Customizable chitosan microfiber-based water sponge gel and preparation method and application thereof

Abstract

The application discloses a customizable chitosan microfiber-based water sponge gel and a preparation method and application thereof, and the preparation method is as follows: shearing treatment is conducted on an acid solution of chitosan, the pH of the system is adjusted to be alkaline, and a chitosan microfiber solution is obtained. An epoxy crosslinking agent is added to crosslink the chitosan microfiber by means of covalent bonds and hydrogen bonds, the obtained precipitate after centrifugal treatment is subjected to multiple freeze-thaw treatments, and the water sponge gel is prepared. The customizable chitosan microfiber-based water sponge gel prepared by the method has rich macroporous structures, can effectively increase the transmission efficiency of nutrients and waste, and can be extruded by means of a syringe to obtain a chitosan microfiber suspension with different shapes of two-dimensional and three-dimensional customizable structures. The customizable chitosan microfiber-based water sponge gel has excellent elasticity, fatigue resistance, low hysteresis, biocompatibility and degradability, can meet the demand of large-scale preparation of various complex structure scaffolds, and is expected to be used for repairing and reconstructing large-volume soft tissue defects as a customizable implantable scaffold.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of soft tissue repair engineering material development, and relates to a customizable chitosan microfiber-based water sponge hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Accidents or surgical operations (such as debridement and tumor resection) can cause skin, fat, muscle, skin flap or combined soft tissue injury, resulting in large volume soft tissue defects. The treatment methods for repairing and reconstructing large volume soft tissue are usually autologous skin flap transplantation, autologous fat transplantation and prosthesis implantation. However, these treatment methods cause serious complications. Autologous skin flap transplantation requires transplantation of soft tissue from other parts of the body to the defect site, resulting in donor site defects; a large amount of transplanted autologous fat is easily absorbed by the human body, and usually needs to be injected repeatedly; prosthesis implantation is prone to foreign body reaction, resulting in fibrosis, hematoma and capsular contracture. At present, there is still a lack of scaffold materials that can perfectly treat large volume soft tissue defects. Therefore, the design of a strategy for repairing and reconstructing large volume soft tissue defects is still a bottleneck problem that needs to be solved in the field of tissue engineering, highlighting the importance of developing customizable viscoelastic biomaterials.

[0003] Hydrogels have become the most promising implantable scaffold materials due to their similar properties to soft tissues. However, the small mesh size (about tens of nanometers) and bound water in the three-dimensional polymer network structure of traditional hydrogels limit the long-distance (> 150 μm) transport of gases and nutrients, making it impossible to meet the repair needs of large volume soft tissue defects. Therefore, it is urgent to design and prepare new flexible implantable scaffolds with large pores to meet the needs of repairing and reconstructing large volume soft tissue defects. New bulk water sponge hydrogel (hydrogel / sponge) materials with highly porous structure and excellent water holding performance have both the viscoelasticity of hydrogels and the reversible compression performance of sponges, and are expected to play a role in repairing and reconstructing large volume soft tissue defects. However, most water sponge hydrogels are derived from petrochemical-derived synthetic materials, which have potential toxicity. Natural polymer materials have the advantages of wide source, low cost, biocompatibility and biodegradability. However, it is still challenging to balance the structure customizability and mechanical properties of natural biopolymer-based water sponge hydrogels. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application aims to provide a customizable chitosan microfiber-based water sponge hydrogel as well as a preparation method and application thereof, so as to solve the technical problems of small mesh size in the three-dimensional polymer network of traditional hydrogels, affecting nutrient transport, and non-customizability.

[0005] The present application is realized by the following technical solutions:

[0006] A method for preparing a customizable chitosan microfiber-based hydrogel includes the following steps:

[0007] S1: The acid solution of chitosan is sheared and the pH is adjusted to alkaline to prepare a chitosan microfiber solution. After adding an epoxy crosslinking agent to the chitosan microfiber, a hydrogel precursor is obtained.

[0008] S2: The hydrogel precursor is centrifuged, and the resulting precipitate is subjected to multiple freeze-thaw cycles to prepare the customizable chitosan microfiber-based hydrosponge gel.

[0009] Preferably, in step S1, the acid solution of chitosan is prepared by dispersing chitosan in hydrochloric acid solution and stirring; the volume concentration of the hydrochloric acid solution is 0.4%~0.5%.

