A shell-core microporous extrusion device and a method for preparing composite insoluble collagen fibers

Abstract

This application relates to a shell-core microporous extrusion device and a method for preparing composite poorly soluble collagen fibers. The shell-core microporous extrusion device includes an injection pump for extruding the core layer material, a stepping motor, an extrusion die, wherein the stepping motor is connected to the extrusion die for extruding the ICFs material, a post-treatment mechanism, and a conduit. One end of the conduit is connected to the injection pump, the other end is connected to the post-treatment mechanism, and the middle of the conduit is connected to the extrusion die. The orientation porous composite collagen fibers can be realized by using the self-developed shell-core microporous extrusion device.

Description

Technical Field

[0001] This application belongs to the technical field of collagen preparation, and particularly relates to a shell-core microporous extrusion device and a method for preparing poorly soluble fibrous collagen using the same. Background Art

[0002] China's leather industry generates a large amount of waste raw materials for glue (such as trimmings from raw hides and alkali-treated hide trimmings) every year. The main component of these trimmings is collagen, and they do not contain heavy metal ions such as chromium, making it relatively easy to extract and utilize collagen. If they are directly discarded, it will undoubtedly be a huge waste of biomass resources. Therefore, how to extract high-quality collagen from leather trimmings and develop it into high-value-added products has important economic and social significance.

[0003] Collagen is a high-quality natural biomass resource with advantages such as good biocompatibility, strong hemostatic and coagulation effects. It can be used to cover the wound surface for a long time, thereby promoting platelet aggregation, and is an ideal natural polymer material for preparing wound dressings. Therefore, applying collagen to the preparation of multifunctional wound dressings is one of the ways to increase the added value of waste collagen resources. During the interaction between the dressing and the tissue, its unique three-dimensional structure can provide a good microenvironment for cell growth and metabolism, regulate the fate of cells, and then induce tissue regeneration. Research shows that the oriented collagen fibers in the dermis layer can regulate the arrangement of cells and the expression of specific phenotypes. Oriented fibers can shorten the inflammation cycle, induce macrophages to polarize towards M2, and promote tissue repair. In addition, due to the siphon principle, the porous structure can quickly absorb the blood and tissue exudate from the wound surface, rapidly adsorb platelets, and release coagulation factors, achieving the effect of rapid hemostasis. Based on the above analysis, oriented porous fibers can simultaneously possess functions such as rapid hemostasis, absorption of exudate, and appropriate regulation of cell phenotypes, and are relatively ideal multifunctional dressings.

[0004] At present, most studies use electrospinning and 3D printing to prepare oriented porous collagen fibers, etc. These technologies can achieve macroscopic and microscopic orientation of collagen microfibers to a certain extent, but they can only use soluble collagen (SC) as the forming material. Compared with SC, the structures of procollagen, microfibers, fibers, and fiber bundles in insoluble collagen fibers (ICFs) are arranged conformationally after hierarchical self-assembly, and their degree of assembly is much higher than that of SC, with better structural stability and mechanical properties. Therefore, ICFs are more suitable for the preparation of wound dressings, but there are few reports on the processing technology of ICFs at present. At present, in the existing reports, the counter-rotating extrusion (CRE) and injection extrusion (IE) can realize the processing and orientation of ICFs. However, the films / fibers processed by these two technologies are all dense and pore-free structures, and the mechanical properties of the film / fiber materials processed solely with ICFs raw materials are limited. Summary of the Invention

[0005] The present application is provided to solve the technical problems mentioned in the background art. Therefore, a shell-core microporous extrusion device and a method for preparing composite insoluble collagen fibers are needed. For the purpose of efficiently and reasonably utilizing biomass resources, adopting the strategy of transforming high-value-added tissue engineering products, a set of shell-core microporous extrusion device suitable for ICFs is independently designed and manufactured. Then, microporous extrusion is combined with freeze-drying, and relevant parameters are optimized to prepare oriented porous collagen fibers. Finally, inspired by the natural epidermal and dermal layer structures, the shell-core microporous extrusion is combined with electrospinning technology to prepare an asymmetric wettable dressing, and the application of this material in the field of multifunctional medical dressings is preliminarily explored. The relevant results open up a new and effective way for the utilization of resources such as collagen-rich by-products.

[0006] One object of the present application is to provide a microporous extrusion device, comprising:

[0007] An injection pump for extruding the core layer material;

[0008] A stepper motor;

[0009] An extrusion die, the stepper motor is connected to the extrusion die for extruding ICFs materials;

[0010] A post-treatment mechanism;

[0011] A catheter, one end of the catheter is connected to the injection pump, the other end is connected to the post-treatment mechanism, and the middle part of the catheter is connected to the extrusion die.

[0012] Further, the extrusion die includes:

[0013] A pressure head, which is connected to the output end of the stepper motor;

[0014] A pressure body for filling ICFs material, and the pressure head is movably arranged in the pressure body;

[0015] A base, and the pressure body is arranged at the upper end of the base;

[0016] An extrusion needle, which is arranged at the lower end of the base, and the extrusion needle is internally communicated with the pressure body, so as to extrude the ICFs material in the pressure body through the pressure head and discharge it into the conduit.

[0017] Furthermore, it further includes:

[0018] An L-shaped extrusion plate, which is arranged on the slide rail of the stepper motor and is used for extruding the extrusion die to extrude ICFs material;

[0019] A die bracket, and the extrusion die is installed on the die bracket;

[0020] A stepper motor card slot box body, and the stepper motor is installed on the stepper motor card slot box body.

[0021] Further, the post-treatment mechanism includes a constant temperature water bath, and a fiber-forming buffer solution or deionized water is arranged in the constant temperature water bath.

[0022] Further, the constant temperature water bath includes a first pot body and a second pot body which are connected by pipelines in sequence. The fiber-forming buffer solution is arranged in the first pot body, and the deionized water is arranged in the second pot body.

[0023] The second object of the present application is to provide a method for preparing poorly soluble fiber collagen. Based on the microporous extrusion device as described above, the method includes:

[0024] Taking a cowhide dermis sample and putting it into an NaCl solution;

[0025] Putting it into acetone for defatting, and then performing ventilation and standing;

[0026] After freezing, chopping it with a slicing machine, taking an appropriate amount of the chopped cowhide, putting it into deionized water, and adjusting the pH with HAc;

[0027] After acidification, homogenizing the cowhide until there are no fragments, adding NaCl and pepsin after homogenization, and putting it into enzymatic hydrolysis at 4°C;

[0028] Centrifuging the enzymatically hydrolyzed material, discarding the supernatant, adding the material to deionized water, and homogenizing until it is completely dispersed, adjusting the pH to 6.8 - 7.2, and refrigerating and standing;

[0029] Centrifuge again, discard the supernatant, add the remaining material to the HAc solution, homogenize until completely dispersed, and let it stand refrigerated or frozen.

[0030] Adjust the pH to around 7.0 - 7.4 and let it stand refrigerated.

