CN114618017B

CN114618017B - Collagen film with highly oriented and crystalline collagen fiber structure and preparation method thereof

Info

Publication number: CN114618017B
Application number: CN202210102039.9A
Authority: CN
Inventors: 屈雪; 万浩然; 雷淼; 刘昌胜
Original assignee: East China University of Science and Technology
Current assignee: Jiangsu Bochuang Biotechnology Co ltd
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2023-06-02
Anticipated expiration: 2042-01-27
Also published as: CN114618017A

Abstract

The invention belongs to the field of biological macromolecule assembly, and relates to a preparation method of a collagen membrane, which comprises the following steps: firstly, taking a collagen microfibril film with short-range orientation, stretching along the length direction of the collagen film, and enabling microfibrils in the collagen film to be further oriented along the stress direction to form a highly oriented collagen material; secondly, carrying out ion incubation on the highly oriented collagen material, and inducing internal microfibril structure rearrangement to form large-diameter collagen fibers with a D band characteristic in a crystalline state; finally, chemical crosslinking is performed by photocrosslinking, glutaraldehyde crosslinking or genipin or polyphenols. The invention can obtain the collagen material with long-range orientation, and the characteristics of appearance, microcosmic appearance, young modulus, crystal structure and the like are similar to those of natural tendons. The preparation method of the invention is simple and convenient to operate, does not need complex instruments and equipment, and can rapidly and effectively prepare the collagen membrane material with the height similar to that of the natural tendon or ligament.

Description

Collagen film with highly oriented and crystalline collagen fiber structure and preparation method thereof

Technical Field

The invention belongs to the field of biological macromolecule assembly, and relates to a collagen membrane preparation technology with a highly oriented and crystalline collagen fiber structure, an obtained collagen membrane and application thereof.

Background

Collagen is one of the most abundant proteins in vertebrates. Because of its low immunogenicity, high biocompatibility, and the ability to promote cell proliferation and wound healing, it has been widely used in various biomedical materials. The collagen molecule is a triple helix structure, and can be assembled in a grading and ordered way under the guidance of some endogenous signals in the body, namely, the collagen molecule starts from the triple helix structure and is subjected to different hierarchical structures such as collagen microfibers, collagen fibrils, collagen fibers and the like. The specific hierarchy of collagen molecules determines the unique properties of each specific tissue. For example, in tendons and ligaments, rod-like collagen molecules self-assemble in a crystalline fashion through different modes of stacking, preferentially align in a certain direction and form covalent crosslinks, resulting in tissues exhibiting good load bearing properties in that direction. Controlled molecular assembly is a process used in nature to form extracellular matrix with anisotropic collagen fibers.

However, the in vitro controlled assembly of collagen molecules remains a major challenge for the next generation of tissue engineering. In vitro assembly of collagen is affected by temperature, pH, ionic strength, hydrophilic-hydrophobic interactions, and thus, current collagen "gels" are typically random networks of loosely packed fibers, resulting in very low mechanical strength.

There have been researchers who use electrochemical deposition technology EDP to prepare highly oriented engineered collagen materials. The principle is that an electric field is applied to the acidic solution of collagen to drive collagen molecules to migrate towards the cathode region in an electrophoresis way, and meanwhile, the electrochemical reaction (usually the electrolytic water reaction) on the cathode can raise the pH value of the solution at the local part of the cathode, so that the collagen which migrates to the isoelectric point region is separated out, and collagen gel is formed. This process can help rod-like collagen molecules to develop a certain alignment, exhibiting a certain anisotropy. Further through ion incubation, collagen molecules in the material are orderly arranged to generate a certain degree of crystallization. Finally, chemical crosslinking is carried out, and the ultimate tensile strength (about 30 MPa) and the tensile modulus (about 400 MPa) of the natural tendon are greatly improved compared with those of randomly crosslinked collagen, so that the natural tendon can be halved.

However, one of the disadvantages of the currently reported techniques is that the isoelectric point region is usually located at a certain position in the electrolyte, the material cannot be obtained inconveniently, the shape of the electrode cannot be used to shape the collagen material (for example, a tubular collagen material with irregular shape is obtained), and the orientation is completely dependent on the electric field, so that the orientation degree is limited, and therefore, the mechanics cannot approach to the natural tendon. Thirdly, the space between the two electrodes is very small, generally 1-2mm, which is inconvenient to operate (An electrochemical fabrication process for the assembly of anisotropically oriented collagen bundles, biomaterials 29 (2008) 3278-3288;Tenogenic Induction of Human MSCs by Anisotropically Aligned Collagen Biotextiles,Adv.Funct.Mater.2014,24,5762-5770).

Disclosure of Invention

The invention aims to provide a preparation method for obtaining a collagen material composed of highly oriented crystalline collagen fibers.