[0010] Preferably, in step S1, during the shearing process, the shearing rate is 8000~11000 rad / min, the shearing time is 4~7 min, and the shearing temperature is 25~37 °C.

[0011] Preferably, the epoxy crosslinking agent is any one of epichlorohydrin, polyethylene glycol diglycidyl ether, glycerol triglycidyl ether, polypropylene glycol glycidyl ether, and sorbitol glycidyl ether.

[0012] Preferably, the chitosan has a deacetylation value of 75% to 85% and a molecular weight of 9000 to 50000 Da.

[0013] Preferably, the ratio of chitosan to epoxy crosslinking agent in the acidic solution of chitosan is (5~1) g: 1 mL.

[0014] Preferably, in step S2, the freeze-thaw process includes a freezing process and a thawing process, wherein the freezing temperature is -20 to -24 °C and the freezing time is 12 to 18 h; the thawing temperature is 25 to 37 °C and the thawing time is 2 to 4 h.

[0015] Preferably, in step S2, after centrifuging the hydrogel precursor, the precipitate obtained by centrifugation is squeezed out by a syringe to form two-dimensional or three-dimensional structures of different shapes, and multiple freeze-thaw processes are performed to obtain chitosan microfiber-based hydrosponge gels of different shapes with two-dimensional or three-dimensional structures.

[0016] A customizable chitosan microfiber-based hydrosponge gel is prepared by the above method; the average diameter of the pore structure in the customizable chitosan microfiber-based hydrosponge gel is 20~40 μm.

[0017] The above-mentioned customizable chitosan microfiber-based water sponge gel is used in the repair and reconstruction of large-volume soft tissue defects.

[0018] Compared with the prior art, the present invention has the following beneficial technical effects:

[0019] This invention discloses a method for preparing customizable chitosan microfiber-based hydrogels. First, a chitosan acid solution is sheared to ensure uniform orientation of the chitosan long chains, shortening their molecular chain length and yielding chitosan microfibers. The shortened microfiber length increases the specific surface area and the number of active functional groups, creating favorable conditions for subsequent cross-linking reactions. This process also improves the dispersibility of chitosan in water. Subsequently, under alkaline conditions, an epoxy cross-linking agent is added to initiate an epoxy ring-opening reaction, forming covalent bonds with the amino and hydroxyl groups on the chitosan microfibers, creating a larger porous structure between the microfibers. The hydrogel precursor is then frozen. During freezing, water within the precursor forms ice crystals, and the free chitosan microfibers undergo secondary reorganization and regeneration. A subsequent thawing process allows the regenerated free chitosan in the hydrogel to precipitate and undergo phase transitions, further facilitating network cross-linking in the hydrogel system. After multiple freeze-thaw cycles, a hydrogel with a large network structure is formed. The customizable chitosan microfiber-based water sponge gel prepared by the method of this invention has a rich porous structure, which effectively increases the transport efficiency of nutrients and waste. In addition, different two-dimensional and three-dimensional customizable structures can be obtained by extruding the chitosan microfiber suspension with a syringe. It has excellent elasticity, fatigue resistance, low hysteresis, biocompatibility and biodegradability, and is expected to be used to repair and reconstruct large-volume soft tissue defects.

[0020] In addition, the present invention also discloses a customizable chitosan microfiber-based water sponge gel prepared by the above method, wherein the average diameter of the mesopore structure of the customizable chitosan microfiber-based water sponge gel is 20~40 μm, which can meet the requirements for effective nutrient transport. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the bonding between chitosan and epoxy crosslinking agents according to the present invention;

[0023] Figure 2 The XRD patterns of the customizable chitosan microfiber-based hydrogels prepared in Examples 1-4 of this invention are shown below.

[0024] Figure 3 The FTIR spectra of the customizable chitosan microfiber-based hydrosponges prepared in Examples 1-4 of this invention are shown below.

[0025] Figure 4 XPS spectra of the customizable chitosan microfiber-based hydrogels prepared in Examples 1-4 of this invention;

[0026] Figure 5 This is a SEM image of the customizable chitosan microfiber-based hydrogel prepared in this invention.

[0027] Figure 6 The mechanical property test results of the customizable chitosan microfiber-based hydrosponge gels prepared in Examples 1-4 of this invention are shown. Among them, (a) is the comparison result of storage modulus (G') and loss modulus (G''), and (b) is the comparison result of elasticity of the customizable chitosan microfiber hydrosponge gels.

