Method for producing collagen fiber membrane and collagen fiber membrane

Abstract

The application provides a preparation method of a collagen fiber membrane with orientation, which accelerates the time of polymer distribution rearrangement and greatly improves the efficiency through a collagen micro-orientation control technology of negative pressure gravity assisted fluid mechanics or a certain directional electric field or magnetic field; therefore, through the technology in the application, the collagen fibers in the collagen biomaterial can be more completely and uniformly oriented and regularly arranged, and the optical performance, mechanical performance and many other aspects are significantly optimized and improved, and the application range of the collagen membrane is greatly expanded.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and particularly to a method for preparing a collagen fiber membrane and the collagen fiber membrane itself. Background Technology

[0002] Collagen, a major component of many organs in the human body (such as the cornea and cartilage), exhibits orientation, enabling it to perform different functions in different organs. Therefore, as an important biomaterial, the ability to design the orientation and arrangement of collagen raw materials according to specific needs is crucial. However, current technologies cannot yet fully achieve this goal.

[0003] The main existing methods for preparing oriented collagen are as follows:

[0004] 1. Oriented Collagen Gel (Patent No.: CN 102341436 A)

[0005] General approach: Utilize fluid dynamics to influence the aggregation of collagen fibers, thereby preparing flow-oriented collagen gels.

[0006] The main drawback is that inducing collagen fiber alignment through shear and stretching flow when a high-concentration collagen monomer solution is directed onto the substrate is too random and may lead to uneven stress. At the same time, this force is too weak to completely change the orientation and alignment of collagen fibers, and the effect may not be ideal.

[0007] 2. A high-strength tissue regeneration membrane and its preparation method (Patent No.: CN 104857578 A)

[0008] The general approach is to use collagen as the raw material and obtain a certain degree of ordered arrangement of collagen fibers through gradient dialysis, thereby improving the mechanical properties of the material.

[0009] The main drawbacks are: 1. Although the mechanical properties of the collagen membrane prepared by gradient dialysis are improved, it is not transparent, indicating that only a certain degree of orderly arrangement has been achieved, and it is far from meeting the application requirements of multiple clinical scenarios; 2. The gradient dialysis method is time-consuming and requires frequent operation, which is not conducive to large-scale production.

[0010] 3. Adhesive raw materials and manufacturing methods of adhesive raw materials (Patent No.: CN 103384536 A)

[0011] The general approach involves extruding collagen through a nozzle (e.g., 0.38mm) and shaping it into straight lines, spirals, or other forms. The main drawback is that it only provides coarse millimeter-level control over the collagen fibers, lacking the precise nanoscale control (stripe structure and orientation of individual collagen fibers) required for the collagen fibers. Summary of the Invention

[0012] This invention aims to achieve orientation control and regular arrangement of collagen fibers, thereby optimizing the mechanical and optical properties of collagen biomaterials and addressing their main shortcomings in application.

[0013] To achieve the above objectives, the present invention provides a method for preparing a collagen fiber membrane, comprising the following steps:

[0014] 1) Obtaining collagen fibers;

[0015] 2) Prepare a collagen monomer solution to form a collagen liquid crystal, wherein the collagen liquid crystal has a collagen nanoscale striation structure;

[0016] 3) Collagen filament formation: Collagen filaments are formed by buffering the collagen monomer solution with a pH value greater than or equal to 7. The buffer solution is not specifically limited; common phosphate-containing buffer solutions are acceptable. In this article, "high pH value" refers to a pH value greater than or equal to 7.

[0017] 4) Ultrasonic treatment of collagen fibers;

[0018] 5) Collagen fiber orientation: using negative pressure-assisted fluid dynamics or applying an electric or magnetic field in a certain direction;

[0019] The orientation and arrangement of collagen fibers were observed under a microscope until the length of the non-linear segments did not exceed 5% of the total length and the orientation consistency reached more than 90%.

[0020] 6) The orientation of collagen fibers is fixed by means of collagen cross-linking.

[0021] Furthermore, in step 2), the concentration of the collagen monomer solution is 80 mg / ml-120 mg / ml.

[0022] Further, in step 4), the collagen solution is subjected to ultrasonic treatment at 4°C. The ultrasonic treatment consists of two 10-minute ultrasonic pulses with a 10-minute pause in between.

[0023] Furthermore, in step 5), a negative pressure suction device is used to generate external force, which helps the collagen fibers to orient and rearrange by creating a flowing liquid phase in the collagen fiber solution.

[0024] Furthermore, in step 5), when applying a magnetic field in a certain direction, the magnetic field direction is set to be circular, and the magnetic field strength is equal at each point of the circle.

