Chitosan-collagen synergistic self-assembly fiber material and preparation method thereof

Abstract

The invention relates to a chitosan-collagen synergistic self-assembly fiber material and a preparation method thereof, and belongs to the technical field of biological fiber materials. According to the method, the mass ratio of chitosan to collagen is controlled, co-assembly is induced through hydrogen bonds and electrostatic interaction in a low-temperature PBS solution, and then glutaraldehyde crosslinking, gradient dehydration and supercritical drying are performed to prepare the freeze-dried fiber. The obtained material completely retains the triple helix conformation of collagen, and has excellent conformation stability and enzymolysis resistance. The mechanical strength of the composite fiber is remarkably improved, and the composite fiber has excellent antibacterial property and cell affinity. The process does not need a toxic cross-linking agent, the operation is simple and convenient, and the prepared fiber is suitable for the high-end biomedical fields of tissue engineering scaffolds, intelligent antibacterial dressings, degradable nerve conduits and the like.

Description

Technical Field

[0001] This invention relates to a chitosan-collagen synergistic self-assembled fiber material and its preparation method, belonging to the field of biofiber material technology. Background Technology

[0002] Collagen, a natural high-molecular-weight structural protein, is widely distributed in connective tissues such as animal skin, cartilage, and tendons, accounting for approximately 30% of total human protein. Its unique triple-helix structure, formed by three polypeptide chains linked by hydrogen bonds, endows it with excellent biomechanical properties and structural stability. Thanks to its superior biocompatibility, tunable degradation, and low immunogenicity, collagen has become a core raw material in tissue engineering (such as cartilage regeneration scaffolds and artificial blood vessel construction), wound dressings, and biomedical materials. However, natural collagen faces significant challenges in in vitro applications: it is prone to structural unwinding and degradation under non-physiological conditions (fluctuations in temperature, pH, or ionic strength), leading to loss of bioactivity; simultaneously, due to its high water solubility and poor thermal stability, it is difficult to maintain a biomimetic fiber conformation in a dry or in vitro state, resulting in a sharp decline in mechanical strength, failing to meet the mechanical support requirements of load-bearing or dynamic environments. Therefore, simultaneously improving the structural stability, mechanical properties, and biofunctionality of collagen while preserving its active triple-helix conformation is currently a core bottleneck in biomaterial development.

[0003] Chitosan, a cationic polysaccharide derived from the deacetylation of natural chitin, possesses excellent biocompatibility, controllable degradation, broad-spectrum antibacterial activity, and molecular modifiability (rich in active amino / hydroxyl groups). It can construct stable composite systems through hydrogen bonding, electrostatic interactions, or coordination complexation, demonstrating core value in hemostatic materials, smart wound dressings, targeted drug carriers, and tissue engineering repair. However, while chitosan exhibits certain film-forming and mechanical properties, its natural state lacks a clear three-dimensional structural feature, failing to provide the biomimetic scaffold function of collagen. Although research on chitosan / collagen composite systems has made progress (such as gel hemostatic agents, thin-film dressings, and porous scaffolds), current technology still has significant limitations: existing research mainly focuses on macroscopic mixed morphologies (sponge-like / thin-film), with insufficient exploration of fiber-level ordered self-assembly mechanisms; the charge matching rules and conformational synergistic effects between the two at the molecular level are not yet clear, making it difficult for materials to simultaneously achieve the stability of the collagen triple helix active domain and the functional advantages of chitosan (such as antibacterial properties), resulting in an imbalance between bioactivity and mechanical properties. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for constructing synergistic fibers of chitosan and collagen through a simple preparation process. Based on this method, composite fiber materials with excellent conformational stability, mechanical properties, and biomimetic functions are obtained, which can not only maintain the bioactivity of collagen but also combine the functionality of chitosan.