[0031] Centrifuge again (10000 r / min, 10 min, 4 °C), discard the supernatant, wash the precipitate multiple times, dialyze, and then freeze-dry to obtain insoluble fibrous collagen.

[0032] Furthermore, the acidification pH value adjusted with HAc is determined by the following method:

[0033] Set three gradients for the acidification pH value, which are 2.5, 3.0, and 3.5 respectively. Using the final collagen extraction rate as the optimization index, screen out the optimal pH for the acidification of cowhide.

[0034] Furthermore, the enzymatic hydrolysis concentration is determined by the following method:

[0035] Set three gradients for the enzymatic hydrolysis concentration, which are 1%, 1.5%, and 2% respectively. Using the final collagen extraction rate as the optimization index, screen out the optimal enzymatic hydrolysis concentration.

[0036] Furthermore, the enzymatic hydrolysis time is determined by the following method:

[0037] Set three gradients for the enzymatic hydrolysis time: 24 h, 36 h, and 48 h. Using the final collagen extraction rate as the optimization index, screen out the optimal enzymatic hydrolysis time for cowhide.

[0038] Furthermore, the method further includes:

[0039] Using the blend of SA and CLL as the core layer material, and determining the concentration ratio of SA to CLL according to the mechanical properties of the material characterized by calculating stress-strain, breaking strength, elongation at break, and elastic modulus based on the curve.

[0040] Fill the insoluble fibrous collagen into the extrusion die, extrude the insoluble fibrous collagen through the extrusion die, and pump out the core layer material with an injection pump to obtain a core-shell structure.

[0041] Immerse the core-shell structure in the FFB solution for post-treatment. The FFB solution includes at least sodium chloride, ethylsulfonic acid, disodium hydrogen phosphate, and polyethylene glycol.

[0042] The advantages of this application are as follows:

[0043] (1) After optimizing the collagen extraction scheme, the best conditions are: acid pH is 3.0, enzymatic hydrolysis concentration is 1.5%, and enzymatic hydrolysis time is 72 h.

[0044] (2) The extracted collagen material, after being characterized, shows a complete structure, similar to that of commercially available products, and high thermal stability, indicating that the extraction scheme developed is correct.

[0045] (3) After optimizing the extrusion parameters, the optimal values for each are as follows: collagen concentration 6%, extrusion needle S2 - 1: 17G, SA (5% CLL), and the extrusion rate of the core - shell layer 3 mm / s.

[0046] (4) Based on the above - optimized extrusion parameters, an independently developed core - shell micro - pore extrusion device can be used to prepare oriented porous composite insoluble collagen fibers. Description of the Drawings

[0047] Figure 1 This is a schematic diagram of the overall structure of a micro - pore extrusion device according to an embodiment of the present application.

[0048] Figure 2 This is a schematic diagram of the core - shell extrusion die structure according to an embodiment of the present application; (A) 3D modeling diagram of the overall extrusion die; (B) pressure head; (C) body; (D) base; (E) extrusion needle.

[0049] Figure 3 This is a physical diagram of the core extrusion die according to an embodiment of the present application.

[0050] Figure 4 This is a circuit schematic diagram of the shell - layer extrusion control device according to an embodiment of the present application.

[0051] Figure 5 This is an overall modeling diagram of the extrusion die support according to an embodiment of the present application.

[0052] Figure 6 This is a three - view drawing of the main components of the support according to an embodiment of the present application; (A) support box; (B) extrusion die support; (C) L - shaped extrusion plate.

[0053] Figure 7 This is a physical diagram of the micro - pore extrusion device according to an embodiment of the present application.

[0054] Figure 8 This is a mesh division diagram according to an embodiment of the present application.

[0055] Figure 9 This is a pressure distribution nephogram of different pore diameters according to an embodiment of the present application: A - 13G (outer 2.4 mm, inner 1.9 mm); B - 15G (outer 1.8 mm, inner 1.4 mm), C - 17G (outer 1.47 mm, inner 1.07 mm)

[0056] Figure 10 This is a shear force distribution nephogram of different pore diameters according to an embodiment of the present application: A - 13G; B - 15G; C - 17G.

[0057] Figure 11 Parameter optimization result diagram of collagen extraction scheme for embodiments of the present application; (A) Acidification pH optimization; (B) Enzyme concentration optimization; (C) Enzymolysis time optimization; (D) External macroscopic diagram of extracted collagen.

[0058] Figure 12 Infrared spectrum diagram of collagen for embodiments of the present application.

[0059] Figure 13 SDS-PAGE diagram of collagen for embodiments of the present application: (A) Commercially available collagen; (B) ICFs.

[0060] Figure 14 DSC analysis diagram for embodiments of the present application: (A) Commercially available collagen; (B) IGFs.

[0061] Figure 15 Extrusion effect diagram and SEM diagram of collagen concentration optimization for embodiments of the present application; (A) Macroscopic diagrams of collagen extrusion concentrations of 4%, 5%, and 6%; (B) Scanning electron microscope, with concentrations from left to right being 4%, 5%, and 6%; (C) Pore size distribution interval diagram of porous structures with concentrations of 4%, 5%, and 6%.

[0062] Figure 16 Bar chart of water absorption rate and water retention rate of 4%, 5%, and 6% concentrations for embodiments of the present application, *p<0.05, **p<0.01.

[0063] Figure 17 Comparison diagram of extrusion needles with different pore sizes for embodiments of the present application.

[0064] Figure 18 Needle pore size optimization for embodiments of the present application, from left to right are 13G, 15G, and 17G; (A) Macroscopic diagrams of 13G, 15G, and 17G; (B) Scanning electron microscope diagram; (C) Pore structure fiber orientation angle distribution interval diagram.

[0065] Figure 19 Nuclear layer component optimization diagram for embodiments of the present application; (A) Diameter test; (B) Stress-strain curve.

[0066] Figure 20 Extrusion effect diagram of shell-core layer rate optimization for embodiments of the present application; (A) Shell-core layer rate is 6mm / s; (B) Shell-core layer rate is 3mm / s; (C) Shell-core layer rate is 1mm / s.

[0067] Figure 21 Macroscopic morphology diagram (A), microscopic morphology diagram (surface, B), and (cross-section, C) of shell-core structure collagen-based fibers for embodiments of the present application. Detailed implementation manners

[0068] The following listed partial embodiments are only for better explaining the present invention, but the content of the present invention is not limited to the applied embodiments. Therefore, those skilled in the art make non-essential improvements and adjustments to the implementation schemes according to the above invention content and apply them to other embodiments, which are still within the protection scope of the present invention.

[0069] Now, the present invention will be further described in conjunction with the accompanying drawings of the specification.

