046] 2. The method of this invention operates under mild conditions, and the resulting collagen membrane retains the unique triple helix structure of undenatured collagen, ensuring the bioactivity of the collagen. After sterilization, it exhibits high safety, with heavy metal content ≤10 mg / kg, and can be widely applied in the biomedical field.

[0047] 3. The collagen membrane prepared by this invention can exhibit anisotropy. Its natural collagen fiber microarchitecture not only maintains the biological and structural integrity of the tissue, but also displays a dual structural feature of 67 nmD periodicity and ordered directional arrangement of its structural modules. Precise regulation of the biomimetic anisotropic collagen fiber microarchitecture has important guiding significance for its application in cell culture and tissue engineering. Attached Figure Description

[0048] Figure 1 This is a photograph documenting the process of preparing a collagen membrane sample in Example 2 of the present invention. The instrument in the picture is an LB membrane analyzer.

[0049] Figure 2 These are Brewster angle (BAM) images of the collagen membrane samples prepared in Comparative Examples 1 and 3 of this invention.

[0050] Figure 3 The image shows the Brewster angle (BAM) image of the collagen membrane samples prepared in Comparative Example 5 and Example 2 of this invention.

[0051] Figure 4 This is a line graph showing the change in transfer ratio (TR) of collagen membrane samples prepared in Comparative Examples 5 and 6 and Examples 1 and 2 of the present invention as a function of the number of layers.

[0052] Figure 5 The images are atomic force microscopy (AFM) scans of the collagen membrane samples prepared in Comparative Example 5 and Example 2 of this invention.

[0053] Figure 6 The images shown are scanning electron microscope (SEM) images of the collagen membrane samples prepared in Comparative Example 5 and Example 2 of this invention. Detailed Implementation

[0054] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. Although it is believed that those skilled in the art will fully understand the following terms, the following definitions are set forth to help illustrate the subject matter disclosed in the present invention.

[0055] In one aspect, the present invention provides a method for modularly and efficiently assembling a biomimetic collagen membrane with D-period characteristics based on LB technology, the main process steps of which are as follows:

[0056] Preparation of S1 and LB collagen solutions for film forming:

[0057] Collagen was prepared into a collagen solution, and then the pH value of the collagen solution was adjusted and an auxiliary agent including organic solvent was added to prepare a collagen solution for LB film preparation with an average particle size of collagen aggregates not greater than 2000 nm.

[0058] S2. Preparation of the substrate LB film:

[0059] The temperature of the LB collagen solution prepared in step S1 is adjusted to 24-26℃. Using a syringe, 500-1500 μL of the LB collagen solution is uniformly dispersed on the subphase surface of the LB membrane analyzer. After standing until the membrane pressure approaches equilibrium, the membrane is pressed at a sheet movement speed of 1-10 cm / min. Finally, the membrane is pressed onto a surface with a surface area of ​​10-20 cm². 2 When the tent panels stop moving, the surface pressure stabilizes at 16~20 mN / m;

[0060] The subphase includes a metal ion solution with an ionic strength of 0.2 to 0.3, and the temperature is controlled at 24 to 26°C, with a pH value of 5 to 9.

[0061] S3, Static rest and micro-pressure cycling:

[0062] After the surface pressure stabilizes at 16~20 mN / m, let it stand for another 5~15 minutes. After the time is up, move the tent slightly to make the surface pressure cycle between Π-ΔP and Π+ΔP 2~4 times. After the cycle is over, the tent returns to the position before the tent was moved slightly.

[0063] Wherein, the moving speed of the micro-moving tent is 1~10 cm / min; in Π-ΔP and Π+ΔP, Π is the surface pressure in front of the micro-moving tent (16~20 mN / m), and ΔP is 1~4 mN / m;

[0064] S4, Modular assembly of collagen membrane:

[0065] Using the vertical lifting method, collagen is transferred onto the substrate while maintaining stable surface pressure to form an LB monolayer collagen film. After the LB monolayer collagen film on the substrate is washed and dried, the vertical lifting method is used again to repeat the above steps to transfer collagen onto the substrate to form an LB superimposed collagen film, thus obtaining the collagen film.