[0005] The first aspect of the technical solution of the present invention provides a method for preparing chitosan-collagen synergistic self-assembled fiber materials, comprising the following steps: S1. Prepare collagen solution and chitosan solution; S2. Cooperative self-assembly: The chitosan solution obtained in step S1 is added to the collagen solution and mixed. PBS buffer solution is added at 2-8 ℃ to induce cooperative self-assembly of chitosan and collagen to form a stable triple helix composite solution. S3. Low-temperature centrifugation to separate solid and liquid, collect the precipitate, and fix it by cross-linking in glutaraldehyde solution; S4. Gradient dehydration: The cross-linked fiber matrix is ​​placed in ethanol solutions with a concentration gradient of 30%-100% for gradual dehydration; then it is dried by supercritical carbon dioxide to obtain freeze-dried fiber material; In step S2, the mass ratio of chitosan to collagen is 1:4 to 1:1; preferably 1:4 to 1:2.

[0006] Further, in step S1, the collagen solution is prepared by dissolving collagen in a dilute acid solution to obtain a collagen solution; the chitosan solution is prepared by dissolving chitosan with a degree of deacetylation of 85%–95% in glacial acetic acid solution and stirring to dissolve and prepare a solution.

[0007] In one embodiment, chitosan with a degree of deacetylation of 85%–95% is dissolved in a 1% (v / v) glacial acetic acid solution, stirred to dissolve and prepare a solution, and then filtered to sterilize for later use.

[0008] In one embodiment, eggshell membrane protein is dissolved in 0.01 mol / L hydrochloric acid to prepare a collagen solution with a concentration of 5 mg / mL.

[0009] In some embodiments, the molecular weight of the collagen is 8000-14000 Da; In some embodiments, the chitosan has a molecular weight of 200-400 kDa; more preferably 200-300 kDa. Furthermore, in step S2, the total concentration of the chitosan and collagen mixed solution is 4.5-6.5 mg / mL.

[0010] In some embodiments of the present invention, the collagen is eggshell membrane protein; the eggshell membrane protein preparation method is as follows: the eggshell membrane is peeled, washed, enzymatically hydrolyzed, salted out, centrifuged, dialyzed, and freeze-dried to obtain the extracted eggshell membrane protein; the salting out temperature of the eggshell membrane enzymatic hydrolysate is 4-8℃; the freeze-drying time is 48-72h.

[0011] Furthermore, the concentration range of the chitosan solution is 0.5-2.0 mg / mL; Furthermore, the concentration range of the collagen is 3.0-7.0 mg / mL; In some embodiments, the PBS buffer is a phosphate buffer with pH 6.4–6.6 and an ionic strength of 10–20 mmol / L, used to induce the co-assembly of chitosan and collagen under low-temperature conditions.

[0012] In some embodiments, in step S3, the volume fraction of glutaraldehyde is 2%-4%; the fixation time is 2-4 h.

[0013] A second aspect of the present invention provides a fiber material prepared by the preparation method, wherein the fiber material is a chitosan-modified collagen self-assembled nanofiber network with an average diameter of 300-500 nm; further, the average diameter is 300-350 nm, 350-400 nm, 400-450 nm, or 450-500 nm.

[0014] Furthermore, the fiber material exhibits D-periodic banding with a clearly visible structure; the triple helix conformation is detected by CD spectroscopy. pn The value is 0.11-0.13; preferably 0.115-0.130. A third aspect of the invention provides the application of the fibrous material in the fields of tissue engineering, drug delivery, bio-dressing, and biomimetic materials.

[0015] Furthermore, the fibrous material can be applied in the field of tissue engineering for the regeneration and repair of cartilage, bone, or skin tissue; It is used in the field of drug delivery for targeted and sustained release of growth factors or antibiotics; It is used in the field of biological dressings for the repair of functional wounds that have antibacterial and healing properties; It is applied in the field of biomimetic materials to prepare biodegradable scaffolds.

[0016] The beneficial effects of this invention are: This invention utilizes the synergistic self-assembly of chitosan and collagen under mild conditions to form a stable nanofiber network. While simultaneously preserving the triple helix conformation of collagen and the functional properties of chitosan, it significantly improves the conformational stability, resistance to enzymatic degradation, mechanical properties, and environmental adaptability of the fiber material. Specific technical effects are as follows: (1) The nanofibers formed by the synergistic self-assembly of chitosan and collagen in this invention have a smooth surface and completely retain the triple helix conformation of collagen (circular dichroism R). pnThe value reached 0.116-0.128, far higher than the comparative example of 0.114), and electron microscopy revealed typical 65–70 nm D-periodic bands. Long-term physiological environment simulations validated that this structure can maintain high-level collagen triple helix conformational stability, significantly reducing the risk of disintegration and degradation.