[0028] Figure 7 The fatigue resistance test results of the customizable chitosan microfiber-based water sponge gels prepared in Examples 1-4 of this invention are shown. (a) is the test curve, and (b) is the Young's modulus self-recovery rate of the customizable chitosan microfiber-based water sponge gel after resting time.

[0029] Figure 8 The results show the water retention performance of the customizable chitosan microfiber-based water sponge gels prepared in Examples 1-4 of this invention. (a) is a representative weight loss curve of the customizable chitosan microfiber-based water sponge gel under compressive strain (0%~90%). The viscoelastic region and the porosity elastic region are divided at the yield point. (b) is the yield point of the customizable chitosan microfiber-based water sponge gel under compressive pressure.

[0030] Figure 9 To utilize the customizability of the chitosan microfiber-based water sponge gel prepared in Example 1 of this invention, (a) shows a viscosity test, (b) shows a test schematic diagram, and (c) shows the preparation of products with different molding properties.

[0031] Figure 10 The results of the biocompatibility test of the customizable chitosan microfiber-based water sponge gel prepared in Example 1 of the present invention at different time points in vitro; wherein (a) is the staining of live / dead cells (scale bar: 100µm) after co-culturing the control group and the customizable chitosan microfiber-based water sponge gel extract with cells for 24 h, 48 h and 72 h; and (b) is the hemolysis rate data and hemolysis photographs of the customizable chitosan microfiber-based water sponge gel.

[0032] Figure 11The tissue compatibility of the hydrosponge gel is shown in (a) the pathological sections of major organs (heart, liver, spleen, lung and kidney) observed 28 days after subcutaneous implantation in SD rats, and (b) the pathological sections of the surrounding tissues of SD rats implanted with the hydrosponge gel.

[0033] Figure 12 The results of in vivo blood toxicity tests for customizable chitosan microfiber-based hydrogels are shown, where (a) represents liver function indicators and (b) represents kidney function indicators. Detailed Implementation

[0034] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0035] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0036] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0037] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0038] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0039] This invention provides a method for preparing a customizable chitosan microfiber-based hydrogel, comprising the following steps:

[0040] S1: The chitosan acid solution is sheared and the pH is adjusted to alkaline. The epoxy crosslinking agent undergoes an epoxy ring-opening reaction and crosslinks with the chitosan microfibers through covalent and hydrogen bonds. After stirring the reaction, a hydrogel precursor is obtained.

[0041] The acidic solution of chitosan is prepared by dispersing chitosan in hydrochloric acid solution and stirring; the volume concentration of the hydrochloric acid solution is 0.4%~0.5%.

[0042] During the shearing process, the shearing speed is 8000~11000 rad / min, the shearing time is 4~7 min, and the shearing temperature is 25~37 °C.

[0043] The epoxy crosslinking agent is any one of epichlorohydrin, polyethylene glycol diglycidyl ether, glycerol triglycidyl ether, polypropylene glycol glycidyl ether, and sorbitol glycidyl ether.

[0044] The chitosan has a deacetylation value of 75% to 85% and a molecular weight of 9,000 to 50,000 Da.

[0045] Preferably, the pH of the system is adjusted to 7-8.5.

[0046] More preferably, the ratio of chitosan to epoxy crosslinking agent in the acidic solution of chitosan is (1~5) g: 1 mL.

[0047] S2: The hydrogel precursor is centrifuged, and the resulting precipitate is subjected to multiple freeze-thaw cycles to prepare the customizable chitosan microfiber hydrosponge gel.

[0048] During centrifugation, the rotation speed is 5000~6000 rad / min and the time is 10~15 min.

[0049] Freeze-thaw treatment includes a freezing process and a thawing process. The freezing temperature is -20 to -24 °C and the freezing time is 12 to 18 h. The thawing temperature is 25 to 37 °C and the thawing time is 2 to 4 h.

[0050] In the preferred embodiment, the freeze-thaw cycle is 1 to 4 times.

[0051] like Figure 1As shown, this invention discloses a method for preparing a customizable chitosan microfiber-based hydrosponge gel. First, chitosan is selected as the main component, and epichlorohydrin, polyethylene glycol diglycidyl ether, glycerol triglycidyl ether, polypropylene glycol glycidyl ether, or sorbitol glycidyl ether are selected as epoxy crosslinking agents. High-speed homogenous shearing treatment is used to ensure consistent shear orientation of the chitosan long chains. Under alkaline conditions, the epoxy crosslinking agent undergoes an epoxy ring-opening reaction, forming covalent bonds with the amino or hydroxyl groups on the chitosan microfiber chains. Further freeze-thaw treatment allows for secondary recombination and regeneration of the chitosan microfiber chains, endowing the hydrosponge gel with a porous structure and excellent self-recovery properties. The customizable chitosan microfiber-based hydrosponge gel prepared by this method possesses a rich porous structure, excellent elasticity, fatigue resistance, low hysteresis, biocompatibility, and biodegradability, and is expected to serve as an implantable scaffold for repairing and reconstructing large-volume soft tissue defects.