[0066] In this document, the collagen mentioned in step S1 is animal-derived collagen. Those skilled in the art can choose from the types of industrial / experimental animal-derived collagen described in the prior art. It can be purchased commercially or obtained by self-production. Preferably, it is an animal tissue part rich in natural collagen. In one embodiment, it includes, but is not limited to, any one of cowhide, pigskin, fish skin, sheepskin, bullfrog skin, rat tail, cow Achilles tendon, pig Achilles tendon and sheep Achilles tendon.

[0067] In one embodiment, the collagen solution used for LB film formation in step S1 can have a specific collagen concentration that can be found in existing technical documents related to collagen LB film formation in the art, or can be arbitrarily selected within the mass concentration range of 0.1 to 0.7 mg / mL, such as 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, or any range or point value between them.

[0068] In one embodiment, the pH value of the collagen solution adjusted in step S1 is specifically selected based on the collagen self-assembly described in the subject matter and technical objectives of this invention. Those skilled in the art can readily ascertain the applicable pH range for conventional collagen self-assembly based on existing literature, such as adjusting the pH to 5-9, for example, 5, 6, 7, 8, 9, or any range or point value between them. They should also be aware of conventional collagen pH adjustment reagents, such as acetic acid solution, used without damaging the collagen structure. Furthermore, the steps of preparing the collagen solution and adjusting its pH value can be combined, i.e., adding a quantitative concentration of a collagen pH adjustment reagent, for example, directly adding the collagen to an aqueous acetic acid solution for dissolution and preparation.

[0069] In one embodiment, the addition of the auxiliary agent including an organic solvent in step S1 is an auxiliary agent that helps the collagen solution to be uniformly dispersed on the subphase. The specific selection can refer to existing literature on collagen LB film formation in the art, and can be a small molecule alcohol solvent (carbon chain length not higher than C4), including but not limited to methanol, ethanol, n-propanol, isopropanol, glycerol, and n-butanol. Further, the addition of the auxiliary agent including an organic solvent in step S1 can be the addition of only an organic solvent, or the addition of an organic solvent and other auxiliary agents. Other auxiliary agents include, but are not limited to, formic acid, trichloroacetic acid, and amide auxiliary agents (e.g., N-methylacetamide). The usage methods and technical effects of other auxiliary agents are as described in existing technical documents in the art.

[0070] Please note that the specific selection and addition amount of collagen, collagen pH adjustment reagent, organic solvent, and other additives mentioned above should be based on meeting the characterization range of the aggregation state index, that is, the average particle size of collagen aggregates should not be greater than 2000 nm. Those skilled in the art can select appropriate collagen, collagen pH adjustment reagent, organic solvent, other additives and their addition amount based on this technical purpose. Based on the specific indicators already given, the above specific selection and addition amount are all common knowledge in the field.

[0071] In one embodiment, the subphase in step S2 includes a metal ion solution with an ionic strength of 0.2-0.3. This metal ion solution can constitute the subphase alone, or it can be combined with other subphase aids. Based on the collagen self-assembly objective of this invention, the metal ion solution is selected from transition / non-transition metal ions that can act on collagen self-assembly, preferably non-transition metal ions such as sodium, potassium, calcium, and magnesium ions. The other subphase aids are those commonly used in LB film fabrication processes, such as PBS buffer and HEPES buffer. The usage methods and technical effects of these other subphase aids are as described in existing technical documents in the field.

[0072] In this document, the LB membrane analyzer referred to is an instrument employing Langmuir-Blodgett and Langmuir-Schäfer technologies.

[0073] In one embodiment, the LB film analyzer described herein uses a substrate held vertically according to standard operating procedures for the vertical pull-up method. During the repeated vertical pull-up process to form the LB-layered collagen film, the substrate's clamping orientation can be maintained to form LB-layered collagen films with consistent orientation; alternatively, the substrate's clamping orientation can be adjusted, i.e., the substrate can be rolled before transferring the next layer of collagen film (i.e., the substrate's clamping orientation is adjusted before each collagen transfer to the substrate), thereby achieving the effect of constructing a 3D structured collagen fiber scaffold.