[0017] (2) The fiber material prepared by the present invention has significantly improved mechanical and anti-degradation properties, good elasticity and structural stability, and significantly enhanced load-bearing capacity; at the same time, it has strong anti-enzymatic properties (the residual mass after 48 hours of collagenase degradation is >65%, compared to <20% in the comparative example), and has excellent structural anti-destruction ability.

[0018] (3) The fiber material prepared by the present invention has excellent antibacterial properties (antibacterial rate > 80%), cell adhesion (adhesion rate > 90%), and biocompatibility. By regulating the reaction conditions, the fiber diameter (300-500nm), orientation and network topology can be precisely controlled to meet diverse biomimetic needs.

[0019] (4) The preparation method of the present invention is mild and simple to operate. It does not use toxic crosslinking agents. The resulting material has high processing adaptability and can be directly applied to tissue engineering scaffolds, composite hydrogel skeletons and smart dressings to support regenerative medicine scenarios such as soft tissue repair and trauma management. Attached Figure Description

[0020] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 A schematic diagram of the preparation process of a chitosan-collagen synergistic self-assembled fiber material; Figure 2 The turbidity test of the composite solution prepared in step (2) of Examples 2-5 of the present invention; Figure 3 Tests were conducted on the degree of self-assembly of collagen fibers prepared in Examples 2-5 of this invention. Figure 4 CD circular dichroism spectroscopy test of the composite solution prepared in step (2) of Examples 2-5 of the present invention; Figure 5 R of the collagen prepared in Examples 2-5 of this invention pn value; Figure 6 Transmission electron microscopy (a)-(d) of the materials prepared for Examples 2-5 of the present invention correspond to Examples 2-5 respectively; Figure 7Transmission electron microscopy tests of the materials prepared for the embodiments / comparative examples of the present invention (a)-(d) correspond to Examples 2-5, respectively, and (e)-(f) correspond to Examples 1-2, respectively; Figure 8 Storage modulus testing of hydrogels prepared from the materials in Examples 2-5 of this invention; Figure 9 The loss modulus of the hydrogels prepared from the materials in Examples 2-5 of this invention was tested. Figure 10 Loss factor test of composite solutions of hydrogels prepared from the materials in Examples 2-5 of this invention; Figure 11 Cell adhesion rate test of the dried film prepared from the materials in Examples 2-5 of this invention; Figure 12 The antibacterial rate of the dried film prepared from the materials in Examples 2-5 of this invention was tested. Figure 13 The collagenase degradation experiments of the materials in Examples 2-5 of this invention are shown. Detailed Implementation

[0021] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification. Many specific details are set forth in the following description to provide a thorough understanding of the present invention; however, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials are obtained from poultry eggshell membranes through enzymatic hydrolysis with proteases.

[0023] Raw materials used in the examples: Chitosan: Industrial-grade high-purity chitosan with a degree of deacetylation of 90% ± 2% and an average molecular weight of 200–300 kDa.

[0024] Example 1: Figure 1 This application illustrates a schematic flowchart of a chitosan-collagen synergistic self-assembly fiber material and its preparation method, provided by an exemplary embodiment of the present application. The method includes: Step 1: The eggshell membrane is subjected to peeling, washing, enzymatic hydrolysis, salting out, centrifugation, dialysis, and freeze-drying to obtain the extracted eggshell membrane protein; Step 2: By controlling the mass ratio of chitosan to collagen, PBS solution is added under low temperature conditions to induce co-assembly and form a stable triple helix composite solution. Step 3: Discard the supernatant by low-temperature centrifugation, collect the precipitate, and fix it with glutaraldehyde solution; Step 4: Gradual dehydration is carried out in ethanol solutions of different concentration gradients, and finally dried with critical point carbon dioxide to obtain the prepared freeze-dried fiber material.