[0052] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0053] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0054] Example 1

[0055] A method for preparing a customizable chitosan microfiber-based hydrogel, comprising the following steps:

[0056] Weigh 1.08 g of chitosan and add it to 90 mL of 0.46% dilute hydrochloric acid. Stir until completely dissolved, then stir at 9000 r for 5 min in a high-speed homogenizer. Slowly add NaOH solution (1 mol / L) to adjust the pH to 7.5-8, and simultaneously add 0.36 mL of epichlorohydrin (ECH) as an epoxy crosslinking agent. Let stand at room temperature for 5 h. Then centrifuge at 9000 r for 10 min in a high-speed centrifuge, remove the supernatant, and place the resulting precipitate in a cylindrical mold and freeze at -24 °C for 12 h. Thaw at room temperature to prepare a customizable chitosan microfiber-based hydrogel.

[0057] Example 2

[0058] The difference between this embodiment and Example 1 is that the added epoxy crosslinking agent is polyethylene glycol diglycidyl ether, wherein the molecular weight of polyethylene glycol diglycidyl ether is 200 Da (PEGDGE-200).

[0059] Example 3

[0060] The difference between this embodiment and Example 1 is that the added epoxy crosslinking agent is polyethylene glycol diglycidyl ether, wherein the molecular weight of polyethylene glycol diglycidyl ether is 500 Da (PEGDGE-500).

[0061] Example 4

[0062] The difference between this embodiment and Embodiment 1 is that the added epoxy crosslinking agent is glycerol triglycidyl ether (GTE).

[0063] Example 5

[0064] The difference between this embodiment and Embodiment 1 is that the added epoxy crosslinking agent is polypropylene glycol glycidyl ether (PPGDGE).

[0065] Example 6

[0066] The difference between this embodiment and Embodiment 1 is that the added epoxy crosslinking agent is sorbitol glycidyl ether (SPE).

[0067] Example 7

[0068] A method for preparing a customizable chitosan microfiber-based hydrogel includes the following steps:

[0069] S1: Chitosan with a deacetylation value of 75% and a molecular weight of 9000 Da was dissolved in a 0.4% (v / v) hydrochloric acid solution and sheared. The pH was then adjusted to 7, and an epoxy crosslinking agent was added. After stirring and reacting, a hydrogel precursor was obtained. During the shearing process, the shearing speed was 8000 rad / min, the shearing time was 7 min, and the shearing temperature was 25 °C. The epoxy crosslinking agent was epichlorohydrin.

[0070] S2: The hydrogel precursor was centrifuged at 5000 rad / min for 15 min, and the resulting precipitate was subjected to a cyclic freeze-thaw process, which included a freezing process and a thawing process. The freezing temperature was -20 °C and the freezing time was 18 h; the thawing temperature was 25 °C and the thawing time was 4 h, thus preparing the customizable chitosan microfiber-based hydrosponge gel.

[0071] Example 8

[0072] A method for preparing a customizable chitosan microfiber-based hydrogel includes the following steps:

[0073] S1: Chitosan with a deacetylation value of 85% and a molecular weight of 50,000 Da was dissolved in a 0.5% (v / v) hydrochloric acid solution and sheared. The pH was then adjusted to 8.5. An epoxy crosslinking agent was added, and the mixture was stirred to obtain a hydrogel precursor. During the shearing process, the shearing speed was 11,000 rad / min, the shearing time was 4 min, and the shearing temperature was 37 °C. The epoxy crosslinking agent was polyethylene glycol diglycidyl ether, and the molecular weight of the polyethylene glycol diglycidyl ether was 400.

[0074] S2: The hydrogel precursor was centrifuged at 6000 rad / min for 10 min, and the resulting precipitate was subjected to multiple freeze-thaw cycles. The freeze-thaw cycle included freezing and thawing processes. The freezing temperature was -24 °C and the freezing time was 12 h. The thawing temperature was 37 °C and the thawing time was 2 h. The freeze-thaw cycle was repeated 4 times to prepare the customizable chitosan microfiber-based hydrosponge gel.