In order to solve the technical problems, the invention adopts four steps of improved EDP assembly, mechanical stretching, ion incubation and chemical crosslinking to obtain the long-range oriented crystalline collagen fiber material, and the mechanical property of the material is close to that of a natural tendon.

The preparation method of the collagen membrane comprises the following steps:

(1) Mechanical stretching: taking a collagen microfibril film with short-range orientation, stretching along the length direction of the collagen film, and enabling microfibrils in the collagen film to be further oriented along the stress direction, so as to form a highly oriented collagen material;

(2) Ion incubation: placing the highly oriented collagen material in the step (1) in a PBS buffer solution with the concentration of 0.05-0.5M for ion incubation for 20-40 hours, and inducing internal microfibril structure rearrangement to form a large-diameter collagen fiber with a crystallization state with the characteristic of D band;

(3) Chemical crosslinking: by photocrosslinking, glutaraldehyde crosslinking or genipin crosslinking.

Optionally, in the step (1), the stretched E-Col collagen film is soaked in ethanol, and the orientation structure is temporarily fixed. The degree of strain in the stretching may be 0-200%, excluding 0.

Optionally, in step (2), the two ends of the collagen membrane material are fixed during the ion incubation, so as to keep the collagen membrane from shrinking due to continuous external force, for example, using adhesive tape to fix the two ends of the collagen membrane in a culture dish or other container capable of storing liquid. Incubation time was 24-36 hours. The incubation temperature was 15-26℃at room temperature.

In the present invention, PBS means phosphate buffer, which can be obtained according to a conventional technique in the art, and for example, the following formulation (8 g of sodium chloride, 0.2g of potassium chloride, 2.90g of sodium dihydrogen phosphate dodecahydrate and 0.2g of potassium dihydrogen phosphate are sufficiently dissolved in 1000ml of water) can be used. Too high or too low a concentration of salt solution may affect the rate of increase in the diameter of the collagen membrane fibers, alternatively, the concentration of salt solution may be 0.05-1.0M in moles/liter. Salt solutions having a concentration of 0.1 to 0.8M, preferably 0.2 to 0.5M, may also be used as desired.

Alternatively, the photo-crosslinking means that the highly oriented and crystallized collagen film obtained in the step (2) is soaked in 0.2-3.0mg/ml riboflavin solution and crosslinked for 1-3 days under ultraviolet irradiation. The riboflavin solution uses 90% v/v ethanol-water as solvent, the concentration is 0.5-2.0mg/ml, and the crosslinking time is 20-40 hours.

Alternatively, glutaraldehyde crosslinking refers to immersing the highly oriented and crystallized collagen film obtained by the treatment in the step (2) in 0.1-1% (volume percentage) glutaraldehyde solution, and crosslinking for 10 minutes-2 hours; the glutaraldehyde component remaining in the collagen film is then removed. Glutaraldehyde solution with 0.5% w/v,90% v/v ethanol-water as solvent, crosslinking time is 30-50 minutes.

Optionally, the genipin crosslinking means that the highly oriented and crystallized collagen film obtained by the treatment in the step (2) is soaked in a genipin solution with the mass percentage of 0.2-2.0% and crosslinked overnight; the residual genipin component in the collagen membrane was then removed. Preferably, the concentration of the genipin solution is 0.5-1.0%, and the crosslinking time is 8-16 hours.

Optionally, the polyphenol crosslinking means that the highly oriented and crystallized collagen film obtained by the treatment in the step (2) is soaked in a procyanidine solution with the mass percent of 1.0-2.0% and the pH value of 8.5, and is crosslinked overnight; the residual polyphenol component in the collagen membrane is then removed. Preferably, the concentration of the procyanidin solution is 1.5-2.0%, and the crosslinking time is 12 hours.

Alternatively, the removal of the residual cross-linker component, such as genipin or glutaraldehyde, from the collagen film may be repeated with ultrapure water. For example, the washing is performed 3 to 5 times with ultrapure water.

In the invention, ultrapure water is also called UP water, and the resistivity of the water reaches 18MΩ cm (25 ℃), and the water has almost no impurities, mineral trace elements and organic matters such as bacteria, viruses, chlorine-containing dioxin and the like except water molecules. When ultrapure water is treated, not only the conductive medium in the water is almost completely removed, but also colloid substances, gases and organic matters which are not dissociated in the water are removed to a very low degree, and four steps of pretreatment, reverse osmosis technology, ultrapure treatment and post-treatment are generally required.