[0025] Example 2: (Comparative example, without chitosan) This embodiment provides a chitosan-collagen synergistic self-assembled fiber material and its preparation method, the specific steps of which are as follows: (1) The eggshell membrane was manually peeled off from the eggshell, rinsed with deionized water, and enzymatically hydrolyzed with pepsin. The supernatant was collected, the pH was adjusted with NaOH solution, NaCl was added for salting out, centrifuged, and purified using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. The dialysis product was freeze-dried to obtain the target eggshell membrane protein extract.

[0026] (2) Dissolve eggshell membrane protein in 0.01 mol / L hydrochloric acid to prepare a collagen solution with a concentration of 5 mg / mL. Add PBS solution at 4℃ to induce co-assembly and form a stable triple helix complex solution. (3) The supernatant was discarded by centrifugation at low temperature (4℃), the precipitate was collected and fixed with glutaraldehyde solution (volume fraction of 2%). (4) The freeze-dried fiber material was obtained by stepwise dehydration at a series of ethanol concentrations (30, 50, 70, 90, 95 and 100%, v / v) and dried with critical point carbon dioxide.

[0027] Example 3: This embodiment provides a chitosan-collagen synergistic self-assembled fiber material and its preparation method, the specific steps of which are as follows: (1) The eggshell membrane was manually peeled off from the eggshell, rinsed with deionized water, and enzymatically hydrolyzed with pepsin. The supernatant was collected, the pH was adjusted with NaOH solution, NaCl was added for salting out, centrifuged, and purified using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. The dialysis product was freeze-dried to obtain the target eggshell membrane protein extract.

[0028] (2) Dissolve eggshell membrane proteins in 0.01 mol / L hydrochloric acid to prepare a collagen solution with a concentration of 5 mg / mL. Take a chitosan solution with a mass concentration of 1.0 mg / mL, which is prepared by dissolving chitosan (degree of deacetylation 90% ± 2%, molecular weight 200-300 kDa) in 1% (v / v) glacial acetic acid solution; add the chitosan solution to the collagen solution, controlling the ratio of chitosan to collagen to be 1:4 (w / w) mixed solution. Add PBS solution at 4℃ to induce co-assembly, forming a stable triple-helix composite solution; (3) The supernatant was discarded by centrifugation at low temperature (4℃), the precipitate was collected and fixed with glutaraldehyde solution (volume fraction of 2%). (4) The freeze-dried fiber material was obtained by stepwise dehydration at a series of ethanol concentrations (30, 50, 70, 90, 95 and 100%, v / v) and dried with critical point carbon dioxide.

[0029] Example 4: This embodiment provides a chitosan-collagen synergistic self-assembled fiber material and its preparation method, the specific steps of which are as follows: (1) The eggshell membrane was manually peeled off from the eggshell, rinsed with deionized water, and enzymatically hydrolyzed with pepsin. The supernatant was collected, the pH was adjusted with NaOH solution, NaCl was added for salting out, centrifuged, and purified using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. The dialysis product was freeze-dried to obtain the target eggshell membrane protein extract.

[0030] (2) Eggshell membrane proteins were dissolved in 0.01 mol / L hydrochloric acid to prepare a collagen solution with a concentration of 5 mg / mL. A chitosan solution with a mass concentration of 1.5 mg / mL was added to the collagen solution, controlling the ratio of chitosan to collagen to be 1:3 (w / w). PBS solution was added at 4℃ to induce co-assembly, forming a stable triple-helix composite solution. (3) The supernatant was discarded by centrifugation at low temperature (4℃), the precipitate was collected and fixed with glutaraldehyde solution (volume fraction of 2%). (4) The freeze-dried fiber material was obtained by stepwise dehydration at a series of ethanol concentrations (30, 50, 70, 90, 95 and 100%, v / v) and dried with critical point carbon dioxide.

[0031] Example 5: This embodiment provides a chitosan-collagen synergistic self-assembled fiber material and its preparation method, the specific steps of which are as follows: (1) The eggshell membrane was manually peeled off from the eggshell, rinsed with deionized water, and enzymatically hydrolyzed with pepsin. The supernatant was collected, the pH was adjusted with NaOH solution, NaCl was added for salting out, centrifuged, and purified using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. The dialysis product was freeze-dried to obtain the target eggshell membrane protein extract.