[0075] Example 9

[0076] A method for preparing a customizable chitosan microfiber-based hydrogel includes the following steps:

[0077] S1: Chitosan with a deacetylation value of 80% and a molecular weight of 20,000 Da was dissolved in a 0.45% (v / v) hydrochloric acid solution and sheared. The pH was then adjusted to 8, and an epoxy crosslinking agent was added. After stirring and reacting, a hydrogel precursor was obtained. During the shearing process, the shearing speed was 10,000 rad / min, the shearing time was 5 min, and the shearing temperature was 30℃. The epoxy crosslinking agent was glycerol triglycidyl ether.

[0078] S2: The hydrogel precursor was centrifuged at 5500 rad / min for 12 min. The resulting precipitate was subjected to multiple freeze-thaw cycles, including freezing and thawing. The freezing temperature was -22 °C for 15 h, and the thawing temperature was 30 °C for 3 h. The freeze-thaw cycle was repeated twice to prepare the customizable chitosan microfiber-based hydrogel. To illustrate the performance of the customizable chitosan microfiber-based hydrogel prepared in this invention, the following tests were conducted:

[0079] Figure 2The XRD patterns of the customizable chitosan microfiber-based water sponge gels prepared in Examples 1-4 of this invention are shown, and their results are compared with those obtained by high-speed shearing of chitosan into chitosan microfibers (Pure CMFs). During the testing, the crystal structure of the products was analyzed using X-ray diffraction (XRD) from Bruker GmbH, Germany, under 40 kV and 100 mA Cu Kα radiation (wavelength λ = 0.154 nm). Figure 2 It can be seen that pure chitosan exhibits the characteristic peak of chitosan at 2θ = 20°. This is because there are intermolecular hydrogen bonds between the hydroxyl (-OH) and amino (-NH2) groups in the chitosan structure, resulting in crystals with different chemical structure regions. Although the epoxy crosslinking agents in Examples 1 to 4 are different and the degree of crosslinking of the prepared products is different, all samples have similar diffraction peaks, indicating that the crosslinking agent does not affect the crystallinity of chitosan microfibers.

[0080] Figure 3 The figures show the FTIR spectra (Vertex70, Bruker Corporation, Germany) of the customizable chitosan microfiber-based hydrosponge gels prepared in Examples 1-4 of this invention, and are compared with those of pure chitosan microfibers. As can be seen from the figures, the pure chitosan microfiber hydrosponge gel exhibits a lower FTIR value at 1600 cm⁻¹. −1 1100cm −1 and 1000 cm −1 Vibrational peaks appeared, which were attributed to the primary COC stretching vibration, secondary CO, and primary CO stretching vibration, respectively. These vibrational peaks also appeared in the FTIR spectra of epichlorohydrin, polyethylene glycol diglycidyl ether-200, polyethylene glycol diglycidyl ether-500, and glycerol triglycidyl ether aqueous sponge gels. All customizable chitosan microfiber-based aqueous sponge gels showed peaks at 3468 cm⁻¹. -1 The presence of -OH and -NH2 stretching vibrations on both sides indicates that the epoxy crosslinking agent is effectively crosslinked with chitosan microfibers. In other words, a chemical network structure can be effectively formed by crosslinking chitosan microfibers with different epoxy crosslinking agents.

[0081] In addition, X-ray photoelectron spectroscopy (XPS) was used to examine the elimination and chemical composition of the products. Figure 4XPS spectra of the customizable chitosan microfiber-based hydrosponge gels prepared in Examples 1-4 of this invention are shown. (a) is the O 1s spectrum, and (b) is the N 1s spectrum. The results were compared with those of pure chitosan microfiber hydrosponge gel. The deconvolution peak analysis of O 1s indicates the presence of COC, -OH, and -C=O bonds. Similarly, the deconvolution peak analysis of N 1s at 399.09 eV indicates the presence of neutral amino groups, while the peak at 400.85 eV corresponds to a protonated amino group. The COC deconvolution peak analysis of O 1s is correlated with the high proportion of peaks, and the -NH3 deconvolution peak analysis of N 1s reveals the highest crosslinking efficiency between glycerol triglycidyl ether and chitosan microfibers, demonstrating the high reaction efficiency between the epoxy crosslinking agent and chitosan amino groups in the customizable chitosan microfiber-based hydrosponge gel.