[0074] In one embodiment, the substrate of the LB membrane analyzer described herein can be an inorganic material substrate, including but not limited to any one of silicon wafers, mica sheets, glass sheets, cell membranes, aluminum sheets, and titanium sheets; or it can be an organic material substrate, including but not limited to any one of collagen membrane sheets, polyvinyl alcohol (PVA) membrane sheets, and gelatin sponge sheets.

[0075] In one embodiment, the substrate of the LB membrane analyzer described herein can be of any shape, such as rectangular, square, trapezoidal, circular, elliptical, or spherical.

[0076] Please note that, based on the basic principles of LB film fabrication, the surface area of ​​one side of the substrate should be less than or equal to the surface area set for lamination.

[0077] In one embodiment, the tent moving speed in steps S2 and S3 is 1 to 10 cm / min, for example, 1 cm / min, 2 cm / min, 3 cm / min, 4 cm / min, 5 cm / min, 6 cm / min, 7 cm / min, 8 cm / min, 9 cm / min, 10 cm / min or any range or point value between them.

[0078] In one embodiment, the surface pressure described in step S2 is stabilized at 16 to 20 mN / m, for example, 16 mN / m, 17 mN / m, 18 mN / m, 19 mN / m, 20 mN / m or any range or point value between them.

[0079] In one embodiment, after the surface pressure stabilizes at 16~20 mN / m in step S3, the surface is allowed to stand for another 5~15 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes or any range or point value between them.

[0080] In this paper, the vertical lifting method described is a routine operation of the LB membrane analyzer. For details, please refer to the operating instructions of the LB membrane analyzer or existing literature on collagen LB membrane preparation in this field.

[0081] In one embodiment, step S4 uses a vertical lifting method. The specific lifting speed can be referred to in existing literature on collagen LB film formation in the art, or it can be arbitrarily selected within the lifting speed range of 1~10 mm / min.

[0082] In one embodiment, after the LB monolayer collagen film on the substrate is washed and dried in step S4, the specific washing and drying method can be selected from conventional collagen drying methods or techniques in the art, such as low temperature drying or vacuum drying; the washing method is similar, such as washing with deionized water.

[0083] In one preferred embodiment, the vertical lifting method described in step S4, which maintains the clamping orientation of the substrate, and repeatedly forms 3 to 5 layers of LB-layered collagen membrane, can be applied in the biomedical field, for example, as a skin excipient.

[0084] In one preferred embodiment, the vertical lifting method described in step S4 maintains the clamping orientation of the substrate and repeatedly forms an LB-layered collagen membrane with 10 to 15 layers, which can be applied in the biomedical field, for example, as an artificial corneal tissue.

[0085] It should be added that the number of LB-layered collagen membranes described in the above preferred technical solutions is a limitation based on its application purpose, rather than a limitation on the number of layers that can be superimposed in this method.

[0086] In one preferred embodiment, the vertical lifting method described in step S4 defines the substrate clamping orientation as 0° when transferring the collagen membrane for the odd number of times, and the substrate rolling angle as 90° when transferring the collagen membrane for the even number of times, by adjusting the clamping orientation of the substrate, and repeatedly forming LB stacked collagen membranes with 15 to 20 layers, which can be applied in the biomedical field, for example, as artificial tendon tissue.

[0087] In one embodiment, considering the repeated operation in step S4, after forming the LB superimposed collagen film, a sterilization treatment of the formed LB superimposed collagen film is also included. This sterilization treatment can be a conventional sterilization treatment method in the field of biomaterials, such as irradiation sterilization.