[0032] (2) Dissolve eggshell membrane proteins in 0.01 mol / L hydrochloric acid to prepare a collagen solution with a concentration of 5 mg / mL. Take a chitosan solution with a mass concentration of 2.0 mg / mL; add the chitosan solution to the collagen solution, controlling the ratio of chitosan to collagen to be 1:2 (w / w) of the mixture. Add PBS solution at 4℃ to induce co-assembly and form a stable triple-helix composite solution; (3) The supernatant was discarded by centrifugation at low temperature (4℃), the precipitate was collected and fixed with glutaraldehyde solution (volume fraction of 2%). (4) The freeze-dried fiber material was obtained by stepwise dehydration at a series of ethanol concentrations (30, 50, 70, 90, 95 and 100%, v / v) and dried with critical point carbon dioxide.

[0033] Comparative Example 1: The preparation steps are the same as in Example 5, except that the chitosan used has an average molecular weight of 500 kDa.

[0034] Comparative Example 2 The preparation steps are the same as in Example 5, except that the chitosan used has an average molecular weight of 150 kDa.

[0035] Figure 2 The turbidity of the solutions prepared in Examples 2-5 of this invention is tested. Turbidity measurement is a typical method for evaluating the collagen self-assembly process, characterized by a typical "S"-shaped turbidity-time curve, which includes three main phases: retention period, growth period, and stationary period. Similar to the pure collagen solution (Example 2), the chitosan / collagen composite solution system also exhibits a typical "S"-shaped curve, indicating that the introduction of chitosan did not alter the collagen nucleation mechanism. The quantitative analysis results of the turbidity curves are shown in Table 1.

[0036] like Figure 2 As shown in Table 1, the appropriate introduction of chitosan can shorten the lag time of collagen fiber formation compared with pure collagen solution. Furthermore, the initial turbidity level and turbidity value changes (Δh) in Examples 3, 4, and 5 are relatively high, which may be due to the electrostatic interaction between collagen and chitosan, resulting in the formation of a collagen-chitosan polyelectrolyte complex.

[0037] Table 1 Quantitative data of turbidity curves Fiber formation is an entropy-driven process involving hydrophobic and electrostatic interactions between nonpolar regions of adjacent molecules, hydrogen bonding between polar residues, and specificity of molecular recognition. These interactions minimize the surface area to volume ratio by removing solvent molecules from the collagen surface, resulting in a circular cross-section.

[0038] like Figure 3 As shown, the degree of self-assembly of collagen fibers in Examples 3-5 was increased compared to that in pure collagen solution (Example 2). These results indicate that appropriate addition of chitosan is beneficial to the fiber formation process. This observation is consistent with the turbidity curve analysis. This phenomenon can be attributed to the synergistic electrostatic interaction and chain entanglement behavior between chitosan and collagen. The introduction of an appropriate amount of chitosan not only enhances the mutual adsorption between cations and negatively charged collagen groups, thereby promoting the initial aggregation and nucleation of triple-helix collagen molecules; at the same time, the molecular chain structure of chitosan can also act as a spatial scaffold during collagen fiber formation, assisting in collagen chain entanglement and orderly extension, thereby accelerating the axial growth process of fibers.

[0039] Figure 4 The circular dichroism (CD) spectroscopy of the composite solutions prepared in Examples 2-5 of this invention is shown. CD spectroscopy is mainly used for the analysis of protein secondary structure. Specifically, the CD spectrum in the far-ultraviolet band is used as an indicator to assess changes in the secondary structure of collagen after interaction with chitosan. Typically, the CD spectrum of natural collagen contains a positive absorption peak at 220 nm and a negative absorption peak at 197 nm, which can be attributed to the n-π* transition and the π-π* amide transition, respectively. Figure 4 As shown, the spectrum of collagen (Example 2) exhibits a positive peak at 220 nm and a negative peak at 197 nm, which is attributed to the unique supercoiled secondary structure of collagen. Furthermore, the CD spectrum of the collagen and chitosan mixture shows a negative absorption band at 198 nm and a positive absorption band at 220 nm, indicating that the composite sample retains the characteristic structure of collagen after the addition of chitosan. The ratio of the intensity of the positive peak to the intensity of the negative peak in the CD spectrum is called Rc. pn It is the characteristic ratio of a triple helix structure. The R of natural collagen... pn The value is 0.11-0.15. For example... Figure 5 As shown, collagen's R pn A value of 0.114 indicates that the triple helix structure is well preserved. Furthermore, the R values ​​in Examples 3, 4, and 5... pnThe values ​​all increased, indicating that an appropriate amount of chitosan can effectively stabilize the triple helix conformation through electrostatic adsorption and chain entanglement.