In the invention, the EDP assembly (short-range order) is a collagen gel film with very uniform appearance, is highly transparent in both dry state and wet state, and is formed by connecting collagen microfibers oriented in short range by non-covalent bonds; the collagen is densely arranged; the material has transparent appearance and uniform structure; the collagen material can be dissolved again by the solvent and can be circularly prepared. The collagen gel film can be directly obtained on an electrode, can be peeled off from the electrode after the preparation is finished, and can be prepared according to the following steps:

s1, preparing a collagen solution: adding acetic acid into collagen solution to dissolve collagen completely, regulating pH value of the final solution to 1.5-4, concentrating to obtain collagen solution with concentration of 1-20 mg/ml.

S2, adding hydrogen peroxide into the collagen solution obtained in the step S1 to ensure that the final concentration of the hydrogen peroxide in the solution is 5-200 mu l/ml, stirring, removing bubbles, and standing at 0-10 ℃ for standby; the amount of hydrogen peroxide optionally added is 5-17%.

S3, titanium sheets are used as working electrodes, platinum is used as counter electrodes, the distance between the two electrodes placed in parallel in the electrolytic cell is controlled to be 0.5-3.0cm, and the collagen solution prepared in the step S2 is slowly added into the electrolytic cell.

And S4, performing electrochemical reaction by adopting constant voltage or constant current deposition for 10-120 minutes to obtain the collagen gel film deposited on the cathode.

The invention provides a collagen membrane material, which mainly consists of collagen fibers which are oriented in a long way and have obvious D-band characteristics, and the Young modulus of the collagen membrane material is close to that of natural tendons. Alternatively, the long-range ordered collagen membrane material is obtained by the preparation method of the collagen material composed of the highly oriented crystalline collagen fibers obtained in the invention.

The collagen membrane preparation method of the present invention can obtain a collagen membrane mainly composed of long-range oriented collagen fibers having remarkable D-band characteristics and having young's modulus close to that of natural tendons. The collagen membrane material is further modified by a short-range ordered collagen material prepared on the electrode, so that the collagen membrane material can be shaped according to the shape of the electrode. Electrodes of different shapes can also be used according to the requirements of subsequent applications, for example, in a preferred embodiment of the invention, the prepared collagen membrane with long-range order has similar performance to natural tendons, not only has a similar crystal structure, but also has a corresponding Young's modulus.

The collagen material composed of the long-range oriented crystalline collagen fibers obtained by the preparation method has mechanical properties close to those of natural tendons. The advantages of the invention include:

1. mechanical stretching achieves high orientation, forming collagen microfibers with long-range orientation.

2. Ion incubation is performed in a stretched state to obtain a long-range oriented and crystallized collagen material.

3. The EDP technique was modified such that the collagen material was shaped according to the electrode shape, not just to the collagen bundles.

4. The collagen material has the characteristics of D band, appearance, microcosmic appearance, young modulus, crystal structure and the like which are similar to those of natural tendons in height.

5. The preparation method of the invention is simple and convenient to operate, does not need complex instruments and equipment, and can rapidly and effectively prepare the collagen membrane material with the height similar to that of the natural tendon or ligament.

Drawings

Fig. 1 is a schematic diagram of an EDP technology assembly process.

The mounting modes of the electrodes are two types: one is to place two electrodes vertically parallel in the cell (as in fig. 1 a) and the other is to place two electrodes horizontally parallel in the cell (as in fig. 1 b).

Fig. 2 is a display view of a collagen gel film.

Fig. 2 (a) shows a short-range ordered collagen gel film, the E-Col collagen material of which has a very uniform appearance and is highly transparent in both the dry and wet state, as shown in fig. 2 (b).

Fig. 3 is a schematic view of the process for preparing a collagen film having a highly oriented and crystalline collagen fibril structure according to the present invention.

The method mainly comprises the steps of mechanical stretching, ion incubation and chemical crosslinking on E-Col.

Fig. 4 is a graph comparing the appearance of collagen materials under various tensile forces and controls.

Fig. 4 (a) shows the appearance of each group of collagen films. Fig. 4 (b) shows the observation result of a polarized light microscope, and it can be seen that: the S-Col control group film has no obvious optical birefringence phenomenon and is in an isotropic structure; the optical birefringence phenomenon appears in partial areas of the E-Col film which is not stretched and deformed, which indicates that the partial areas have ordered structures; when E-Col is stretched to a greater degree of strain, a distinct optical birefringence is observed throughout the region of E-Col, and when the degree of deformation is further increased to 200%, the birefringence color is more vivid, indicating the formation of highly oriented structures within E-Col. The TEM image of fig. 4 (c) shows that the S-Col control film has a loose isotropic structure (red circles indicate fibrils perpendicular to the cross section), whereas the degree of densification and the degree of orientation of the E-Col film can be significantly improved by mechanical stretching, and the greater the deformation induced by mechanical stretching, the denser the internal microfiber arrangement and the higher the degree of orientation. FIG. 4 (D) is a graph of 2D SAXS showing a nearly uniform intensity ring for the S-Col control film, consistent with its internally isotropic structure; whereas the 2DSAXS pattern of E-Col showed a distinct elongated ring, demonstrating the appearance of anisotropically aligned nanofiber structures.