[0070] Embodiment 1:

[0071] As is well known, the fibers of the ECM in most human tissues (such as skin, blood vessels, tendons, etc.) have obvious orientation. In the field of tissue engineering, there is a considerable demand for biocompatible materials that exhibit properties similar to those of the ECM. Collagen is a structural protein of the ECM in human tissues, with good mechanical properties, biodegradability, low immunogenicity, etc., as well as the ability of good cell attachment and growth, and is a promising biomaterial in the fields of wound healing and tissue regeneration. And how to make collagen mimic natural tissue ECM so that it can regulate the fate of cells is the key to tissue regeneration.

[0072] A large amount of raw skin and limed hide scraps are generated in the leather-making field in our country every year, and their main component is collagen. If it is extracted and converted into high-value-added medical dressings, it has great economic and social value. Collagen is divided into soluble collagen and insoluble collagen (Insoluble Collagen Fibers, ICFs). The latter has a self-assembly degree and mechanical properties closer to natural collagen ECM than the former, and is more suitable for the preparation of medical dressings. At present, the relatively mature technologies for processing collagen in the market are mainly 3D printing and electrospinning technologies, but they are more suitable for soluble collagen molecules. In previous reports, the counter-rotating extrusion technology and injection technology can achieve the shaping and orientation control of ICFs, but the shaped materials of these two technologies are both dense and pore-free structures, and the mechanical properties of the film / fiber materials processed solely with ICFs raw materials are limited.

[0073] Based on this, in this embodiment, a fully automatic shell-core microporous extrusion device suitable for ICFs is designed and developed. We further use ANSYS software to perform hydrodynamic simulation analysis on the shear force and pressure of micropores with different pore diameters (13G, 15G, 17G), laying a certain theoretical foundation for guiding the subsequent experiments on the regulation of the orientation of ICFs fibers by microporous extrusion.

[0074] Inspired by a medical syringe and improved on this basis, such as Figure 1As shown in the figure, a micro-hole extrusion device is proposed. The extrusion die is designed as a shell-core structure. The shell layer is a natural polymer material based on ICFs in an aqueous solution system, and the core layer is any other polymer material or inorganic non-metallic material suitable for extrusion molding. They can be combined with the ICFs in the shell layer to improve its mechanical properties, and an integrally formed collagen-based fiber can be obtained through extrusion. Among them, the stepper motor is the extrusion device for the shell layer material, the injection pump is the extrusion device for the core layer material, and the extrusion devices are all automated, reducing the experimental errors caused by uneven rates during the manual extrusion process. After extrusion, in order to further improve the mechanical strength and stability of the collagen-based fiber, post-treatments such as fiber self-assembly, cross-linking, and cleaning are required. Therefore, a fiber-forming buffer bath and a deionized water bath are set below the extrusion device, and the temperature is automatically controlled by a constant temperature water bath.

[0075] The structure of the extrusion die is as Figure 2 shown. The upper part adopts the principle of a syringe to make the ICFs extruded and formed. Among them, the plunger head and the barrel body form the upper part of the die (as Figure 2 , shown in B and C), and the barrel body of the die is the filling area for the ICFs material. The lower part is divided into a base and an extrusion needle (as Figure 2 , shown in D and E), and the needle has a shell-core structure. In the initial design, the needle was a straight cylinder at the front end of the shell layer, and the base and the needle were not detachable, making it extremely easy for the ICFs to become blocked during the extrusion process, greatly affecting the experimental progress. Then, on the basis of the initial design, the extrusion needle was changed to a detachable type, and the needle and the base can also be disassembled. While facilitating cleaning, it can also prevent the viscoelastic core layer material and the ICFs from being blocked, effectively promoting the experimental progress. Based on the material properties of the ICFs (high viscosity, acidic), 316L stainless steel material is used, and the physical diagram is as Figure 3 shown.

[0076] This device can achieve automation. As Figure 4 shown, a circuit schematic diagram for automatic control is provided. The automatic extrusion device mainly consists of a control power supply, a driver, a motion controller, a two-phase stepper motor, a linear module slide, and a travel proximity switch. Assemble each part according to the circuit schematic diagram.

[0077] Furthermore, the connection support and fixing device are designed and processed according to the dimensions of the stepper motor and the extrusion die. As Figure 5 and Figure 6 shown, the whole is divided into three parts: an L-shaped extrusion plate, an extrusion die bracket, and a stepper motor card slot box body. The L-shaped extrusion plate is embedded on the stepper motor slide rail and is used to extrude the ICFs material. The extrusion die bracket is used to place and fix the extrusion die to prevent the die from falling off during the extrusion process.

[0078] The overall length of the mold support equipment is about 50 cm, the width is about 30 cm, and the height is about 23 cm. The middle square groove is the placement point for the stepper motor. In the front is the extrusion mold support, which is generally in a U-shape, with a length of about 30 cm, a width of about 18 cm, and a height of about 22 cm. The surface of the support is a circular groove for the extrusion mold. The extrusion mold is fixed by turning the nut on the Y-shaped screw. The needle of the extrusion mold can pass through the middle long strip-shaped hole slot.

[0079] The rear box is the placement point for the stepper motor driver, control power supply, controller, etc., to prevent circuit failures caused by improper placement of electrical components during the operation of the motor. The physical diagram of the overall extrusion equipment is as Figure 7 shown.

[0080] The hydrodynamic simulation analysis of the micro-hole extrusion of this device is as follows:

[0081] 1. Mesh analysis

[0082] Mesh generation is an essential and crucial part of finite element simulation analysis. In order to improve the calculation accuracy and efficiency, it is necessary to generate a mesh. The quality of the effective mesh will affect the calculation result accuracy, solution convergence, and solution speed. The geometric model is as Figure 8 shown. Two viscous fluids converge near the outlet position through two flow channels. In order to carry out the simulation smoothly, the internal fluid region is extracted, and the inlet boundary is set at the fluid inlet position, and the outlet boundary condition is set at the fluid outlet position.

[0083] In this mesh generation, polyhedral meshes are generated using Fluent Meshing. The generated polyhedral mesh is a method of dividing the surface of an object with polygons (such as quadrilaterals, pentagons, etc.) in three-dimensional space to form a mesh. Compared with traditional tetrahedral meshes, it has the following advantages:

[0084] (1) Higher mesh quality: Polyhedral meshes can adaptively generate various polygons with different shapes and sizes on the surface and inside, so as to better capture the geometry of the object and reduce mesh distortion and distortion.

[0085] (2) Higher mesh efficiency: Polyhedral meshes can provide higher mesh resolution with the same number of mesh elements, thus reducing the demand for computing resources.

[0086] (3) Easier to generate: Compared with tetrahedral meshes, the polyhedral mesh generation method is simpler and easier to generate automatically.

[0087] (4) More suitable for complex geometries: When dealing with complex geometries, polyhedral meshes usually have more advantages than tetrahedral meshes because they can better adapt to the surface shape and can ensure the accuracy of geometric details by adjusting the element size.