[0025] Furthermore, when collagen fibers are oriented by applying a magnetic field or electric field in a certain direction, in step 1) when obtaining collagen fibers, the collagen fibers are converted into short segments.

[0026] Furthermore, after the orientation is completed in step 5), the short collagen fibers with consistent orientation are reassembled into long collagen fibers by liquid crystal technology and PBS dialysis. During this process, a magnetic field and electric field of a certain direction are maintained.

[0027] Furthermore, after ultrasonic treatment, electromagnetic induction particles are added to the collagen fibers, and then step 5) is performed to apply a magnetic field or electric field in a certain direction to complete the orientation of the collagen fibers.

[0028] Furthermore, by adding electromagnetically induced particles and combining them with 3D printing technology, a multi-layered structure is formed, with each layer of the collagen membrane having a different orientation.

[0029] Specifically, the present invention achieves collagen fiber orientation in the following ways:

[0030] The first implementation method is as follows:

[0031] 1) Acquisition of collagen fibers: There is no particular limitation on the source of collagen: Collagen can be extracted from human or animal tissues, collagen obtained by gene recombination technology, or untreated terminal collagen for the purpose of inhibiting antigenicity.

[0032] 2) Prepare a high-concentration collagen monomer solution to form a collagen liquid crystal: thereby forming a microscopic striation structure (nanoscale) of collagen, which is the basis of the mechanical properties of collagen fibers.

[0033] 3) Collagen fiber formation: The collagen solution is guided into the microchannels and buffered with a high pH or neutral solution (the buffer solution is not specifically limited here, including phosphate buffer solution, etc.), thereby causing collagen fiber formation. At this time, the collagen fibers are affected by shear force and tensile flow.

[0034] 4) Ultrasonic treatment of collagen fibers to prevent them from tangling.

[0035] 5) Negative pressure-assisted fluid dynamics promotes collagen fiber orientation: Introducing a negative pressure suction device into the above system, the external force generated by the suction device is used to accelerate the flow of collagen monomer solution in a liquid phase, thereby strengthening the shear and tensile forces in a directional direction, which greatly improves the degree of relaxation of collagen fibers under shear and tensile flow.

[0036] 6) By repeatedly guiding the stretching under negative pressure and observing the orientation and arrangement of collagen fibers under a microscope, until the collagen fibers are fully stretched (the length of non-straight segments does not exceed 5% of the total length) and the orientation consistency reaches more than 90% (uniform orientation), the fine control of collagen fiber orientation at the nanometer level is achieved.

[0037] 7) The orientation of the collagen formed above is fixed by means of collagen cross-linking.

[0038] Furthermore, a negative pressure suction device is used to generate external force, which helps to orient and rearrange the collagen fibers by creating a flowing liquid phase in the collagen fiber solution.

[0039] Second implementation method:

[0040] 1) Acquisition of collagen fibers: There is no particular limitation on the source of collagen: Collagen can be extracted from human or animal tissues, collagen obtained by gene recombination technology, or untreated terminal collagen for the purpose of inhibiting antigenicity.

[0041] 2) Collagen fibers are prepared into short-segment fibers by enzymatic digestion and / or acid dissolution. Short-segment fibers are more rigid than long-segment fibers, have better response to electric and magnetic fields, and are easier to control in orientation.

[0042] 3) Prepare a high-concentration collagen monomer solution to form collagen liquid crystal: Liquid crystals are easily deformed and are very sensitive to electric and magnetic fields. That is, when liquid crystals are affected by electric and magnetic fields, they undergo considerable deformation, and the orientation of liquid crystal molecules also changes.

[0043] 4) Applying a strong magnetic field or electric field in a certain direction to the collagen short-segment liquid crystal fiber solution drives the collagen short-segment fibers to change orientation in the electromagnetic field, which can precisely control the orientation of short-segment collagen fibers at the nanoscale.

[0044] 5) Collagen self-assembly and formation of oriented collagen fibers: Building upon the above, short collagen fibers with consistent orientation are reassembled into long collagen fibers using liquid crystal technology and PBS dialysis. During this process, the original electric / magnetic field directions are maintained, thus achieving precise nanoscale control over the orientation of collagen fibers.

[0045] 6) The orientation of the collagen formed above is fixed by means of collagen cross-linking.

[0046] To further enhance the orientation and sorting properties of collagen fibers, we chose to add electromagnetic induction particles to pre-chemically modify the collagen fibers, thereby enhancing their responsiveness in electric or magnetic fields and achieving better efficiency in the orientation and regular sorting of collagen fibers.