[0088] Based on the technical solution described in the inventor's prior patent "Method for Modular Assembly of Collagen Membranes Using LB Technology" (CN115137882B), there is a clear need for process-level improvements to move from experimental feasibility to engineering manufacturability and large-scale production. Within a known parameter window, interval narrowing and coupling optimization are performed. Without altering the intrinsic properties of the material, the pairing relationship between temperature, ionic strength, and surface pressure path is clearly defined within executable intervals / levels to reduce the probability of interface defects and improve the statistical success rate of D-cycle formation. Based on satisfying the surface pressure balance criterion, two key process steps are introduced: continued static setting after balanced molding and micro-pressure cycling to create a "compression-relaxation" effect. This improves the sufficiency of interfacial molecule spreading and rearrangement, releases residual interfacial stress, and thus significantly improves the integrity of the membrane structure and the stability of the preparation. The statistical caliber and recording dimensions of the "successful membrane" criteria (coverage, defect threshold) and the D-cycle criteria (strip / FFT main peak identification) are unified to improve the comparability and traceability of control experiments.

[0089] By employing the above-described process of the present invention, the occurrence rate of interface defects such as molecular stacking and voids can be effectively reduced without changing the collagen system formulation, thereby improving film yield and D-cycle formation success rate, thus improving batch repeatability and meeting the needs of large-scale and standardized preparation.

[0090] The present application will be further explained in detail below with reference to embodiments. However, those skilled in the art should understand that these embodiments are provided for illustrative purposes only and are not intended to limit the present application.

[0091] Example

[0092] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of this application. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all commercially available conventional products. This application should not be construed as being limited to the specific embodiments described.

[0093] 1. Raw materials and instruments

[0094] Collagen, acid-soluble type I bovine skin-derived collagen;

[0095] The LB membrane analyzer (JML04) includes a Langmuir tank, a compression grid, a Wilhelmy plate surface pressure sensor, a temperature and wind protection system, and a substrate transfer mechanism.

[0096] 2. Preparation method

[0097] Preparation of S1 and LB collagen solutions for film forming:

[0098] By weight, 1 part of collagen (theoretical weight, which can also be regarded as dry weight) was mixed with 1800 parts of 0.1 mol / L aqueous acetic acid solution and 200 parts of isopropanol, and stirred at 4 ℃ for 8 h to prepare a collagen solution for LB film preparation with an average particle size of collagen aggregates of about 1500~1600 nm.

[0099] S2. Preparation of the substrate LB film:

[0100] The temperature of the LB collagen solution prepared in step S1 was adjusted to 24-26 °C. Using a syringe, 1000 μL of the LB collagen solution was uniformly dispersed on the subphase surface of the LB membrane analyzer. After standing until the membrane pressure approached equilibrium, the membrane was pressed at a sheet movement speed of 5 cm / min. Finally, a membrane with a surface area of ​​16 cm² was formed. 2 When the tent panels stop moving, the surface pressure stabilizes at 20 mN / m;

[0101] The subphase is a sodium ion solution with an ionic strength of 0.2 to 0.3, and the temperature is controlled at 24 to 26 °C with a pH of 7.4.

[0102] S3, Static rest and micro-pressure cycling:

[0103] After the surface pressure stabilizes at 20 mN / m, let it stand for another 5 to 15 minutes. After the time is up, move the tent slightly to make the surface pressure cycle between Π-ΔP and Π+ΔP 2 to 4 times. After the cycle is over, the tent returns to the position before the slight movement of the tent.

[0104] Wherein, the moving speed of the micro-moving tent is 5 cm / min; in Π-ΔP and Π+ΔP, Π is the surface pressure in front of the micro-moving tent (5 mN / m), and ΔP is 1~4 mN / m;

[0105] S4, Modular assembly of collagen membrane:

[0106] Using the vertical lifting method, collagen is transferred onto the substrate while maintaining stable surface pressure to form an LB monolayer collagen film. After the LB monolayer collagen film on the substrate is washed and dried, the vertical lifting method is used again to repeat the above steps to transfer collagen onto the substrate to form an LB superimposed collagen film, thus obtaining the collagen film.