[0082] Furthermore, Figure 5 The image shows the internal pore structure of the customizable chitosan microfiber-based water sponge gel as observed by scanning electron microscopy (SEM). As can be seen from the image, the customizable chitosan microfiber-based water sponge gel has a rich porous structure with pore sizes of about 20-40 μm, which is conducive to the transport of oxygen and nutrients.

[0083] Furthermore, the mechanical properties of the customizable chitosan microfiber-based hydrosponge gel prepared in this invention were tested using an electronic universal testing machine (CMT Shenzhen Sansi, Shenzhen, China) at room temperature (25 °C), a compression rate of 20 mm / min, and a maximum compressive strain of 85%. During the testing process, the customizable chitosan microfiber-based hydrosponge gel was fabricated into a cylindrical shape (15 mm in diameter and 18 mm in height). The test results are as follows: Figure 6 As shown. Figure 6 The mechanical properties of the customizable chitosan microfiber-based hydrosponge gels prepared in Examples 1-4 of this invention are shown, where (a) is a comparison of storage modulus (G') and loss modulus (G''), and (b) is a comparison of elasticity of the customizable chitosan microfiber-based hydrosponge gels. The rheological properties of the customizable chitosan microfiber-based hydrosponge gels (cylindrical shape, 10 mm in diameter and 2 mm in height) were tested at 25 °C using a rheometer (DHR-1, TA Instruments, American). The test results are as follows. Figure 7As shown, G' is consistently higher than G'' across the entire frequency range (0.1 rad / s-100 rad / s), indicating that the hydrosponge gel is stable and behaves as an elastic solid. According to G'=kTv (where k is the Boltzmann constant, T is the temperature, and v is the crosslinking density of the ideal crosslinked network), the high G' of the customizable chitosan microfiber-based hydrosponge gel indicates that the chitosan microfibers have a high interfiber crosslinking density. By comparing the G' of hydrosponge gels crosslinked with epichlorohydrin, polyethylene glycol diglycidyl ether-200, and polyethylene glycol diglycidyl ether-500 epoxy crosslinking agents, it can be found that G' increases with increasing aliphatic hydrocarbon chain length. This may be because epoxy crosslinking agents with shorter aliphatic hydrocarbon chains cannot form effective crosslinks between the larger gaps of the chitosan microfibers.

[0084] Furthermore, the fatigue resistance of the customizable chitosan microfiber-based hydrogel prepared in this invention was tested using an electronic universal testing machine (CMT Shenzhen Sansi, Shenzhen, China) at room temperature (25 °C), a compression rate of 20 mm / min, and a maximum compressive strain of 85%. The results are as follows: Figure 7 As shown, in the loading test, residual strain refers to the irreversible strain generated after the material enters the plastic stage. The smaller the residual strain, the better the elastic properties. The residual strain of epichlorohydrin hydrosponge gel (24.8%) is close to that of pure chitosan microfiber hydrosponge gel (26.4%), because only one epoxy group and short-chain epichlorohydrin do not form effective crosslinks between chitosan microfibers. The residual strains of glycerol triglycidyl ether (5.3%), polyethylene glycol diglycidyl ether-200 (16.9%), and polyethylene glycol diglycidyl ether-500 (14.4%) hydrosponge gels are all lower than those of epichlorohydrin and pure chitosan microfiber hydrosponge gels. The lower the residual strain, the better the crosslinking efficiency. Therefore, glycerol triglycidyl ether, polyethylene glycol diglycidyl ether-200, and polyethylene glycol diglycidyl ether-500 have better crosslinking efficiency. With the increase of epoxy functional groups and aliphatic hydrocarbon chain length, the chemical cross-linking effect of customizable chitosan microfiber-based hydrosponge gels increases.