FIG. 5 is a graph showing the results of the test for the degree of stretch orientation of E-Col films.

Fig. 5 (a) is an azimuthal integrated intensity distribution curve. The results show that the azimuthal integrated intensity distribution curve of the E-Col film becomes narrower as the degree of strain increases. Hulman orientation parameter (f) _c ) Is a quantitative indicator describing the degree of orientation, which can be calculated from the azimuthal integrated intensity distribution curve. FIG. 5(b) It was revealed that the degree of orientation of the S-Col film control group was almost 0 (f _c =0.02), while an unstretched E-Col film has a low degree of orientation (f _c =0.15). As the tensile strain of the E-Col film is increased, the Huffman orientation parameter of the E-Col film is reduced from lower f _c =0.15 gradually increasing to f _c =0.93 (when the strain is 200%). These results indicate that stretching of the E-Col film will induce the creation of oriented structures along the direction of strain.

FIG. 6 is a graph comparing macroscopic and microscopic structures of E-Col films and natural tendons.

Among them, fig. 6 (a) and 6 (c) are macro and micro images of the E-Col film, and fig. 6 (b) and 6 (d) are macro and micro images of the E-Col film. As shown in fig. 6 (a), the E-Col, which was initially highly transparent, became milky opaque after mechanical stretching and ion incubation, and the surface exhibited millimeter-sized oriented stripes, similar to natural tendons (fig. 6 (b)), probably due to the formation of higher order structures (i.e., large diameter fibers) that caused a change in optical transparency. FIG. 6 (c-d) shows SEM images at low and high magnification, and it can be seen that E-Col shows a higher order hierarchy, i.e., densely arranged fibers with diameters of 5-10 μm, after PBS incubation.

FIG. 7 is a graph showing the degree of orientation and crystal form of E-Col films.

As can be seen from the 2D SAXS spectrum of FIG. 7 (a), after 200% pretensioning and ion incubation, E-Col still has a significantly stretched ring, indicating that the anisotropic alignment structure remains after ion incubation, f _c Calculated to be about 0.52 to about 0.53. And a distinct D band diffraction ring appears simultaneously in the 2D SAXS spectrum, indicating that collagen molecules are ordered after ion incubation. FIG. 7 (b) 2D SAXS pattern of natural tendons also shows a pronounced D band diffraction ring, f _c Calculated to be about 0.69-0.72. As can be seen from the 1DSAXS spectrum in FIG. 7 (c), E-Col after ion incubation produced a crystalline structure similar to that of the natural tendon.

FIG. 8 is a graph of static mechanical properties of E-Col films.

The test was performed under dry conditions, and fig. 8 (a) and (b) show the breaking stress and elastic modulus of the material, respectively. The breaking stress of the E-Col material is about 108+/-6 MPa and slightly lower than the breaking stress of the natural tendon (128+/-14 MPa); while the Young's modulus of E-Col materials (0.795+ -0.060 GPa) substantially reaches the level of natural tendons (0.890+ -0.118 GPa).

Detailed Description

The invention is further illustrated by the following examples.

Example 1: EDP technology for assembling amorphous collagen material with short-range orientation

(1) Preparation of collagen solution: accurately weighing 400mg of type I collagen in 40mL of ultrapure water, dropwise adding glacial acetic acid, fully stirring to promote complete dissolution of collagen, and regulating the pH value of the final solution to 3.5. Putting it into dialysis bag (M) _{Wcut off} =7.0 kDa) and put into a beaker containing 1000ml of water and 15ml of glacial acetic acid, dialyzed at 4 ℃ for 72h to remove small molecule impurities. After dialysis, 10mg/ml of a collagen viscous liquid was obtained. The concentration of the finally obtained collagen solution can be adjusted to be changed within the range of 1-20mg/ml by changing the mass of the collagen raw material, and the solution loses fluidity when the concentration exceeds 20 mg/ml.

(2) Adding 100 μl/ml hydrogen peroxide into the collagen solution obtained in step (1), stirring, centrifuging at 8000rpm/min at 4deg.C to remove bubbles, and standing the centrifuged collagen solution in ice water mixed bath to prevent hydrogen peroxide decomposition. The concentration of hydrogen peroxide to be added may be selected in the range of 5 to 200. Mu.l/ml, and hydrogen peroxide exceeding the maximum of 200. Mu.l/ml is liable to be decomposed directly in the electrolyte to generate bubbles.