[0040] Figure 6 Transmission electron microscopy (TEM) tests were performed on the solutions prepared in Examples 2-5 of this invention. Figure 6 As shown, relatively small fibers were observed in the pure collagen sample (Example 2), indicating that although collagen molecules have self-assembly capabilities, the degree of fibrillation is limited without an auxiliary system. In contrast, the fiber morphology in Examples 3, 4, and 5 was significantly improved, forming a dense, continuous, and robust three-dimensional fiber network structure, demonstrating higher fiber forming ability and network stability. The appropriate introduction of chitosan can significantly promote the lateral entanglement and fiber aggregation of collagen molecules, improving the continuity and biomimicry of the macroscopic morphology.

[0041] To further evaluate the effect of chitosan on the maturity of collagen self-assembly, the microstructure of each sample was observed, with a focus on analyzing the D-periodic structure of collagen fibers. D-periodicity is a typical structural feature of mature collagen fibers, typically manifested as axially repeating stripes of approximately 67 nm, formed by the end-to-end arrangement of collagen triple helix molecules, and is an important indicator for measuring the integrity of collagen self-assembly.

[0042] As shown in Figure 7, although pure collagen samples can form fibrous structures, they exhibit unclear and irregular D-periodic stripes in TEM images. After introducing chitosan with different proportions and molecular weights of 200-400 kDa, the resulting composite nanofiber structures all exhibited clear and continuous D-periodic stripe structures, indicating that the triple helix conformation was well preserved and the axially ordered aggregation of collagen molecules was not disrupted. Simultaneously, it can be observed that as the chitosan proportion increases, the fiber diameter increases from below 300 nm in pure collagen samples to 300-500 nm, with thicker fibers and a still regular arrangement. This suggests that the introduction of an appropriate amount of chitosan can enhance the lateral entanglement ability and structural stability of collagen molecules through synergistic effects, thereby forming dense nanofibers with strong resistance to deconstruction. In contrast, Figure 7 (e) and Figure 7 (f) These are composite samples prepared using chitosan with a content greater than 400 kDa (Fig. e) and less than 200 kDa (Fig. f) respectively, as compared to Comparative Examples 1-2, showing significantly different structural characteristics. Figure 7 As shown in (e), although the striped structure is still visible in the sample with added high molecular weight chitosan, the overall image is darker, the stripe clarity is reduced, and there is a certain degree of discontinuity. Furthermore, the fiber structure shows a trend of blurred edges and loose axial aggregation. This is because high molecular weight chitosan has high viscosity, excessive molecular chain entanglement, and is prone to forming irregular cross-links, interfering with the orderly self-assembly of collagen molecules. Figure 7As shown in (f), the D-periodic bands of the sample with added low molecular weight chitosan are extremely blurry, and some areas are even completely invisible. Furthermore, the fiber structure is loose, even exhibiting a dispersed state or "flocculated aggregation." This may be because the chitosan chain length is insufficient to provide an effective hydrogen bond network and charge adsorption support, leading to a weakened synergistic effect and difficulty in stabilizing the triple helix structure. This indicates that under the preparation conditions of this invention, the introduction of an appropriate amount of chitosan did not interfere with the axial aggregation of collagen fibers. Instead, it stabilized the triple helix conformation through non-covalent interactions such as hydrogen bonding and electrostatic adsorption, improving the uniformity and orderliness of fiber assembly. Besides its effect on the D-periodic structure, the introduction of chitosan also significantly modulated the diameter of the self-assembled collagen fibers. The fibers formed from pure collagen were thinner and denser; while the fiber diameters in Examples 3, 4, and 5 gradually increased, and the structure remained regular and ordered. This suggests that an appropriate amount of chitosan may enhance lateral entanglement and aggregation capabilities through synergistic interaction with collagen molecules, thereby forming a thicker and more stable fiber structure.