[0085] Furthermore, the water-holding capacity of the customizable chitosan microfiber-based water sponge gel was tested by weighing the water lost under different stress compressions. The test structure is shown in [reference needed]. Figure 8 , Figure 8The results of the water-holding capacity test of the customizable chitosan microfiber-based water sponge gel prepared in Examples 1-4 of this invention are shown. (a) shows the representative weight loss curve of the customizable chitosan microfiber-based water sponge gel under compressive strain, ranging from 0% to 90%. The viscoelastic and porosilicate regions are delineated at the yield point. (b) shows the yield point of the customizable chitosan microfiber-based water sponge gel under compressive pressure. As shown in the figure, the quantitative strain boundary between bulk elastic deformation and porous elastic deformation is called the yield point. Below the yield point, the customizable chitosan microfiber-based water sponge gel undergoes viscoelastic deformation, leading to conformational rearrangement. Above the yield point, the sample undergoes porous elastic deformation, and water is squeezed out of the customizable chitosan microfiber-based water sponge gel. The weight change depending on the strain of the customizable chitosan microfiber-based water sponge gel was measured during the compression test to find the yield point of the chitosan microfiber-based water sponge gel. Figure 8 a) As expected, the compressive strain at the yield point increases with increasing interfiber crosslinking density. Figure 8 As shown in b, the glycerol triglycidyl ether aqueous sponge gel exhibits the highest strain at the yield point (14%), while the pure chitosan microfiber-based aqueous sponge gel shows the lowest strain (7.2%). Furthermore, the yield point strain gradually increases when the aqueous sponge gel is crosslinked with epichlorohydrin (8.9%), polyethylene glycol diglycidyl ether-200 (9.2%), and polyethylene glycol diglycidyl ether-500 (9.6%). The yield point increases with increasing chain length. These phenomena indicate that the formation of crosslinks between chemical fibers can enhance the bulk elasticity of the aqueous sponge gel and prevent morphological changes during volumetric deformation below the yield point.

[0086] To demonstrate the customizability of the hydrogel in this invention, a customizable chitosan microfiber-based hydrogel solid suspension was extruded into the desired shape using a 25G syringe. The results are as follows. Figure 9 As shown, the chitosan microfiber suspension is in solid form, with a viscosity as high as 10 at low shear rates. 4 Pa·s. As the shear rate increased from 0.1 rad / s to 100 rad / s, the viscosity decreased linearly, dropping sharply to only 10 Pa·s at 100 rad / s. This is due to the orientation of the microcrystalline fibers under shear stress. Figure 9 a). This shear-thinning behavior endows chitosan microfiber suspensions with extrusion molding capabilities. Solid chitosan microfiber suspensions can be smoothly extruded from a syringe under pressure, forming independent patterns without altering their structure and composition. Figure 9 b). Based on the outstanding extrusion molding capability of chitosan microfiber suspensions, we can prepare various two-dimensional and three-dimensional network structures after chemical cross-linking to obtain customizable chitosan microfiber-based hydrosponge gels with different structures. Figure 9c shows rectangular networks, planar circular grids, and triangular networks for customizable chitosan microfiber-based hydrosponges. This demonstrates that the present invention can yield chitosan microfiber-based hydrosponges with customizable structures.

[0087] To demonstrate the in vitro biocompatibility of the customizable chitosan microfiber-based hydrogel of this invention, evaluation was conducted by combining cell viability, proliferation, and blood compatibility. Specifically, human umbilical vein endothelial cells (HUVECs) after digestion and centrifugation were resuspended in 1 mL of fresh F-12K complete medium and extraction medium of different composite hydrogels, respectively. 100 μL of the cell suspension was aspirated and diluted 10-fold, then 10 μL was used for hemocytometer counting, and the cell suspension density was diluted to 1 × 10⁻⁶. 4 Cells / mL. Subsequently, each 100 μL contains 1×10⁻⁶ cells / mL. 3 Human umbilical vein endothelial cells were evenly seeded into 96-well plates and blocked with PBS. The plates were incubated in a CO2 incubator (37 °C, 5% CO2) with medium changes every 48 h. After 24, 48, and 72 h of culture, the culture medium was discarded, and 100 μL of CCK-8 mixture (90 μL culture medium and 10 μL CCK-8 staining solution) was added to each well. The plates were then transferred to an incubator for 2 h of incubation, followed by absorption measurement at 450 nm using a microplate reader. Each experiment was performed in triplicate. Third-generation human umbilical vein endothelial cells were used, and cell toxicity was assessed using a Live / Dead staining kit. First, cells containing 5 × 10⁶ cells were seeded into each well. 5 Cell suspensions of individual umbilical vein endothelial cells were seeded into 24-well plates containing 1 mL of F-12K complete medium and incubated in a CO2 incubator (37 °C, 5% CO2) for 24 h. Subsequently, the complete medium was replaced with 990 μL of hydrogel extract and supplemented with 10 μL of FBS. The control group was cultured in 1 mL of fresh F-12K complete medium. In vitro cytotoxicity was evaluated using a Live / Dead staining kit after 24, 48, and 72 h of incubation at 37 °C and 5% CO2. Two mL of anticoagulated rat blood was taken, and 2 mL of hydrogel extract at different concentration gradients (50, 100, 500, and 1000 g / mL) was added to each well. Simultaneously, 2 mL of anticoagulated blood mixed with 2 mL of physiological saline served as a negative control, and 2 mL of anticoagulated blood mixed with 2 mL of deionized water served as a positive control. After thorough mixing, each mixture was placed in a 37 °C water bath for 1–3 h and then allowed to stand. Two mL of supernatant was taken from each sample, and the optical density of each supernatant was measured using a UV spectrophotometer to calculate the hemolysis rate. The results are as follows: Figure 10 As shown, the customizable chitosan microfiber-based water sponge gel is non-cytotoxic to cells and does not exhibit in vitro hemolytic reactions.