[0088] The brief steps for meshing using Fluent Meshing are as follows: Import the geometric model that needs to be meshed in Fluent Meshing, import the UG file, and create the geometric model; Use the initial mesh generator to create the initial mesh; Before subsequent meshing, trim and separate the initial mesh (remove the unnecessary parts and separate the domains); Use the polyhedral mesher to mesh the grid; Adjust grid parameters such as grid size and grid resolution; After completing the meshing, perform quality checks on the mesh using mesh warping, mesh size distribution, and mesh element quality to ensure that the generated mesh quality meets the requirements; Export the mesh file for subsequent Fluent simulations.

[0089] 2. Aperture analysis

[0090] (1) Pressure simulation analysis

[0091] The CFD numerical calculation method is adopted. The pressure contour maps of extrusion needles of each specification are as Figure 9 shown. In this simulation analysis, the extrusion needle can be analogized to the pipe model in the polymer flow model. Due to the viscous resistance between polymer fluid layers and the frictional resistance with the pipe, pressure changes will occur during the flow along the pipe, and changes in the cross-sectional shape and size of the flow channel will also cause changes in pressure, volume flow rate, etc. in the polymer fluid. For example, the change in aperture in this simulation analysis will cause a change in the pressure of the glue raw material. It can be seen from the figure that the models with different aperture sizes have a distribution pattern. Both the core layer needle and the shell layer needle show a pattern of gradually decreasing pressure from the inlet to the outlet because the pressure difference between the inlet and outlet provides the kinetic energy for fluid flow, and the pressure energy is converted into fluid kinetic energy during the fluid flow process.

[0092] The inlet pressure of the core layer needle of the 17G model is 16433.05 Pa, and the inlet pressure of the shell layer needle is 58221.46 Pa; the inlet pressure of the core layer needle of the 15G model is 16371.96 Pa, and the inlet pressure of the shell layer needle is 22226.69 Pa; the inlet pressure of the core layer needle of the 13G model is 16412.25 Pa, and the inlet pressure of the shell layer needle is 17067.12 Pa. It can be seen that as the aperture of the shell layer needle increases, the inlet pressure of the shell layer needle gradually decreases, and the inlet pressure of the core layer needle does not change significantly. This may be because the inner needle aperture is not changed. When the inlet fluid flow rate is kept constant, the inlet pressure and outlet pressure of the model do not change significantly. The outlet pressure of the shell layer needle gradually increases with the increase of the aperture, which is 19.60 Pa at 13G, 87.73 Pa at 15G, and 645.84 Pa at 17G.

[0093] (2) Shear force simulation analysis

[0094] Using the CFD numerical calculation method, the shear force distribution nephograms of extrusion needles of each specification are as Figure 10 shown. The flow pattern of the collagen material in the conical extrusion needle is a converging flow, that is, the polymer fluid flows in a channel with a gradually decreasing cross-sectional area. This flow is not only affected by shear but also by tensile forces. It can be seen from the figure that the wall shear forces at different apertures show a similar pattern, that is, a larger shear force is more likely to be formed at positions with a smaller pipe diameter value. This is because the fluid flow follows the law of mass conservation. The fluid flow rate is higher at smaller apertures, resulting in a stronger shear effect on the wall and a larger shear force. The wall shear force distributions of the models at each aperture are studied separately. The shear force at the shell layer outlet position gradually increases with the decrease of the aperture, which is 87.73 Pa at 13G, 241.20 Pa at 15G, and 3236.87 Pa at 17G.

[0095] The influence of the extrusion aperture on the extrusion of the collagen material: In most cases, the polymer fluid exhibits stable laminar flow and obeys the power law. Under the condition of conforming to this law, the shear stress is linearly related to the extrusion hole radius and is independent of the type of fluid. That is, within a certain range, the smaller the extrusion aperture, the greater the shear force on the collagen, and the better the orientation of the collagen.

[0096] The proposed device in this embodiment mainly consists of an injection pump, a stepping motor extrusion device, an extrusion die, a die connection and a fixing bracket, and a post-treatment bath. The injection pump is an extrusion device for the core layer material in the extrusion shell-core structure. The equipment stroke distance is 10 cm. When extruding the core layer material, the experimental rate can be set in the range of 1 mm / s - 100 mm / s. The stepping motor is an extrusion device for the shell layer ICFs in the extrusion shell-core structure, which consists of a driver, a controller, a control power supply, a motor, a module slide, a travel switch, etc. The equipment stroke distance is 20 cm. When extruding the collagen raw material, the experimental rate is 0.1 mm / s - 100 mm / s.

[0097] Set the temperature of the post-treatment bath in advance. After it stabilizes, the extrusion and molding of the material can be carried out. Load the ICFs gel into the shell-core microporous extrusion die (such as Figure 3 ) and place the die in the circular groove of the bracket (such as Figure 6 ), and fix the die by turning the nut on the Y-shaped screw. At the same time, load the core layer material into a medical syringe and fix it in the injection pump. Then, set the rates of the stepping motor and the injection pump respectively and start the program. Extrude the collagen-based fiber material into the post-treatment bath and turn off the power supply after the fiber reaches the required length.

[0098] This device has the following advantages:

[0099] (1) According to the material properties of ICFs, a set of detachable and automated shell-core microporous extrusion device is designed and manufactured with 316L stainless steel as the raw material, the extrusion die as the core, the stepping motor and the injection pump as the extrusion drive modules, and the constant temperature water bath as the post-treatment temperature control equipment, which can realize the integrated molding of collagen-based fibers with a shell-core structure.

[0100] (2) Establish a three-dimensional model of the shell-core micropores, use ANSYS Fluent Meshing for polyhedral mesh generation and boundary condition setting, and then conduct simulation analysis of the pressure and shear force of the collagen extrusion micropores by Fluent. The results show that: when the inlet flow rate is constant, with the decrease of the pore diameter, the pressure and shear force gradually increase.

[0101] Example 2:

[0102] According to the extraction methods, collagen is mainly divided into soluble collagen (SC) and insoluble collagen fibrils (ICFs). Among them, the extraction methods of SC are relatively mature, mainly including acid method, alkali method, salt dissolution method, enzymatic hydrolysis method, acid-enzymatic combination method, etc. In addition, ultrasonic and microwave treatments can assist in improving the extraction rate of collagen and shortening the extraction time. Among them, the extraction rate of the salt dissolution method is relatively low, and it is affected by various factors such as the type, concentration, and temperature of the salt. The collagen extracted by the alkali method is severely hydrolyzed and may be teratogenic and carcinogenic. Therefore, the products extracted by these two methods cannot meet the requirements in the field of biomedical applications. For the acid method and the enzymatic hydrolysis method, the former achieves the purpose of solubilization extraction by breaking the ionic bonds and Shiff bonds of collagen molecules; while the latter can specifically act on the telopeptides of collagen molecules to achieve the purpose of extraction. The collagen extracted by both methods can completely retain its triple helix structure and meet the requirements of the biomedical field. As the name implies, the acid-enzymatic combination method is an extraction method that combines the acid method and the enzymatic hydrolysis method, which can combine the advantages of both and is more widely used. At present, there are few reports on the extraction and forming preparation of ICFs. Compared with SC, ICFs have a higher degree of assembly, better structural stability and mechanical properties, and are more suitable for the preparation of medical dressings. Therefore, in this example, bovine dermis is used as the raw material, and the acid-enzymatic combination method is adopted to explore the extraction conditions of ICFs (mainly including acidification pH, enzymatic hydrolysis concentration, and enzymatic hydrolysis time), and optimize the parameters to extract ICFs.