[0107] For ease of testing, the collagen membranes prepared in the following examples and comparative examples were prepared by repeatedly forming 10-layer LB stacked collagen membranes while maintaining the clamping orientation of the substrate, and were then vacuum dried at 25 °C for 24 h to remove residual moisture, as well as irradiated for sterilization.

[0108] 3. Testing Methods

[0109] The surface pressure and transfer ratio (TR) of collagen membranes were determined using an LB membrane analyzer (JML04).

[0110] Film yield (%): Number of successful film samples / Total number of samples × 100. Criteria for successful film: Coverage of ≥90% on the effective area, with no large-area peeling / breakage; Defect area ratio ≤10% according to image binarization statistics.

[0111] D-cycle success rate (%): Area of ​​field of view with approximately 67 nm bands in the AFM image field of view / total area × 100; ≥10 independent AFM images are counted per sample.

[0112] Orientation consistency: The coherency index is calculated based on OrientationJ (or equivalent software); the statistical methods used in the reports are consistent.

[0113] Transfer ratio (TR) and multilayer homogeneity: TR was recorded layer by layer; if necessary, UV-Vis (e.g., 318 nm) was used to assess the linear correlation between the number of layers and absorption.

[0114] Comparative Examples 1-4

[0115] First, comparative examples 1-4 were used to verify whether to continue the settling process after surface pressure stabilization in step S3 and the effect of the settling time on the film yield (%) and the success rate of the D cycle (%). No micro-pressure cycling (i.e., no micro-movement of the tent sheet) was performed in any of the comparative examples. LB-layered collagen films were prepared as samples. The specific process parameters used in each comparative example are shown in Table 1 below.

[0116] Table 1. Effect of settling time on film yield and D-cycle success rate in Comparative Examples 1-4

[0117]

[0118] In Table 1, the temperature (°C) refers to the temperature of the collagen solution used for LB film preparation and the subphase temperature in step S2. Comparative Example 1, with a settling time (min) of 0, means that no settling was performed and step S4 was executed directly. The number of samples n is n samples prepared for each comparative example.

[0119] Based on the results in Table 1, under the common conditions of a temperature of 25 ℃ and a subphase ionic strength of 0.20, the two indicators of Comparative Examples 1-3 generally showed an upward trend from no settling to settling for 10 min. The reason for this is speculated to be that appropriate settling after equilibration allows interfacial molecules to continue spreading and rearranging in an orderly manner, reducing the influence of interfacial micro-disturbances, thereby reducing the number of stacked and void regions, making the strips more continuous, and leading to a simultaneous increase in D-cycle success rate and film yield. However, in Comparative Example 4, when the settling time was extended to 15 min, excessive aging / local aggregation or slight dehydration due to changes in interfacial moisture content may occur, which is inconsistent with the subsequent transfer sequence and easily generates new micro-defects, causing the two indicators to decrease compared to Comparative Example 3. The above conclusions are used to determine the appropriate range of settling time (preferably about 10 min).

[0120] Examples 1-2, Comparative Examples 5-6

[0121] Based on the optimal 10-minute settling time in Comparative Examples 1-4, Examples 1-2 and Comparative Examples 5-6 verified the effects of whether micro-pressure cycling was performed and the magnitude of surface pressure cycling variation after 10 minutes of settling in step S3 on the film yield (%) and D-cycle success rate (%). LB-layered collagen films were prepared as samples. The specific process parameters used in each example and comparative example are shown in Table 2 below:

[0122] Table 2. Effects of micro-pressure cycling on film yield and orientation consistency in Examples 1-2 and Comparative Examples 5-6.

[0123]

[0124] In Table 2, the temperature (°C) represents the temperature of the collagen solution used for LB film formation and the subphase temperature in step S2. Comparative Example 5, where ΔP (mN / m) is 0, means that micro-pressure cycling was not performed, and step S4 was executed directly. The sample number n is n samples prepared for each comparative example and embodiment.