[0047] 1) Acquisition of collagen fibers: There is no particular limitation on the source of collagen: Collagen can be extracted from human or animal tissues, collagen obtained by gene recombination technology, or untreated terminal collagen for the purpose of inhibiting antigenicity.

[0048] 2) Dialysis with PBS yielded a collagen fiber solution with a microscopic striation structure (nanoscale).

[0049] 3) Ultrasonic treatment of collagen fibers to prevent them from tangling.

[0050] 4) Electro- or magnetically responsive particles are loaded onto collagen fibers through methods such as direct adsorption or chemical modification.

[0051] 5) By designing and adjusting the direction and pattern of electric or magnetic fields, the extension and rotation of collagen fibers can be precisely controlled at the nanoscale, while the micro-orientation of collagen liquid crystals can be controlled, so that the collagen orientation can exhibit diverse characteristics that can be adjusted according to changes in demand.

[0052] 6) The orientation of the collagen formed above is fixed by means of collagen cross-linking.

[0053] Furthermore, the present invention also provides a collagen fiber membrane, which is obtained by the above preparation method. This collagen fiber membrane can be used in corneas, ligaments, tendons, etc.

[0054] Compared with the prior art, the present invention has the following advantages:

[0055] The preparation method of this invention first involves preparing a high-concentration collagen fiber liquid to form a liquid crystal and then using a high-pH buffer solution to allow it to self-assemble into a microscopic striation state. Next, it is ultrasonically treated to prevent the collagen fibers from tangling. Then, it is oriented using an orientation method, and finally, the collagen orientation is fixed by collagen cross-linking, thereby obtaining a collagen membrane with high orientation and stability.

[0056] Traditional fluid dynamics methods for inducing collagen fiber alignment are often inefficient, time-consuming, and result in uneven fiber orientation. This invention accelerates polymer distribution and rearrangement time by using a negative pressure-assisted flowing liquid phase, significantly improving efficiency. Furthermore, the continuous presence of negative pressure allows collagen fibers to spread more fully and uniformly in the flowing phase. Therefore, through the technology of this invention, collagen fibers in collagen biomaterials can achieve more thorough and uniform orientation and regular arrangement, resulting in significant optimization and improvement in optical and mechanical properties, greatly expanding the application range of collagen.

[0057] This invention also combines collagen liquid crystal technology with electric / magnetic field technology, making full use of the sensitivity of liquid crystal to electromagnetic fields and its easy deformation, so that the degree of control of the electromagnetic field on the orientation of collagen is significantly improved compared with traditional methods; at the same time, liquid crystal technology makes it easier for collagen fibers to form micro-stripe structures, which are the most basic structures for the mechanical properties of collagen fibers.

[0058] Furthermore, this invention introduces electromagnetic nanoparticles and combines them with 3D printing technology, enabling variability and on-demand diversity in collagen fiber orientation (such as orthogonal grid patterns in the cornea, multi-ring shapes in cartilage, etc.). Simultaneously, its integration with mass-production technologies like 3D printing makes industrial-scale production possible. Therefore, this invention solves a universal problem and can be applied to all collagen orientation-related fields, including the cornea, cartilage, ligaments, and tendons, with a wide-ranging impact.

[0059] To make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0060] Figure 1 It has a cross-striped structure of collagen liquid crystal fibers.

[0061] Figure 2 This is a diagram showing the state of collagen fibers from an unused state to an oriented state.

[0062] Figure 3 This is a diagram showing the oriented and ordered arrangement of liquid crystal collagen fibers at a concentration of 120 mg / mL.

[0063] Figure 4 This is a graph that assesses the relationship between negative pressure and collagen orientation.

[0064] Figure 5 This is a graph that assesses the relationship between other factors and the collagen orientation effect. Detailed Implementation

[0065] Example 1: Negative pressure assisted fluid dynamics technology → Regular linear arrangement

[0066] 1. Obtaining in vitro recombinant human collagen

[0067] Human recombinant collagen is most suitable for this embodiment, but collagen from other sources can also be used; the method for preparing human recombinant collagen is a common method and is not particularly limited.

[0068] 2. Preparation of collagen fiber liquid crystal solution and formation of collagen fiber filaments: The striation microstructure of collagen was stably constructed through two key steps: preparation of high-concentration collagen solution and PBS dialysis.