[0103] Furthermore, on the basis of having designed and manufactured a shell-core microporous extrusion device, an extrusion scheme is designed. Sodium Alginate (SA) and Cellulose (CLL) are both natural polymer materials, which have the advantages of low price, good biocompatibility, biodegradability, and mechanical properties. And after SA comes into contact with a polyvalent cation (such as Ca 2+ ) solution, it can crosslink to form a network structure and instantaneously gel. By mixing the two components (SA / CLL) and forming through extrusion technology, a fiber material with high strength and toughness can be prepared. Therefore, SA / CLL is suitable as the core layer component for shell-core microporous extrusion. Based on this, in this example, ICFs gel is used as the shell layer component, and SA / CLL is used as the core layer component. The variable parameters are optimized (mainly including the collagen concentration in the shell layer, the extrusion pore diameter in the shell layer, the component concentration in the core layer, and the extrusion rate of the shell-core layer), and combined with freeze-drying technology to prepare oriented porous collagen-based fibers with a shell-core structure, laying a foundation for further application in medical dressings.

[0104] The experimental materials and experimental instruments are shown in Tables 1 and 2:

[0105] Table 1. Experimental Materials

[0106] Material Manufacturer Cowhide Slaughterhouse Sodium chloride (NaCl) Chongqing Wansheng Chuandong Chemical Industry <![CDATA[Acetone (C3H6O)]]> Chongqing Wansheng Chuandong Chemical Industry Glacial acetic acid (HAc) Chongqing Wansheng Chuandong Chemical Industry Sodium hydroxide (NaOH) Chengdu Kelong Chemicals Pepsin Beijing Solarbio Science & Technology Ethanesulfonic acid Shanghai Macklin Biochemical Co., Ltd. <![CDATA[Sodium hydrogen phosphate (Na2HPO4)]]> Shanghai Macklin Biochemical Co., Ltd. Absolute ethanol Chongqing Wansheng Chuandong Chemical Industry Collagen Shanghai Macklin Biochemical Co., Ltd. Hydrochloric acid (HCl) Chongqing Orient Chemical Reagent Phosphate buffered saline (PBS) Beijing Biosharp Sodium alginate (SA) Beijing Solarbio Cellulose (CLL) Beijing Solarbio <![CDATA[Calcium chloride (CaCl2)]]> Beijing Solarbio

[0107] Table 2. Experimental Instruments

[0108]

[0109] Experimental Method:

[0110] 1) Extraction of Insoluble Collagen Fibers

[0111] (1) Take the cowhide dermis sample, remove the surface impurities, wash it with deionized water, put it into 0.9% NaCl solution, and place it at 4°C for 24 h;

[0112] (2) Wash it with deionized water, put it into acetone for 24 h for defatting, and then place it in the fume hood to stand overnight;

[0113] (3) Put it into the refrigerator, freeze it and then chop it with a slicer (about 1 mm particles). Take 5 g of the chopped cowhide, put it into 300 mL of deionized water, adjust the pH with 0.5 mol / L HAc (the acidification pH to be optimized), and after acidification for 24 h;

[0114] (4) After acidification, put the cowhide into a high-speed emulsifier for homogenization until there are no particles left (about 30 min). After homogenization, add NaCl and pepsin (the enzymolysis concentration to be optimized) to make the solution concentration 1 mol / L NaCl, and place it at 4°C for enzymolysis (the enzymolysis time to be optimized);

[0115] (5) Put the enzymolyzed material into a low-temperature centrifuge for centrifugation (10000 r / min, 10 min, 4°C), discard the supernatant, add the material to 300 mL of deionized water, and then use a high-speed emulsifier to homogenize the material until it is completely dispersed (about 10 min). Then adjust the pH to about 7.0 with 1 mol / L NaOH solution, and place it in the refrigerator to stand overnight;

[0116] (6) Centrifuge again (10000 r / min, 10 min, 4°C), discard the supernatant, add the remaining material to 300 mL of 0.1 mol / L HAc solution, use a high-speed emulsifier to homogenize the material until it is completely dispersed (about 10 min), and place it in the refrigerator to stand overnight;

[0117] (7) Adjust the pH to about 7.2 with 1 mol / L NaOH solution, and place it in the refrigerator to stand overnight;

[0118] (8) Centrifuge again (10000 r / min, 10 min, 4°C), discard the supernatant, wash the precipitate with deionized water multiple times and then dialyze for 3 d, and finally freeze-dry.

[0119] 2) Optimization of the Extraction Conditions of Insoluble Collagen Fibers

[0120] (1) Optimization of the Acidification pH:

[0121] Set the acidification pH at three gradients: 2.5, 3.0, and 3.5, and use the final collagen extraction rate as the optimization index to screen out the optimal pH for bovine hide acidification.

[0122] (2) Optimization of enzymatic hydrolysis concentration:

[0123] Set the enzymatic hydrolysis concentration at three gradients: 1%, 1.5%, and 2%, and use the final collagen extraction rate as the optimization index to screen out the optimal enzymatic hydrolysis concentration for bovine hide.

[0124] (3) Optimization of enzymatic hydrolysis time:

[0125] Set the enzymatic hydrolysis time at three gradients: 24 h, 36 h, and 48 h, and use the final collagen extraction rate as the optimization index to screen out the optimal enzymatic hydrolysis time for bovine hide.

[0126] 3) Characterization of the structure and composition of insoluble collagen fibers

[0127] Based on the above optimized conditions, the extracted collagen material and commercially available collagen (Macklin, Shanghai) were characterized as follows and analyzed by comparison.

[0128] (1) FTIR

[0129] Take 1 - 2 mg of ICFs sample, cut it into pieces, mix it with 200 mg of KBr powder, grind it finely in an agate mortar, and then press it into a tablet (16 MPa, 2 min). Record the spectrum in the range of 4000 - 400 cm -1 range.

[0130] (2) SDS - PAGE

[0131] Prepare a collagen solution with a concentration of 1 mg / mL using 0.5 mol / L HAc for standby; prepare the gel (6% separating gel and 5% stacking gel), and then perform sample loading, electrophoresis, Coomassie brilliant blue staining, and analyze the molecular weights of each band in sequence.

[0132] (3) Amino acid analysis

[0133] Accurately weigh 15 - 20 mg of collagen fibers, place them in an ampoule, add an appropriate amount of 6 mol / L HCl solution to dissolve them, seal the tube mouth, hydrolyze at 110 °C for 18 h - 24 h, and evaporate to dryness. Add the mobile phase for reaction, then perform n - hexane extraction, dilution, filtration through a membrane, etc., and use HPLC for amino acid composition analysis.