(3) Titanium sheets (cathode) were chosen as working electrode (electrode size 2cm x 3 cm), platinum wire or sheet (anode) as counter electrode. The mounting modes of the electrodes are two: one is to place two electrodes vertically in parallel in the cell (fig. 1 (a)), the distance between the electrodes being controlled to be 1.5cm (the electrode distance being adjustable in the range of 0.05-3.0 cm); the other is to place two electrodes horizontally in parallel in the electrolytic cell (FIG. 1 (b)), the distance between the electrodes is controlled to be 1.5cm (the electrode distance can be adjusted in the range of 0.05-3.0 cm). The collagen solution (concentration is 10 mg/ml) prepared in the step (2) is carefully added into an electrolytic cell, and the addition is slow, so that bubbles caused by excessive solution viscosity are prevented.

(4) The electrode was then connected to an electrochemical workstation CHI 660E, a cathodic voltage was applied, and constant current deposition was used with a current density of 6.67mA/cm ² The voltage variation range is 1-1.5V/cm ² The deposition time was 2000 seconds and the electrode half-reactions that occurred were as follows.

Anode: 2H (H) ₂ O-4e ^- →4H ⁺ +O ₂

And (3) cathode: 4H (4H) ₂ O+4e ^- →4OH ^- +2H ₂

After the end of the experiment, a collagen gel film was present on the cathode as shown in fig. 2 (a). The working electrode with the collagen hydrogel film was washed with ultrapure water a plurality of times, and then the collagen material E-Col was peeled off from the electrode. Both the horizontal electrode and the vertical electrode can prepare collagen materials, but the materials prepared by the vertical electrode are thin and thick at the top due to gravity, so that the situation can be avoided by adopting the horizontal electrode. The E-Col collagen material was very uniform in appearance and highly transparent in both the dry and wet state, as shown in FIG. 2 (b).

Example 2: preparation example I of collagen film having Long-range orientation and crystalline collagen fiber Structure

(1) Mechanical stretching

A collagen film E-Col was prepared as in example 1, and cut into rectangular bars having a length of 30mm and a width of 10 mm. And (3) immersing a plurality of rectangular E-Cols in ultrapure water for 5min, and then stretching the E-Cols to a strain degree of 200% along the length direction of the collagen film by adopting an Electro-Force3200 type biological power tester, so that microfibers inside the collagen film are further oriented along the stress direction, and a long-range oriented collagen material is formed. Finally, the stretched E-Col is soaked in ethanol to temporarily fix the orientation structure.

(2) Ion incubation

Ion incubating the long-range oriented collagen material of step (1): the two ends of the strip-shaped collagen membrane material are fixed in a culture dish by using adhesive tape, the collagen membrane is kept to be continuously and externally acted without shrinkage, then 0.1M PBS buffer solution is added into the culture dish, and the culture dish is incubated for 24 hours at room temperature, so that internal microfiber structure rearrangement is induced, and the large-diameter collagen fiber with the D band characteristic in the crystalline state is formed.

(3) Chemical crosslinking

Crosslinking by adopting a photocrosslinking method: the highly oriented and crystallized collagen film obtained by the treatment in the step 2 is soaked in 1mg/ml riboflavin solution (90% v/v ethanol-water) and crosslinked for 24 hours under 365nm ultraviolet light irradiation, so as to further enhance the mechanical properties of the material.

FIG. 3 is a schematic illustration of mechanical stretching-ion incubation-chemical crosslinking of E-Col.

Example 3: preparation example II of collagen film with highly oriented and crystalline collagen fiber Structure

(1) Mechanical stretching

A collagen film E-Col was prepared as in example 1, and cut into rectangular bars having a length of 20mm and a width of 20 mm. And (3) immersing a plurality of rectangular E-Cols in ultrapure water for 10min, and then stretching the E-Cols to a strain degree of 50% along the length direction of the collagen film by adopting an Electro-Force3200 type biological power tester, so that microfibers inside the collagen film are further oriented along the stress direction, and a long-range oriented collagen material is formed. Then, the stretched E-Col was immersed in ethanol to temporarily fix the alignment structure.

(2) Ion incubation

Ion incubating the highly oriented collagen material of step (1): the two ends of the strip-shaped collagen membrane material were fixed in a petri dish with an adhesive tape, the collagen membrane was kept from shrinking by a continuous external force, then 0.05M PBS buffer was added to the petri dish, and the internal microfibril structure was induced to rearrange at room temperature for 30 hours, to form a large-diameter collagen fiber in a crystalline state having D-band characteristics.