[0069] 1) We adjusted the concentration of the recombinant human collagen solution to 80 mg / mL (based on our experiments, we found that when the concentration reaches 80 mg / mL, it can form an ordered collagen-like liquid crystal film through self-assembly; the acetic acid solution of collagen exhibits a cholesteric liquid crystal texture in the high concentration range), as shown in the attached figure. Figure 1 As shown.

[0070] 2) The collagen acid solution was adjusted to pH 7.0-7.4 by dialysis with phosphate-buffered saline (PBS) and gelled at 37°C. The collagen solution was transferred onto a substrate and buffered with a high-pH solution. During the PBS dialysis process to obtain the collagen gel, collagen molecules have the ability to self-assemble, forming a structure similar to the regular collagen fibers in living organisms. The process of collagen inducing growth and aggregation into fibers in vitro was observed by placing small amounts on glass slides at different time points and incubating them at 30-37°C, then observing their growth under a microscope.

[0071] Experiments revealed that during dialysis of collagen acid solution with PBS, the collagen solution gradually transitioned from acidic to neutral. The original random coil structure of collagen fibers was gradually opened up. As external conditions shifted towards biomimetic conditions, collagen molecules continuously formed axial connections, and their transverse interactions gradually strengthened, ultimately forming collagen fibers with a striation structure. (See attached image) Figure 2 As shown.

[0072] The PBS-collagen gel obtained in this example, observed under an electron microscope, exhibits a rod-like structure, with individual fibers mostly ranging from 30 to 60 nm in diameter. Periodic striations are visible on the fibers, consistent with collagen fibers found in living organisms. The rod-like structure of collagen fibers possesses high strength and modulus, thus the gel exhibits high tensile strength on a macroscopic scale.

[0073] 3. Ultrasonic treatment to prevent collagen fibers from tangling.

[0074] To reduce collagen fiber tangling, the collagen solution was sonicated at 4°C. The sonication consisted of two 10-minute ultrasonic pulses with a 10-minute pause in between for observation, thus reducing the difficulty of subsequent processing. Furthermore, the literature indicates that the sonication process does not damage the triple helix characteristics of the molecules.

[0075] 4. Utilizing negative pressure-assisted fluid dynamics to influence collagen fibers

[0076] First, the collagen solution is transferred onto the substrate and buffered with a high-pH solution (the buffer solution is not specifically limited and can include phosphate buffer, etc.). Then, a negative pressure aspirator (0.1-1000 kPa) is introduced into the system, with a water injection rate of 0.1 ml / h-1,000,000 ml / h. The negative pressure and injection rate are determined based on the viscosity and volume of the collagen solution to be treated, as well as the available equipment.

[0077] The flow of collagen monomer solution is accelerated by using the external force generated by a negative pressure suction device. By adjusting the negative pressure value, the shear and tensile forces are strengthened in a directional direction, thereby greatly improving the relaxation of collagen fibers under shear and tensile flow, thus achieving the orientation and alignment of collagen fibers. The fibers can be oriented with the flow direction of PBS. By controlling the direction of PBS flow, the orientation of collagen fibers can be controlled. The orientation direction of collagen fibers in PBS is affected by factors such as injection speed (e.g., the higher the injection speed, the greater the fluid shear force, and the faster the orientation control) and the viscosity of collagen fibers (the higher the viscosity, the more difficult the orientation control; we address this issue by increasing the injection speed).

[0078] The key components of the negative pressure-assisted orientation control device include at least: a negative pressure component, a microchannel module, a fluid sealing chamber component, a waste liquid tank component, and corresponding connecting pipes.

[0079] 1) Negative pressure assembly: Includes all types of negative pressure pumps and manual negative pressure devices, which are connected to fluid sealing chamber assemblies and micropipe assemblies.

[0080] 2) Microchannel assembly: Various shapes as needed: straight, ring, etc.; slope: 0° to 90°; the microchannels should be as smooth and even as possible to minimize the adverse effects of surface finish and geometry on fiber orientation; the microchannel assembly is placed in a fluid-sealed chamber assembly.

[0081] 3) Fluid-sealed chamber assembly: It consists of three parts: inlet, chamber body and outlet, and can be fitted and installed with different micro-pipe assembly; it is connected to the negative pressure assembly through the outlet.

[0082] 4) Waste liquid tank assembly: used to collect waste liquid flowing through the system and connected to the negative pressure assembly.

[0083] 5. Repeated negative pressure guided stretching to achieve orientation control: Due to the flexibility and deformability of collagen, repeated negative pressure guided stretching was performed more than 20 times in the experiment until the collagen fibers were fully stretched and the orientation consistency reached more than 90%.