[0043] The mechanical properties of the hydrogels obtained by casting after self-assembly of natural collagen and CS / Col were determined by frequency-dependent rheological measurements. As shown in Figures 8 and 9, the storage modulus (G') of natural collagen and CS / Col was consistently higher than the loss modulus (G''), indicating that all collagen and its composite gel systems exhibited gel properties, suggesting that the system formed a certain degree of three-dimensional network structure. The G' values ​​in Examples 3, 4, and 5 all showed an increasing trend, indicating enhanced structural stability of the gel at this ratio. This suggests that the collagen triple helix structure was stable, the degree of fiber self-assembly was improved, and a dense and uniform three-dimensional cross-linked network was formed, thus significantly enhancing the mechanical properties of the gel. Figure 10 The curves show the variation of the loss factor (tanδ) of each sample with frequency. When the value of tanδ is less than 1, it indicates that the system mainly exhibits elastic behavior; while when the value of tanδ is greater than 1, it means that the system mainly exhibits viscous behavior. The results show that the tanδ of all samples is less than 0.5, indicating that the system still mainly exhibits elastic response. Among them, the tanδ of Examples 3, 4, and 5 shows a decreasing trend, indicating that the proportion of elastic energy storage in their internal structure is relatively high and the network is highly stable.

[0044] The collagen / chitosan composite solution was dried at room temperature, and the cell adhesion rate and antibacterial rate were tested. Figure 11As shown, the collagen / chitosan composite fiber material exhibits excellent cell adhesion properties to osteoblasts (MC3T3-E1). Compared to pure collagen material, the 24-hour cell adhesion rate was approximately 62%; however, with the appropriate introduction of chitosan, the cell adhesion rate significantly increased, reaching 82%, 89%, and 93% in Examples 3, 4, and 5, respectively. This indicates that the introduction of chitosan significantly improves the cationic charge density and roughness of the fiber material surface, which is beneficial for integrin-mediated cell adhesion factor expression, thereby promoting osteoblast adhesion behavior. Figure 12 As shown, the collagen / chitosan composite fiber material exhibits excellent antibacterial properties against Staphylococcus aureus. Compared to pure collagen fiber without chitosan, the 24-hour inhibition rate was only 43%; however, the antibacterial performance of the samples with added chitosan was significantly enhanced, with the sample in Example 5 achieving an inhibition rate of 98%. This result verifies that the cationic amino groups in chitosan can interact with negatively charged components of bacterial cell membranes, disrupting their membrane structure and inhibiting their proliferation, thereby endowing the material with excellent antibacterial function.

[0045] like Figure 13 As shown, the collagenase degradation experiments of Examples 2-5 were conducted. All samples showed a gradual decrease in residual mass with increasing degradation time, but different chitosan doping ratios significantly affected their resistance to enzymatic degradation. The Example 1 (pure collagen) sample degraded rapidly, with the residual mass decreasing to approximately 36% after 12 hours, and almost completely decomposed by 48 hours, leaving less than 10% of the mass, indicating weak resistance to enzymatic degradation. The Example 2 sample showed a reduced degradation rate, with approximately 21% of the residual mass remaining after 48 hours, indicating that the introduction of chitosan provided initial protection to the collagen structure. The Example 3 sample exhibited stronger structural stability, maintaining a residual mass of approximately 38% after 48 hours, nearly four times higher than Example 1, indicating that a high proportion of chitosan significantly inhibited collagenase degradation. The Example 4 sample performed best, with a residual mass exceeding 68% after 48 hours, extremely low structural decomposition, and a slow overall decline in the degradation curve within the first 24 hours, demonstrating excellent resistance to enzymatic degradation.