(3) Chemical crosslinking

Crosslinking by glutaraldehyde crosslinking method: preparing glutaraldehyde solution (0.5% w/v,90% v/v ethanol-water), immersing the highly oriented and crystallized collagen film obtained by the treatment in the step (2) in glutaraldehyde solution, and crosslinking for 30min. And then repeatedly washing with ultrapure water to remove the glutaraldehyde component remained in the collagen film, thereby obtaining the collagen film with highly oriented and crystalline collagen fiber structure.

Example 4: preparation example III of collagen film with highly oriented and crystalline collagen fibrillar Structure

(1) Mechanical stretching

A collagen film E-Col was prepared as in example 1, and cut into rectangular bars having a length of 20mm and a width of 10 mm. And (3) immersing a plurality of rectangular E-Cols in ultrapure water for 3min, and then stretching the E-Cols to 100% strain degree along the length direction of the collagen film by adopting an Electro-Force3200 type biological power tester, so that microfibers inside the collagen film are further oriented along the stress direction, and a highly oriented collagen material is formed. Finally, the stretched E-Col is soaked in ethanol to temporarily fix the orientation structure.

(2) Ion incubation

Ion incubating the highly oriented collagen material of step (1): the two ends of the strip-shaped collagen membrane material are fixed in a culture dish by using adhesive tape, the collagen membrane is kept to be subjected to continuous external force without shrinkage, then 0.2M PBS buffer solution is added into the culture dish, and the culture dish is incubated for 20 hours at room temperature, so that internal microfiber structure rearrangement is induced, and the large-diameter collagen fiber with the D band characteristic in the crystalline state is formed.

(3) Chemical crosslinking

Crosslinking by adopting a genipin crosslinking method: preparing a 1% genipin solution, and soaking the highly oriented and crystallized collagen film obtained by the treatment in the step (2) in the genipin solution for crosslinking for 10 hours. And then repeatedly cleaning with ultrapure water to remove the residual genipin component in the collagen membrane, thereby obtaining the collagen membrane with long-range order.

Example 5: preparation example IV of collagen film with highly oriented and crystalline collagen fiber Structure

(1) Mechanical stretching

(2) Ion incubation: ion incubation of the highly oriented collagen material (subjected to 50% stretching) of step (1): the two ends of the strip-shaped collagen membrane material were fixed in a petri dish with an adhesive tape, the collagen membrane was kept from shrinking by a continuous external force, then 0.05M PBS buffer was added to the petri dish, and incubation was performed at 37 ℃ for 18 hours, to induce rearrangement of the internal microfibril structure, thereby forming a large-diameter collagen fiber in a crystalline state having D-band characteristics.

(3) Chemical crosslinking: crosslinking by adopting a procyanidine crosslinking method: 1.5% procyanidine aqueous solution was prepared and pH was adjusted to 8.5 with NaOH. Immersing the highly oriented and crystallized collagen film obtained in the step (2) in a procyanidine solution, and crosslinking for 12 hours. And then repeatedly washing with ultrapure water to remove the residual polyphenol component in the collagen film, thereby obtaining the collagen film with long-range orientation.

Example 6: preparation example V of collagen film having highly oriented and crystalline collagen fiber Structure

Mechanical stretching and ion incubation were the same as in example 4. The chemical crosslinking adopts photocrosslinking, and is specifically as follows:

immersing the highly oriented and crystallized collagen film obtained by the treatment in the step (2) in 0.5mg/ml riboflavin solution (90% v/v ethanol-water), and crosslinking for 40 hours under 365nm ultraviolet light irradiation to further enhance the mechanical properties of the material.

Example 7: preparation example VI of collagen film with highly oriented and crystalline collagen fibrillar Structure

Ion incubation and chemical crosslinking were the same as in example 2. The mechanical stretching steps are as follows:

a short-range ordered collagen film E-Col was prepared as in example 1, and cut into rectangular bars of 40mm in length and 20mm in width. And (3) immersing a plurality of rectangular E-Cols in ultrapure water for 8min, and then stretching the E-Cols to 150% strain degree along the length direction of the collagen film by adopting an Electro-Force3200 type biological power tester, so that microfibers inside the collagen film are further oriented along the stress direction, and a long-range oriented collagen material is formed. Finally, the stretched E-Col is soaked in ethanol for more than 10 minutes for standby, and the orientation structure is temporarily fixed.

Example 8: orientation characterization of E-Col after mechanical stretching and photo-crosslinking

The internal orientation structure of E-Col obtained by the preparation method of example 2 was examined by a polarized light microscope (POM, nikonEclipseCi-L), synchronous 2D small angle x-ray scattering (2 DSAXS, BL19U 2) and a transmission electron microscope (TEM, JEM-2100, JEOL), respectively.