[0125] Based on the results in Table 2, it can be seen that, under the condition of only changing ΔP, setting a small-amplitude (e.g., 1~4 mN / m) pressure cycle significantly improves both indicators compared to no micro-amplitude pressure cycle. The possible mechanism is that short-term micro-amplitude reciprocating modulation near the target surface pressure can promote interfacial molecular rearrangement and stress release, weakening local shear and edge tension gradients, thereby reducing the incidence of defects such as stacking / voids and making the stripe direction more concentrated, resulting in improved film yield and enhanced orientation consistency. However, in Comparative Example 6, when ΔP is further increased, the interface is excessively disturbed, easily introducing local tearing, ripples, or flow stripes, leading to the disruption of the ordered structure and a decline in related indicators. The above analysis is used to determine the suitable range for ΔP (preferably approximately 4 mN / m).

[0126] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for modularly and efficiently assembling a biomimetic collagen membrane with D-periodic features based on LB technology, characterized in that... The main process steps are as follows: Preparation of S1 and LB collagen solutions for film forming: Collagen was prepared into a collagen solution, and then the pH value of the collagen solution was adjusted and additives including organic solvents were added to prepare a collagen solution for LB film preparation with an average particle size of collagen aggregates not greater than 2000 nm. S2. Preparation of the substrate LB film: The temperature of the LB collagen solution prepared in step S1 is adjusted to 24-26℃. Using a syringe, 500-1500 μL of the LB collagen solution is uniformly dispersed on the subphase surface of the LB membrane analyzer. After standing until the membrane pressure approaches equilibrium, the membrane is pressed at a sheet movement speed of 1-10 cm / min. Finally, the membrane is pressed onto a surface with a surface area of ​​10-20 cm². 2 When the tent panels stop moving, the surface pressure stabilizes at 16~20 mN / m; The subphase includes a metal ion solution with an ionic strength of 0.2 to 0.3, and the temperature is controlled at 24 to 26°C, with a pH value of 5 to 9. S3, Static rest and micro-pressure cycling: After the surface pressure stabilizes at 16~20 mN / m, let it stand for another 5~15 minutes. After the time is up, move the tent slightly to make the surface pressure cycle between Π-ΔP and Π+ΔP 2~4 times. After the cycle is over, the tent returns to the position before the tent was moved slightly. Wherein, the moving speed of the micro-moving tent is 1~10 cm / min; in Π-ΔP and Π+ΔP, Π is the surface pressure in front of the micro-moving tent, and ΔP is 1~4 mN / m; S4, Modular assembly of collagen membrane: Using the vertical lifting method, collagen is transferred onto the substrate while maintaining stable surface pressure to form an LB monolayer collagen film. After the LB monolayer collagen film on the substrate is washed and dried, the vertical lifting method is used again to repeat the above steps to transfer collagen onto the substrate to form an LB superimposed collagen film, thus obtaining the collagen film.

2. The method according to claim 1, characterized in that: The collagen mentioned in step S1 is animal-derived collagen.

3. The method according to claim 1, characterized in that: The collagen mass concentration in the LB film-forming collagen solution described in step S1 is 0.1~0.7 mg / mL.

4. The method according to claim 1, characterized in that: The addition of an auxiliary agent in step S1 includes an organic solvent, which is a small molecule alcohol solvent with a carbon chain length not higher than C4.

5. The method according to claim 1, characterized in that: The addition of an auxiliary agent including an organic solvent in step S1 refers to adding an organic solvent and other auxiliary agents, including at least one of formic acid, trichloroacetic acid, and amides.

6. The method according to claim 1, characterized in that: The subphase mentioned in step S2 includes a metal ion solution with an ionic strength of 0.2 to 0.3, wherein the metal ions in the metal ion solution include at least one of sodium ions, potassium ions, calcium ions, and magnesium ions.

7. The method according to claim 1, characterized in that: The subphase mentioned in step S2 includes a metal ion solution with an ionic strength of 0.2 to 0.

3. The subphase is composed of the metal ion solution and other subphase aids. The other subphase aids include at least one of PBS buffer and HEPES buffer.

8. The method according to claim 1, characterized in that: Step S4, after forming the LB superimposed collagen membrane, also includes sterilization of the formed LB superimposed collagen membrane.

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