[0084] 6. The collagen orientation formed above is fixed by means of collagen cross-linking:

[0085] Add 0.2% concentration of glutaraldehyde (glutaraldehyde crosslinking agent) to the sample to allow it to fully penetrate and react with all collagen fibers inside and on the surface for 2 hours. Glutaraldehyde crosslinks collagen by reacting the aldehyde group with the amino residue to form -C=N- crosslinks, making it a gel and effectively improving the collagen's resistance to degradation.

[0086] There are no specific restrictions on the crosslinking agents and crosslinking procedures here, including glutaraldehyde (GTA), genipin, diphenylphosphine azide (DPPA), dimethylaminopropyl terminal ethyl carbodiimide (DEC) and terminal hydroxy succinimide (NHS), carbodiimide (EDC), butanediol-glycidyl ether (BD), hexamethylene diisocyanate (HMDIC), etc.; however, non-toxic or low-toxic chemical crosslinking agents are preferred.

[0087] 7. Comparison of the effects of negative pressure assisted fluid dynamics technology and ordinary fluid dynamics technology without negative pressure on collagen fiber orientation.

[0088] The application of non-negative pressure conventional fluid dynamics technology in collagen fiber orientation, as described in Oriented Collagen Gel (Patent No.: CN 102341436 A); we repeated the experiments of this patent and found that it is difficult to achieve microscopic control of collagen fibers by relying solely on natural liquid flow or a water column (1 mm) from a syringe, and the obtained collagen fiber orientation consistency is no higher than 20% (multiple repetitions).

[0089] Negative pressure-assisted fluid dynamics achieves a qualitative improvement in both the precision and direction of negative pressure control. It not only significantly expands the negative pressure control range to achieve wide-ranging control of fluid velocity and quantifiable precision control, but also allows for convenient and stable repeated operation until a satisfactory orientation effect is achieved. Observations show that the negative pressure-assisted fluid dynamics technology of this invention can achieve a microscopic orientation uniformity of over 80% for collagen fibers, and shorten the time to achieve collagen fiber uniformity by at least 10% (controllable).

[0090] 8. Evaluation indicators: The most important evaluation indicators for collagen fiber orientation and regular arrangement are fiber orientation consistency (e.g., fiber orientation consistency reaches more than 90%) and stretching degree (e.g., the length of non-straight segments accounts for less than 10% of the total length).

[0091] 9. Evaluate the relationship between negative pressure and collagen orientation effects (e.g., Figure 4 Formula: Negative pressure and collagen orientation are positively correlated. However, after the negative pressure reaches a certain level, the degree of collagen orientation basically stops increasing. This is because after the negative pressure reaches a certain level, the container is basically in a vacuum state, and further increasing the negative pressure cannot continue to increase the liquid flow rate.

[0092] 10. Evaluate the relationship between other factors and the collagen orientation effect (e.g., Figure 5 Formula): It was found that in addition to negative pressure having a direct impact on collagen orientation, the following factors also affect collagen orientation, but their influence is smaller than that of negative pressure: a. Flow rate (affects shear force in the channel system) b. Fiber length c. Solution viscosity (can also affect shear force, but much less than the influence of flow rate).

[0093] Example 2: Strong electromagnetic-assisted orientation control technology → Circular arrangement (corresponding organs: such as cartilage, etc.)

[0094] 1. Collagen derived from human autologous tissue

[0095] Extracting autologous tissue from the patient, such as tendons, fascia lata, Achilles tendons, ligaments, or skin (the source of collagen is not particularly limited here; any extractable tissue containing collagen clinically is suitable for this invention). Of course, there is no particular limitation on the source of collagen; recombinant human collagen or collagen from other suitable sources can be used.

[0096] Using autologous tissue from the patient, any tissue (weighing more than 0.1g) from tendons, fascia lata, Achilles tendons, ligaments, or skin was obtained for collagen extraction. The extraction reaction temperature was controlled at 3℃-5℃. Fat and fascia were removed, and the tissue was chopped and soaked in sodium carbonate solution for 2 hours. After rinsing with distilled water, impurities and proteins were removed with Tris-HCl buffer solution containing 1M NaCl (pH=7.5). Then, the tissue was digested with pepsin in 0.3% acetase solution (P:T=1:10) for 48-72 hours. Afterward, the tissue was centrifuged at high speed (200rpm, 20min, -4℃) to obtain the supernatant, which yielded crude collagen extract.