The collagen film S-Col was prepared using the solution method as a control sample. The preparation method of the S-Col comprises the following steps: acidic collagen solution (5 mg/mL; ph=3.5) was adjusted to neutral ph=7.2 with 0.5M NaOH, then cast in a round thin film petri dish (the collagen content per unit area was the same as the collagen mass per unit area assembled by EDP), incubated at 37 ℃ for 12 hours to completely gel, and then the gel was dehydrated at room temperature for 48 hours to form a milky translucent gel film.

Fig. 4 (a) is the appearance of each group of films. Fig. 4 (b) shows the observation result of a polarized light microscope, and it can be seen that: the S-Col control group film has no obvious optical birefringence phenomenon and is in an isotropic structure; the optical birefringence phenomenon appears in partial areas of the E-Col film which is not stretched and deformed, which indicates that the partial areas have ordered structures; when E-Col is stretched to a greater degree of strain, a distinct optical birefringence is observed throughout the region of E-Col, and when the degree of deformation is further increased to 200%, the birefringence color is more vivid, indicating the formation of highly oriented structures within E-Col.

The TEM image of fig. 4 (c) shows that the S-Col control film has a loose isotropic structure (red circles indicate fibrils perpendicular to the cross section), whereas the degree of densification and the degree of orientation of the E-Col film can be significantly improved by mechanical stretching, and the greater the deformation induced by mechanical stretching, the denser the internal microfiber arrangement and the higher the degree of orientation.

FIG. 4 (D) is a graph of 2D SAXS showing a nearly uniform intensity ring for the S-Col control film, consistent with its internally isotropic structure; whereas the 2DSAXS pattern of E-Col showed a distinct elongated ring, demonstrating the appearance of anisotropically aligned nanofiber structures.

To quantitatively describe the degree of orientation of the collagen membrane, the SAXS profile of fig. 2 (d) was further analyzed, and an azimuthal integrated intensity distribution curve was prepared as shown in fig. 5 (a). The results show that the azimuthal integrated intensity distribution curve of the E-Col film becomes narrower as the degree of strain increases. Hulman orientation parameter (f) _c ) Is a quantitative indicator describing the degree of orientation, which can be calculated from the azimuthal integrated intensity distribution curve. FIG. 5 (b) shows that the degree of orientation of the S-Col film control group was almost 0 (f _c =0.02), while an unstretched E-Col film has a low degree of orientation (f _c =0.15). As the tensile strain of the E-Col film is increased, the Huffman orientation parameter of the E-Col film is reduced from lower f _c =0.15 gradually increasing to f _c =0.93 (when the strain is 200%). These results indicate that stretching of the E-Col film will induce the creation of oriented structures along the direction of strain.

Example 9: characterization of E-Col morphology after mechanical stretching, ion incubation and photo-crosslinking

Using the procedure of example 2, E-Col was mechanically stretched (200% stretch) and ion incubated, in comparison to the macroscopic and microscopic structure of natural tendons. And collecting morphology data by adopting a micro-distance and an SEM.

As shown in fig. 6 (a), the E-Col, which was initially highly transparent, became milky opaque after mechanical stretching and ion incubation, and the surface exhibited millimeter-sized oriented stripes, similar to natural tendons (fig. 6 (b)), probably due to the formation of higher order structures (i.e., large diameter fibers) that caused a change in optical transparency. FIG. 6 (c-d) shows SEM images at low and high magnification, and it can be seen that E-Col shows a higher order hierarchy, i.e., densely arranged fibers with diameters of 5-10 μm, after PBS incubation.

Example 10: orientation degree and crystal form characterization of E-Col after mechanical stretching and ion incubation

Using the procedure of example 2, E-Col was subjected to mechanical stretching (200% stretching), ion incubation and photocrosslinking, and alignment and crystal form comparison with natural tendons were performed.

Example 11: characterization of static mechanical properties of E-Col after mechanical stretching, ion incubation and photo-crosslinking

The collagen membrane of example 2 was subjected to 200% mechanical stretching, ion incubation, and photocrosslinking, and compared with the mechanical properties of natural tendons. Static tensile properties of the two were studied at room temperature using an Electro-Force3200 biodynamic tester.

In order to examine the contribution of the material structure to the material mechanics, the test was chosen to be performed in dry conditions. Fig. 8 (a) and (b) show the breaking stress and elastic modulus, respectively, of the material. The breaking stress of the E-Col material is about 108+/-6 MPa and slightly lower than the breaking stress of the natural tendon (128+/-14 MPa); while the Young's modulus of E-Col materials (0.795+ -0.060 GPa) substantially reaches the level of natural tendons (0.890+ -0.118 GPa). It is speculated that the E-Col material, after mechanical stretching and ionic incubation, exhibits excellent mechanical properties similar to natural tendons due to the creation of oriented structures and crystalline properties highly similar to natural tendons, and can provide a potential biomaterial for tendon/ligament repair.