[0046] In summary, this invention significantly improves the stability of the triple helix conformation of collagen (circular dichroism R) by enabling chitosan and collagen to form a stable complex through hydrogen bonding and electrostatic synergistic self-assembly in a specific ratio. pnWith a pH value of 0.116-0.128 and an anti-enzymatic hydrolysis residual mass >65%, the resulting fibers have a diameter of 300-500 nm and enhanced mechanical properties. This composite fiber simultaneously retains the bioactivity of collagen and the functional properties of chitosan. The fiber morphology and orientation are precisely controlled by adjusting the mass ratio, exhibiting both environmental stability (maintaining high conformational stability even after prolonged incubation with PBS) and excellent processing adaptability. This method eliminates the need for toxic cross-linking agents, and the resulting materials can be directly applied to high-end biomedical fields such as tissue engineering scaffolds, smart dressings, and biodegradable implants, promoting the clinical translation of soft tissue repair and regenerative medicine.

[0047] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing chitosan-collagen fiber material, characterized in that, Includes the following steps: S1. Prepare collagen solution and chitosan solution; S2. Cooperative self-assembly: The chitosan solution obtained in step S1 is added to the collagen solution and mixed. PBS buffer solution is added at 2-8 ℃ to induce cooperative self-assembly of chitosan and collagen to form a stable triple helix composite solution. S3. Separate the solid and liquid phases by low-temperature centrifugation at 0-6℃, collect the precipitate, and fix it by cross-linking in glutaraldehyde solution; S4. Gradient dehydration: The cross-linked fiber matrix is ​​placed in ethanol solutions with a concentration gradient of 30%-100% for gradual dehydration; then it is dried by supercritical carbon dioxide to obtain freeze-dried fiber material; In step S2, the mass ratio of chitosan to collagen is 1:4 to 1:

1.

2. The method for preparing fiber materials according to claim 1, characterized in that, In step S1, Preparation of collagen solution: Dissolve collagen in dilute acid solution to obtain collagen solution; Chitosan solution preparation: Dissolve chitosan with a degree of deacetylation of 85%–95% in glacial acetic acid solution and stir to prepare a solution.

3. The method for preparing fiber materials according to claim 1, characterized in that, The molecular weight of the collagen is 8000-14000 Da; And / or, the chitosan has a molecular weight of 200-400 kDa.

4. The method for preparing fiber materials according to claim 1, characterized in that, The total concentration of the chitosan and collagen mixed solution is 4.5-6.5 mg / mL.

5. The method for preparing fiber materials according to claim 1, characterized in that, The collagen is eggshell membrane protein; The method for preparing eggshell membrane protein involves peeling, washing, enzymatic hydrolysis, salting out, centrifuging, dialysis, and freeze-drying the eggshell membrane to obtain the extracted eggshell membrane protein. The salting-out temperature of the eggshell membrane enzymatic hydrolysate is 4-8℃; the freeze-drying time is 48-72h.

6. The method for preparing fiber materials according to claim 1 or 2, characterized in that, The concentration range of the chitosan solution is 0.5-2.0 mg / mL; And / or, the concentration range of said collagen is 3.0-7.0 mg / mL; And / or, the PBS buffer is a phosphate buffer with pH 6.4–6.6 and an ionic strength of 10–20 mmol / L.

7. The method for preparing fiber materials according to claim 1, characterized in that, In step S3, the volume fraction of glutaraldehyde is 2%-4%; the fixation time is 2-4 h. And / or, the centrifugation temperature is 0 - 6 ℃.

8. The fiber material prepared by the method according to any one of claims 1-7, characterized in that, The fiber material is a chitosan-modified collagen self-assembled nanofiber network with an average diameter of 300-500 nm. The fibers exhibit a clearly visible D-periodic band structure, and the triple helix conformation was detected by CD spectroscopy. pn The value is 0.115-0.

130.

9. The application of the fiber material obtained by the method of any one of claims 1-7 or the fiber material of claim 8, characterized in that, It is applied in the fields of tissue engineering, drug delivery, bio-dressing, and biomimetic materials.

10. The application according to claim 9, characterized in that, It is used in the field of tissue engineering for the regeneration and repair of cartilage, bone, or skin tissues. Or it can be applied in the field of biological dressings for the repair of functional wounds that are antibacterial and promote healing. It can also be applied in the field of biomimetic materials to prepare biodegradable scaffolds.

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