[0097] Crude collagen was purified by a fractional precipitation method involving high-speed centrifugation, NaCl salting-out, and dialysis. The enzymes were inactivated with 1% H₂O₂ solution for 4 hours, followed by NaCl salting-out for 12-18 hours, and then soaking in Tris (pH=7) for 24 hours. Dialysis was performed for 6 days, with the dialysate concentration decreasing each time, and finally, dialysis with distilled water for 3 days to obtain high-purity collagen.

[0098] This section only provides preferred methods for collagen extraction; there are no particular limitations, and all effective extraction methods can be used.

[0099] 2. Preparation of high-concentration liquid crystal solution

[0100] The collagen short-segment fibers prepared by the aforementioned enzymatic digestion and acid dissolution are more rigid than long-segment fibers, have better response to electric and magnetic fields, and are easier to control in orientation. In order to further enhance their electromagnetic induction (so as to minimize the intensity of the applied electric or magnetic field), we prepared the collagen short-segment fibers into a high-concentration solution, making them into a liquid crystal state. Liquid crystal materials are electromagnetically sensitive, easily deformable, and easy to control in orientation.

[0101] We prepared the collagen solution at a concentration of 120 mg / ml (experimental results showed that the high-concentration collagen fiber solution exhibited a more pronounced liquid crystal morphology, and this collagen liquid crystal was more sensitive to magnetic and electric fields). Figure 3As shown in the figure, at this point, the collagen fibers in the collagen solution have already exhibited a liquid crystal state. The short collagen fibers in the liquid crystal state are very sensitive to electric and magnetic fields. That is, the liquid crystal undergoes considerable deformation under the influence of electric and magnetic fields, and the orientation of the liquid crystal molecules also changes.

[0102] 3. Apply a strong magnetic moment to orient the short collagen fibers in the collagen solution (without adding any electrically or magnetically responsive particles, but only by applying a strong electric / magnetic field to affect the orientation of the collagen fibers).

[0103] 1) The above-mentioned collagen short-segment fiber solution was placed between a pair of magnetic cores, with applied magnetic field strengths of 1T, 3T, and 6T for 30 minutes, respectively, to drive the collagen short-segment fibers in the solution. Observations showed that collagen molecules moved and aligned in the magnetic field, and the reaction rate and orientation changes of the collagen liquid crystal short-segment fibers increased with increasing magnetic field strength. Furthermore, we found that in addition to the direct effect of the magnetic field on the collagen fibers, the interaction between collagen fibers also accelerated the alignment process. Therefore, the relative aggregation of collagen molecules is beneficial to enhancing the effect of the magnetic field.

[0104] 2) By adjusting the direction of the electric field strength, magnetic field strength, or external force, the orientation properties of collagen liquid crystals can be controlled, allowing collagen orientation to exhibit diverse characteristics and adjust according to changes in demand. In this example, to construct a ring-shaped collagen morphology, we set the magnetic field direction to be ring-shaped, with the magnetic field strength being equal at every point in the ring. During the experiment, it was observed that the short collagen fibers under the action of the magnetic field underwent deformation and orientation changes, gradually arranging into a ring shape. During the process, appropriate manual or magnetic bead stirring is beneficial to ensure a more uniform distribution of collagen fibers.

[0105] 4. By dialysis with PBS, the short fibers in the collagen solution self-assemble into long fibers based on the previously formed consistent orientation, resulting in more stable and larger-scale cross-stretched collagen long fibers.

[0106] A high-pH buffer is used to buffer it (the buffer is not specifically limited, and can include phosphate buffer, etc.). During the process of obtaining collagen gel by PBS dialysis, collagen molecules have the ability to self-assemble, forming a structure similar to the regular structure of collagen fibers in living organisms. Due to the pretreatment of the orientation consistency of short fiber segments, the orientation consistency and regular arrangement are even better when they self-assemble.

[0107] 5. Fix the orientation of the collagen formed above by means of collagen cross-linking: (This step is the same as in Example 1)

[0108] 6. Comparison of the strong electromagnetic-assisted orientation control technology of the present invention with ordinary electromagnetic orientation control in terms of collagen fiber orientation effect.

[0109] The difference between this invention and conventional electromagnetic orientation control technology lies in the fact that this invention first transforms long collagen fibers into short fibers and liquid crystal states. The short liquid crystal fibers exhibit significantly greater rigidity than the long fibers, meaning they are more susceptible to electromagnetic fields. To this end, we specifically designed a comparative implementation of the two technologies. The results showed that conventional electromagnetic orientation technology has very limited control over the orientation of long collagen fibers; the collagen fibers only undergo localized deformation under the influence of the electromagnetic field, with an orientation consistency of less than 10% (after multiple repetitions). However, when the same electromagnetic field and intensity are applied to the collagen fibers, the orientation consistency can reach over 70%.