[0088] In vivo biocompatibility was evaluated through histological sections, complete blood count, liver function tests, and kidney function tests. Specifically, SD rats were anesthetized with 3% sodium pentobarbital, and a 10 mm diameter, 5 mm high hydrosponge gel was subcutaneously implanted into their backs. In short, the skin was incised along the dorsal midline, and the subcutaneous tissue was dissected using blunt dissection with vascular forceps before the hydrosponge gel was implanted. Results are as follows: Figure 11 As shown, pathological sections of major organs (heart, liver, spleen, lungs, and kidneys) observed after 28 days showed no in vivo toxicity. Blood analysis of the femoral artery in rats, including complete blood count, liver and kidney function tests, also showed no blood toxicity. Figure 12 The customizable chitosan microfiber-based hydrogel prepared by this invention exhibits good biocompatibility.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0090] Finally, 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 the scope of protection of the present invention. 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 essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a customizable chitosan microfiber-based hydrogel, characterized in that, Includes the following steps: S1: The acid solution of chitosan is sheared and the pH is adjusted to 7-8.5 to prepare a chitosan microfiber solution. After adding an epoxy crosslinking agent to the chitosan microfiber, a crosslinking reaction is carried out to obtain a hydrogel precursor. S2: The hydrogel precursor is centrifuged, and the resulting precipitate is subjected to multiple freeze-thaw cycles to prepare the customizable chitosan microfiber-based hydrosponge gel. In step S1, the acidic solution of chitosan is prepared by dispersing chitosan in hydrochloric acid solution and stirring; the volume concentration of the hydrochloric acid solution is 0.4%~0.5%. In step S1, during the shearing process, the shearing rate is 8000~11000 rad / min, the shearing time is 4~7 min, and the shearing temperature is 25~37 °C. The chitosan has a deacetylation value of 75%~85% and a molecular weight of 9000~50000 Da; The ratio of chitosan to epoxy crosslinking agent in the acidic solution of chitosan is (1~5) g: 1 mL; In step S2, after centrifuging the hydrogel precursor, the precipitate obtained by centrifugation is squeezed out by a syringe to form two-dimensional or three-dimensional structures of different shapes. Then, multiple freeze-thaw treatments are performed to prepare chitosan microfiber-based hydrosponge gels of different shapes with two-dimensional or three-dimensional structures. During centrifugation, the rotation speed is 5000~6000 rad / min and the time is 10~15min. The number of freeze-thaw cycles is 2 to 4.

2. The method for preparing a customizable chitosan microfiber-based hydrogel according to claim 1, characterized in that, The epoxy crosslinking agent is any one of epichlorohydrin, polyethylene glycol diglycidyl ether, glycerol triglycidyl ether, polypropylene glycol glycidyl ether, and sorbitol glycidyl ether.

3. The method for preparing a customizable chitosan microfiber-based hydrogel according to claim 1, characterized in that, In step S2, the freeze-thaw process includes a freezing process and a thawing process. The freezing temperature is -20 to -24 °C and the freezing time is 12 to 18 h. The thawing temperature is 25 to 37 °C and the thawing time is 2 to 4 h.

4. A customizable chitosan microfiber-based hydrosponge gel, characterized in that, Prepared by the method described in any one of claims 1 to 3; the customizable chitosan microfiber-based hydrosponge gel has an average pore structure diameter of 20 to 40 μm.

5. The application of the customizable chitosan microfiber-based hydrosponge gel as described in claim 4 in the preparation of implantable scaffolds for repairing and reconstructing large-volume soft tissue defects.

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