[0134] (4) DSC

[0135] Weigh 3 - 5 mg of the collagen fiber sample, with a blank crucible as a control. Under the protection of N2, the heating rate is 10 °C / min, and the temperature range is 20 °C - 160 °C.

[0136] 4) Optimization of Extrusion Parameters

[0137] (1) Optimization of Collagen Concentration

[0138] First, prepare the Fibre Formation Buffer (FFB): 135 mM sodium chloride, 30 mM ethanesulfonic acid, 30 mM disodium hydrogen phosphate, 10% polyethylene glycol.

[0139] Then, refer to the optimized conditions described above to extract ICFs. Using 0.5% mol / L HAc as the solvent, prepare ICFs gels with concentrations of 4%, 5%, and 6%. Use the self-developed core-shell microporous extrusion device (only the shell layer ICFs gel is passed through, and the core layer material is not passed through), a 15G extrusion needle, and an extrusion rate of 3 mm / s for extrusion molding. Immerse the formed fibers in the FFB solution (35 °C, 60 min), rinse 3 times with deionized water, then immerse in the PBS solution (22 °C, 15 min), rinse 3 times with deionized water, freeze-dry, and perform the following characterizations on the collagen fibers:

[0140] ① SEM

[0141] Attach a 10-mm long fiber material to the stage with conductive tape, sputter with gold for 20 s, and observe and record the image using SEM (voltage 3 kV). Further, randomly select 3 - 5 fields of view, count the pore diameters on the material surface using Image J, and measure the number of pore diameters in each sample > 60. Make a pore diameter distribution graph using Origin and perform Gaussian curve fitting.

[0142] ② Water Absorption Rate

[0143] Place 30 - 40-mm long collagen fibers (initial mass denoted as m0) in a beaker and add 5 mL of PBS. After 30 min, gently dry the liquid on the surface of the dressing with filter paper, measure its mass and record it as m1. The water absorption rate is calculated according to the following formula:

[0144] Water Absorption Rate = (m1 - m0) / m0 × 100%

[0145] ③ Water Retention Rate

[0146] Immerse 30 - 40-mm long collagen fibers in the PBS solution for 30 min, place them in a centrifuge tube, and fill the bottom of the tube with filter paper to absorb the PBS removed by centrifugation. Centrifuge (1200 rpm, 15 min), weigh and record the mass m2, then place the material in an oven and dry it to a constant weight and record its mass m3. The water retention rate is calculated according to the following formula:

[0147] Water Retention Rate = (m2 - m3) / m2 × 100%

[0148] Comprehensively compare the morphologies, SEM images, water absorption rates, water retention rates, and extrusion processes of the three concentration materials after extrusion, and select the most suitable concentration.

[0149] (2) Optimization of the extrusion needle aperture

[0150] Use extrusion needles with different aperture specifications (13G, 15G, 17G), extrusion methods (other parameters: 6% collagen, extrusion rate of 3 mm / s), and post-treatment. The post-treatment has been described previously, and here it is directly carried out according to the post-treatment method described above. Use SEM to observe the surface morphology of the collagen fibers.

[0151] Furthermore, randomly select 3 - 5 fields of view, and use Image J to statistically analyze the orientation of the fibers on the material surface (define the Y-axis direction as 0°, the clockwise included angle as θ, and the counterclockwise included angle as -θ). The measurement quantity for each sample is >60. Use Origin to create an aperture distribution graph and perform Gaussian curve fitting.

[0152] Finally, optimize the needle aperture based on a comprehensive comparison of the extrusion process, cleanability, macroscopic morphology, and microscopic morphology.

[0153] (3) Optimization of the core layer component ratio

[0154] Use the blend of SA and CLL as the core layer material (denoted as SA / CLL). Their different ratios have a significant impact on the mechanical properties, and thus should be optimized. Weigh 2 g of SA and dissolve it in 100 mL of deionized water. Set four groups of CLL with different concentrations (2%, 5%, 8%, 10%) and mix them with 2% SA. Use an injection pump to extrude and form SA / CLL fibers through a syringe (extrusion rate is 6 mm / s). Meanwhile, to facilitate the observation of the stability of the core layer extrusion, add 10 mg of trypan blue to the core layer solution. Furthermore, use a universal mechanical testing machine to conduct mechanical tensile tests on the SA / CLL fibers (the distance between the clamps is 20 mm ± 1 mm, and the tensile rate is 5 mm / min), and record the force-displacement curve. Take pictures under an inverted microscope, use Image J to statistically analyze the average diameter of the fibers, and calculate the cross-sectional area.

[0155] Furthermore, calculate parameters such as stress-strain, fracture strength, fracture elongation rate, and elastic modulus based on the curve to characterize the mechanical properties of the material, and finally comprehensively analyze and select the most suitable concentration ratio.

[0156] (4) Optimization of the core-shell layer extrusion rate

[0157] Based on the above optimization experiments, using 6% ICF gel as the shell material and SA / CLL (5%) as the core material, an extrusion experiment was carried out with a 17G extrusion needle. The shell and core materials were extruded while maintaining the same rate, and three different gradients were set, namely: 1 mm / s, 3 mm / s, and 6 mm / s. Finally, the extrusion rate was optimized according to the macroscopic morphology of the overall extruded core-shell structure material.

[0158] Experimental results:

[0159] As Figure 11 shown, both the acidification pH and enzymatic hydrolysis concentration showed a similar trend of first increasing and then decreasing in the extraction rate of ICFs. When the acidification pH and enzymatic hydrolysis concentration were 3.0 and 1.5% respectively, the extraction rates were the highest, reaching 37.45% and 38.35% respectively. In the experiment of optimizing the enzymatic hydrolysis time, six gradients of 24 h, 36 h, 48 h, 60 h, 72 h, and 84 h were set. As Figure 11 shown, after 60 h of enzymatic hydrolysis time, the upward trend of collagen extraction rate slowed down and reached a peak of 38.18% at 72 h, indicating that 72 h was the optimal enzymatic hydrolysis time during the collagen extraction process. In summary, the optimal collagen extraction conditions in the above several optimization experiments were: acidification pH 3.0, enzyme concentration 1.5%, and enzymatic hydrolysis time 72 h.