[0110] 7. Evaluation indicators: The most important evaluation indicators for collagen fiber orientation and regular arrangement are fiber orientation, uniformity, collagen fiber length and stretch.

[0111] 8. Evaluation of the relationship between magnetic field strength and collagen orientation effect: Experimental observations revealed a positive correlation between the applied magnetic field strength and collagen orientation. However, when the magnetic field strength is too high, it can interfere with the corresponding electronic components. This means that in subsequent industrial production, an excessively strong magnetic field may adversely affect other surrounding production equipment, leading to production difficulties. The suitable magnetic field strength for this experiment was 1-3T.

[0112] 9. Evaluate the relationship between other factors and collagen orientation effect: It was found that in addition to negative pressure having a direct impact on collagen orientation, the concentration of collagen fiber solution also affects collagen orientation, but its influence is smaller than that of negative pressure.

[0113] Example 3: Electromagnetic induction particle-assisted orientation control technology + 3D printing technology → orthogonal grid arrangement (corresponding organs: such as cornea, etc.)

[0114] 1. Extraction of human autologous collagen

[0115] The patient's own tendons, fascia lata, Achilles tendon, ligaments, or skin are extracted, purified, enzymatically digested, and dialyzed to obtain a high-purity collagen short fiber solution (acid-dissolved).

[0116] Human recombinant collagen can also be used, and there are no special restrictions on the materials themselves. The steps are the same as in step one.

[0117] 2. Preparation of Collagen Fiber Liquid Crystal Solution

[0118] (This step is the same as in Example 1)

[0119] 3. Ultrasonic treatment to prevent collagen fibers from tangling.

[0120] To reduce collagen fiber tangling, the collagen solution was sonicated at 4°C. The sonication consisted of two 10-minute ultrasonic pulses with a 10-minute pause in between for observation, thus reducing the difficulty of subsequent processing. Furthermore, the literature indicates that the sonication process does not damage the triple helix characteristics of the molecules.

[0121] 4. Preparation of electro / magnetically sensitive collagen liquid crystals

[0122] To further enhance the orientation and alignment properties of collagen fibers, we fully immersed the collagen fibers in a dispersion of nano-ferric oxide particles. This significantly strengthens the reactivity of the collagen fibers in electric or magnetic fields, resulting in better efficiency in achieving orientation and regular alignment. By applying a magnetic moment to the magnetic collagen fibers after this treatment, orientation control can be achieved with a lower magnetic field strength, and it is easier to achieve orientation control for all fibers.

[0123] 5. Construction of a highly biomimetic corneal model

[0124] The corneal stroma accounts for about 90% of the total thickness of the cornea. Collagen fibers form 200-250 lamellar layers, which overlap each other and have a certain radius of curvature. They are parallel to the corneal surface, ensuring the transparency of the cornea.

[0125] A 3D scanner is used to perform a full-thickness scan of the patient's cornea. The scanned images are then digitized and analyzed to create a spatial model suitable for 3D printing. Simultaneously, ① mimicking the multi-lamellar structure of the natural corneal stroma, the obtained parameters are processed into slices with each layer ranging from 2 to 100 μm in thickness; ② collagen fiber gaps similar to those in the corneal stroma are designed on the 3D corneal model to facilitate the induction of recipient corneal cell generation and nerve fiber growth, promoting the repair and reconstruction of the donor cornea; ③ mimicking the anterior and posterior elastic lamina of the natural cornea, slices similar to these lamina are designed on the 3D corneal model.

[0126] 6. Electromagnetically assisted 3D printing

[0127] The collagen solution prepared above, pre-added with an initiator or photosensitizer (including cleavage initiator, photosensitive initiator, and cationic photoinitiator), is added to the barrel of a 3D femtosecond laser printer. The printer then prints the collagen layer by layer using femtosecond laser technology, following the layered pattern in the corneal 3D model described above (to induce initial cross-linking of the collagen). The printhead diameter is 2-10 μm. The printhead moves along the xy-axis direction at a speed of 10-50 mm / s on the printing platform to form a two-dimensional plane of the scaffold. Simultaneously, a magnetic moment is applied to the magnetic collagen fibers, causing the fibers to unfold and rotate in the liquid matrix to control their orientation. After confirming that the collagen fibers are aligned and have undergone initial cross-linking and shaping (this process is usually quite rapid because each printed layer is very thin), the printing nozzle is moved up along the z-axis (the z-axis is moved up by 2-100μm), and another layer is printed. The printing directions of the 3D nozzles between adjacent layers are perpendicular to each other, and a magnetic moment is applied in the same direction as the printing. This process is repeated layer by layer until the printed scaffold is formed. Ultimately, the collagen fibers between different layers can be arranged in an orthogonal grid pattern.