The foregoing description of the embodiments is provided to facilitate the understanding and appreciation of the invention by those skilled in the art. It will be apparent to those skilled in the art that various modifications can be readily made to these teachings and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the invention is not limited to the above description and the description of the embodiments, and those skilled in the art, based on the disclosure of the invention, should make improvements and modifications without departing from the scope of the invention.

Claims

1. A method for preparing a collagen membrane, comprising the steps of:

(1) Mechanical stretching: taking a collagen microfibril film with short-range orientation, stretching along the length direction of the collagen film, and enabling microfibrils in the collagen film to be further oriented along the stress direction, so as to form a highly oriented collagen material; the degree of strain in stretching was 200%;

(2) Ion incubation: placing the highly oriented collagen material in the step (1) in a PBS buffer solution with the concentration of 0.1M for ion incubation for 24-36 hours, and inducing internal microfibril structure rearrangement to form large-diameter collagen fibers with the characteristic of D band in a crystalline state; fixing two ends of the collagen membrane material during ion incubation, and keeping the collagen membrane from shrinking due to continuous external acting force;

(3) Chemical crosslinking: by photocrosslinking, glutaraldehyde crosslinking, genipin or polyphenols crosslinking.

2. The method of claim 1, wherein in the step (1), the stretched collagen film is immersed in ethanol to temporarily fix the alignment structure.

3. The method of claim 1, wherein in step (2), the incubation time is 24 hours.

4. The method for producing a collagen film according to claim 1, wherein in the step (3),

the photo-crosslinking is to soak the highly oriented and crystallized collagen film obtained by the treatment in the step (2) in 0.2-3.0mg/ml riboflavin solution, and crosslink for 1-3 days under the irradiation of ultraviolet light; or alternatively, the process may be performed,

the glutaraldehyde crosslinking refers to immersing the highly oriented and crystallized collagen film obtained by the treatment in the step (2) in glutaraldehyde solution with the volume percentage of 0.1-1%, and crosslinking for 10 minutes-2 hours; subsequently removing the glutaraldehyde component remaining in the collagen film; or alternatively, the process may be performed,

the genipin crosslinking is to soak the highly oriented and crystallized collagen film obtained by the treatment in the step (2) in a genipin solution with the mass percent of 0.2-2.0%, and crosslink the collagen film overnight; subsequently removing the residual genipin component in the collagen membrane; or alternatively, the process may be performed,

the polyphenol crosslinking is to soak the highly oriented and crystallized collagen film obtained by the treatment in the step (2) into an aqueous solution of procyanidine, tannic acid or gallic acid with the mass percentage of 0.1-2.0%, and crosslink the collagen film overnight; the residual polyphenol component in the collagen membrane is then removed.

5. The method for producing a collagen membrane according to claim 4, wherein in the step (3),

in the photo-crosslinking process, the riboflavin solution takes 90% ethanol solution as a solvent, the concentration is 0.5-2.0mg/ml, and the crosslinking time is 20-40 hours; or alternatively, the process may be performed,

in the genipin crosslinking process, the concentration of the genipin solution is 0.5-1.0% by mass, and the crosslinking time is 8-16 hours; or alternatively, the process may be performed,

in the polyphenol crosslinking process, the pH of the procyanidine aqueous solution is 8-9, and the crosslinking time is 8-12 hours;

the crosslinking agent component remaining in the collagen film was removed, and repeatedly rinsed with ultrapure water.

6. The method of producing a collagen film according to claim 1, wherein the produced collagen film has a highly oriented and crystalline collagen fiber structure.

7. The method of claim 1, wherein the preparation of the short-range oriented collagen microfibril film comprises the following steps:

s1, preparing a collagen solution: adding acetic acid into the collagen solution to completely dissolve collagen, regulating the pH value of the final solution to be 1.5-4, and concentrating to obtain collagen solution with the concentration of 1-20 mg/ml;

s2, adding hydrogen peroxide into the collagen solution obtained in the step S1 to ensure that the final concentration of the hydrogen peroxide in the solution is 5-200 mu l/ml, stirring, removing bubbles, and standing at 0-10 ℃ for standby;

s3, titanium sheets are used as working electrodes, platinum is used as counter electrodes, the distance between the two electrodes placed in parallel in the electrolytic cell is controlled to be 0.5-3.0cm, and the collagen solution prepared in the step S2 is slowly added into the electrolytic cell;

s4, performing electrochemical reaction, and depositing for 10-60 minutes to obtain the collagen gel film deposited on the cathode.

8. A collagen membrane material, characterized in that the preparation method of the collagen membrane material is as described in any one of the collagen membrane preparation methods of claims 1 to 7.

9. Use of the method for producing a collagen film according to claim 1, wherein the produced collagen film is used as an artificial tendon.