[0160] Characterization of the structure and composition of insoluble collagen fibers:

[0161] (1) FTIR

[0162] Fourier transform infrared spectroscopy can reflect the secondary conformation of collagen. To characterize the secondary structure of the collagen extracted this time, its FTIR spectral analysis was compared with commercially available collagen, as Figure 12 shown. The strong absorption at 3270 cm -1 was due to the stretching vibration (hydrogen bond) peak of the N-H group in the amide A band, and the weak absorption near 2934 cm-1 was mainly the characteristic absorption peak caused by the stretching vibration of the C-N group in the amide B band. The amide I band usually appears around 1631 cm -1 and is mainly caused by the stretching vibration of the C=O group, while the amide II band appears around 1529 cm -1 and is caused by the coupling of the C-N stretching vibration and the N-H bending vibration. As Figure 12 shown, similar to commercially available collagen, the extracted ICFs showed the above-mentioned characteristic absorption peaks (amide A band: 3289 cm -1 vs 3270 cm -1 , amide B band: 2934 cm -1 vs 2934 cm -1 , amide I band: 1629 cm -1 vs 1631 cm -1, Amide II band: 1543 cm -1 vs 1529 cm -1 , Amide III band: 1285 cm -1 vs 1243 cm -1 ). The results showed that the characteristic peaks of the two collagen samples were obtained at similar wavenumbers, indicating that the prepared ICFs had the structural characteristics of collagen. The Amide III band is very sensitive to the secondary structure changes of collagen. When there is an absorption peak at 1235 - 1450 cm -1 and the absorbance ratio of the Amide III band to that near 1450 cm -1 is 1, collagen has a complete triple helix structure. As can be seen from the figure, there are absorption peaks at 1285 cm -1 , 1448 cm -1 and the ratio is 0.9926, indicating that the extracted structure of collagen is complete.

[0163] (2) SDS-PAGE

[0164] Figure 13 is the SDS-PAGE analysis diagram of the extracted collagen fiber material, where lane A is Sigma collagen and lane B is insoluble collagen (ICFs). Three separated bands were obtained successively by collagen fiber electrophoresis, namely β, α1 and α2, among which the molecular mass of β is about 170 KDa and the molecular mass of α2 is about 115 KDa. There are no extra electrophoresis bands below the α2 chain, indicating that there are no small molecular collagen polypeptides in the extracted collagen fibers.

[0165] (3) Amino acid analysis

[0166] It can be seen from Table 3 that the amino acid compositions of commercially available collagen and extracted ICFs are similar, that is, they both do not contain tryptophan, and the contents of glycine (31.9% vs 32.5%) and imino acids (hydroxyproline (9.6% vs 10.1%), proline (12.2% vs 13.1%)) are close, which is consistent with the literature reports. The results indicate the primary structure characteristics of the extracted ICFs collagen.

[0167] Table 3. Amino acid composition analysis of collagen

[0168]

[0169]

[0170] (4) DSC

[0171] As Figure 14 shown, differential scanning calorimetry analysis was carried out on Sigma collagen and the extracted insoluble collagen for comparison. From Figure 14As can be seen, the endothermic peaks of both samples are sharp and steep, indicating a high purity of the samples. The thermal denaturation temperature of insoluble collagen is higher than that of Macklin collagen (107.34 °C vs 73.37 °C). The thermal absorption peak of collagen reflects the ease with which the triple helix structure of collagen molecules is gradually destroyed and the molecules change from an extended and ordered state to a disordered coiled state. Therefore, to a certain extent, it can be reflected that the structural stability of insoluble collagen is higher than that of the control group and it will not easily denature during practical applications.

[0172] Collagen concentration:

[0173] When the collagen concentration is 4%, the extrusion process is smooth, but due to the too low viscosity of the collagen, it is easy to break and the extrusion is uneven during extrusion, as shown by the circle in (A) in Figure 15 . When the collagen concentration is 5%, the extrusion process is smooth, the overall extrusion condition of the collagen is good, the overall extrusion shape is good, and the breakage situation is reduced, but there will still be an uneven extrusion condition. When the collagen concentration is 6%, the extrusion is relatively smooth. Due to the relatively high viscosity of the collagen during extrusion, the compactness and shape of the collagen during extrusion are good, the overall texture is relatively uniform, and basically no breakage occurs. According to the SEM results comparison in (B) in Figure 15 , collagens with concentrations of 4%, 5%, and 6% all have a certain degree of orientation. Semi-quantitative analysis of their surface pore sizes (such as (C) in Figure 15 ) shows that the porous structures of 4% and 5% are similar, the pore sizes are mainly distributed in the range of 4 - 6 μm and the connectivity between pores is good; while the pore size range in 6% mainly focuses on 0 - 4 μm. Theoretically, smaller pore sizes are more likely to exhibit microporous siphon action and have a better effect of absorbing exudate. On the other hand, for larger and well-connected pore structures during wound repair, fibroblasts are more likely to migrate into the pore structures, which may cause adhesion between the dressing and the skin wound, resulting in secondary injury.

[0174] Therefore, we further detected the water absorption rate and water retention rate of fiber materials with different concentrations. The results are as shown in Figure 16 . The water absorption rates of 5% and 6% are comparable and are both significantly greater than 4%. As the collagen concentration increases, the water retention rate shows an upward trend, indicating that 6% has a better effect of absorbing exudate, which is consistent with the theoretical analysis. Therefore, considering comprehensive indicators such as the extrusion process, the macroscopic morphology of collagen, the microscopic morphology, as well as the water absorption rate and water retention rate, when the collagen concentration is 6%, the experimental effect is the best and it is suitable for subsequent dressing preparation experiments, etc.

[0175] Extrusion needle aperture:

[0176] In the early stage of the experiment, we welded the extrusion needle to the bottom of the extrusion head (S1) to ensure the airtightness during extrusion. During the experiment, it was found that for the extrusion die made in this way, the needle was not easy to clean and was prone to blockage.

[0177] Furthermore, an adapter was designed and processed, (S2) to facilitate the replacement of needles of different specifications. To better optimize the stent extrusion parameters, we conducted extrusion comparison experiments using extrusion needles of various models. As Figure 17 shown, from left to right, S2-1, S2-2, S2-3, and S2-4 are the conical needle outer diameters of 17G (1.07 mm), conical needle 15G (1.40 mm), conical needle 13G (1.90 mm), and straight needle 17G, respectively. The inner diameter remains unchanged at 22G (0.40 mm). We scored the experimental phenomena that hindered the extrusion and forming of the collagen material according to a 5-level scoring system, such as the extrusion smoothness of the mold, the blockage situation, and the cleaning situation. The degree of hindrance increases with the increase in the number. The collagen forming rate is divided into three levels. The first level is that the collagen forms unevenly or easily spreads; the second level is that the collagen extrudes relatively evenly and has a good adhesive shape; the third level is that the collagen extrudes evenly and the overall collagen extrusion shape is good.