[0128] 7. Fix the orientation of the collagen formed above by means of collagen cross-linking: (This step is the same as in Example 1)

[0129] 8. Complete removal of magnetic particles

[0130] First, the prepared sample containing magnetic particles was placed in a dialysis bag, which was then placed in a beaker of distilled water. Permanent magnets were placed around the beaker to attract the magnetic nanoparticles in the sample. At the same time, the beaker and permanent magnets were placed in a shaker, and fresh distilled water was replaced every 2 hours. After 24 hours, the sample was repeatedly rinsed.

[0131] The difference between this invention and ordinary electromagnetic orientation control technology lies in the fact that this invention introduces electromagnetically inductive nanoparticles and incorporates these particles into collagen fibers, thus giving the latter electromagnetic induction properties. To this end, we specifically designed a comparative study of the two technologies. The results showed that ordinary electromagnetic orientation technology has very limited control over the orientation of collagen fibers (length); the collagen fibers only undergo localized deformation under the influence of the electromagnetic field, with an orientation consistency of less than 10% (after multiple repetitions). However, when the same electromagnetic field and intensity are applied to the collagen fibers, the orientation consistency can reach over 90%.

[0132] 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 should be considered equivalent substitutions and are included within the protection scope of the present invention. Furthermore, all artificial organs such as corneas, skin, cartilage, and ligaments constructed based on this patented technology are within the protection scope of the present invention.

Claims

1. A method for preparing an oriented collagen fiber membrane, characterized in that, 1) Obtaining raw materials for collagen fibers; 2) Prepare collagen monomer solutions to form collagen liquid crystals; 3) Collagen fiber formation: Adding a buffer solution with a pH value greater than or equal to 7 to buffer the collagen monomer solution forms collagen fiber filaments with a collagen nanoscale striation structure; 4) Ultrasonic treatment of collagen fibers; 5) Collagen fiber orientation: using negative pressure-assisted fluid dynamics or applying an electric or magnetic field in a certain direction; The orientation and arrangement of collagen fibers were observed under a microscope until the collagen fibers stretched to the point where the length of the non-linear segments accounted for a significant portion of the total length. The length should not exceed 5%, and the orientation consistency should reach more than 90%. 6) The orientation of collagen fibers is fixed by means of collagen cross-linking.

2. The method for preparing a collagen fiber membrane according to claim 1, characterized in that, In step 2), the concentration of the collagen monomer solution is 80 mg / ml-120 mg / ml.

3. The method for preparing a collagen fiber membrane according to claim 1, characterized in that, Step 4) Sonicate the collagen solution at 4°C. The sonication process consists of two 10-minute ultrasonic pulses with a 10-minute pause in between.

4. The method for preparing a collagen fiber membrane according to claim 1, characterized in that, In step 5), a negative pressure suction device is used to generate external force, and the collagen fiber orientation and rearrangement are directionally assisted by creating a flowing liquid phase in the collagen fiber solution.

5. The method for preparing a collagen fiber membrane according to claim 1, characterized in that, When applying a magnetic field in a certain direction in step 5), the magnetic field direction is set to be circular, and the magnetic field strength is equal at every point in the circle.

6. The method for preparing a collagen fiber membrane according to claim 1, characterized in that, When collagen fibers are oriented by applying a magnetic field or electric field in a certain direction, in step 1), collagen fibers are obtained by converting collagen into short fiber segments.

7. The method for preparing a collagen fiber membrane according to claim 6, characterized in that, Step 5) After orientation is completed, short collagen fibers with consistent orientation are reassembled into long collagen fibers by liquid crystal technology and PBS dialysis. During this process, a magnetic field and electric field of a certain direction are maintained.

8. The method for preparing a collagen fiber membrane according to claim 6, characterized in that, After ultrasonic treatment, electromagnetic induction particles are added to the collagen fibers, and then step 5) is performed to apply a magnetic field or electric field in a certain direction to complete the orientation of the collagen fibers.

9. The method for preparing a collagen fiber membrane according to claim 8, characterized in that, After adding electromagnetically induced particles, and with the help of 3D printing technology, a multi-layered collagen membrane with different orientations is formed in each layer.

10. A collagen fiber membrane, characterized in that, The collagen fiber membrane is obtained by any one of the preparation methods described in claims 1-9.

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