[0178] The experimental results are shown in Table 4, Figure 18 as shown. When the extrusion needle is 13G, the extrusion process is smooth, but due to the large aperture, the pressure during extrusion has a small binding force on the collagen, resulting in easy uneven extrusion. It was found by SEM observation that the collagen fibers are relatively loose and have poor orientation; when the extrusion needle is 15G, the extrusion process is relatively smooth, and the binding force of the extrusion pressure on the collagen increases, reducing uneven extrusion. However, it was found by SEM observation that the overall collagen orientation is not as good as that of 17G; when the extrusion needle is 17G, the extrusion process is relatively difficult, but due to the further increase in the binding force of the extrusion pressure on the collagen, the overall extruded collagen is uniform and the extruded collagen has a good shape. By SEM observation, it has good orientation and high collagen fiber density. Through semi-quantitative statistics (as Figure 18 shown in (C) below), as the needle aperture decreases, the degree of fiber orientation shows an increasing trend, and this result is consistent with the trend of the shear force hydrodynamics results of the micropores in Chapter 2 ( Figure 10 ). In summary, on the premise that S2-1 ensures that the extrusion smoothness, blockage situation, and cleaning situation are all at an upper-middle level, it also has a relatively high collagen forming rate and is the most suitable needle among the five needle models.

[0179] Table 4. Influence of needle models on collagen forming

[0180]

[0181] Optimization of the nuclear layer components:

[0182] As Figure 19The diameter tests for 2%, 5%, 8%, and 10% are as follows: 0.590 mm ± 0.006 mm, 0.585 mm ± 0.008 mm, 0.587 mm ± 0.011 mm, 0.620 mm ± 0.010 mm. The mechanical property parameters are calculated from the stress-strain curve and summarized in Table 5. The results show that the 5% group has the best ratio in terms of breaking tensile force, elastic modulus, and breaking strength among the four groups.

[0183] Table 5. Mechanical tensile properties of the inner sodium alginate-cellulose

[0184]

[0185] Note: Experimental data are expressed as mean ± standard deviation, n≥3

[0186] Optimization of the core-shell layer extrusion rate:

[0187] As Figure 20 shown, when the core-shell layer rate is 6 mm / s, due to the relatively fast rate, the outer layer of collagen has obvious rupture phenomena, as shown by the circles in (A) in Figure 20 , which cannot meet the requirements of the experiment; when the core-shell layer rate is 3 mm / s, the rate is relatively moderate, the core-shell layer material is extruded more evenly, and the overall morphology is good; when the core-shell layer rate is 1 mm / s, the overall extrusion will cause swirling and winding due to the too slow rate, affecting the orientation of the overall core-shell material, as shown by the circles in (C) in Figure 20 . The rate optimization experiment shows that when the core-shell layer rate is about 3 mm / s, the overall material extrusion rate is more appropriate.

[0188] Based on the above optimization results, the core-shell structured collagen fibers were successfully prepared. As shown in (A) in Figure 21 , after adding TB, it can be clearly seen that the middle SA / CLL fibers are well integrated with the shell layer collagen, meeting our preparation expectations. As shown in (B) and (C) in Figure 21 , the fibers on the surface of the collagen are arranged tightly, with obvious fiber orientation and pore structure. From the cross-section observation, it can be found that the internal arrangement of the collagen fibers is loose and has good connectivity, which is beneficial to cell migration and the transport of water and nutrients.

[0189] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and the description of the present invention.

Claims

1. A method for preparing composite insoluble collagen fibers, characterized in that, Based on a shell-core microporous extrusion device, comprising: An injection pump for extruding the core layer material; A stepper motor; An extrusion die, the stepper motor is connected to the extrusion die for extruding ICFs material; A post-treatment mechanism; A conduit, one end of the conduit is connected to the injection pump, the other end is connected to the post-treatment mechanism, and the middle of the conduit is connected to the extrusion die; The method includes: Taking a cowhide dermis sample and putting it into a NaCl solution; Putting it into acetone for defatting, and then ventilating and standing still; After freezing, chopping it with a slicer, taking an appropriate amount of chopped cowhide, putting it into deionized water, and adjusting the pH with HAc; After acidification, homogenizing the cowhide until there are no fragments, adding NaCl and pepsin after homogenization, and enzymolyzing at 4°C; Centrifuging the enzymolyzed material, discarding the supernatant, adding the material to deionized water, and homogenizing it into a homogeneous solution, adjusting the pH to 6.8 - 7.2, and refrigerating and standing still; Centrifuging again, discarding the supernatant, adding the remaining material to an HAc solution, homogenizing it into a homogeneous solution, and refrigerating and standing still; Adjusting the pH to about 7.0 - 7.4, and refrigerating and standing still; Centrifuging again, discarding the supernatant, washing the precipitate multiple times, dialyzing, and then freeze-drying to obtain composite insoluble collagen; Using the blend of SA and CLL as the core layer material, the concentration ratio of SA and CLL is determined according to calculating stress-strain, breaking strength, elongation at break, and elastic modulus to characterize the mechanical properties of the material by a curve; Filling the insoluble fibrous collagen into the extrusion die, extruding the insoluble fibrous collagen through the extrusion die, and pumping out the core layer material by the injection pump to obtain a core-shell structure; Immersing the core-shell structure in an FFB solution for post-treatment, the FFB solution at least includes sodium chloride, ethane sulfonic acid, disodium hydrogen phosphate, and polyethylene glycol.

2. The method according to claim 1, characterized in that, The extrusion die includes: A press head, the press head is connected to the output end of the stepper motor; A press body for filling ICFs material, the press head is movably arranged in the press body; A base, the press body is arranged at the upper end of the base; An extrusion needle, the extrusion needle is arranged at the lower end of the base, and the extrusion needle is internally connected to the press body to discharge the ICFs material in the press body from the extrusion needle into the conduit by squeezing of the press head.

3. The method according to claim 1, characterized in that, It further includes: An L-shaped extrusion plate arranged on the slide rail of the stepper motor for squeezing the extrusion die to extrude ICFs material; A die bracket on which the extrusion die is installed; A stepper motor card slot box body on which the stepper motor is installed.

4. The method according to claim 1, wherein The post-treatment mechanism includes a constant temperature water bath, and a fiber-forming buffer solution or deionized water is arranged in the constant temperature water bath.

5. The method according to claim 4, characterized in that, The constant temperature water bath includes a first pot body and a second pot body connected by pipes in sequence, a fiber-forming buffer solution is arranged in the first pot body, and deionized water is arranged in the second pot body.

6. The method according to claim 5, wherein The acidification pH value adjusted with HAc is determined by the following method: Setting three gradients for the acidification pH value, which are 2.5, 3.0, and 3.5 respectively, and using the final collagen extraction rate as the optimization index to screen out the optimal pH for cowhide acidification.

7. The method according to claim 5, characterized in that The enzymatic hydrolysis concentration was determined by the following method: The enzymatic hydrolysis concentration was set at three levels, namely 1%, 1.5%, and 2%, and the final collagen extraction rate was used as the optimization index to screen out the optimal enzymatic hydrolysis concentration.

8. The method according to claim 5, characterized in that, The enzymatic hydrolysis time was determined by the following method: The enzymatic hydrolysis time was set at three gradients: 24 h, 36 h, and 48 h, and the final collagen extraction rate was used as the optimization index to screen out the optimal time for enzymatic hydrolysis of cowhide.

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