Photo-crosslinked helical polyisocyanate hydrogel matrix for three-dimensional printing, and preparation method therefor

Abstract

A photo-crosslinked helical polyisocyanurate hydrogel matrix for three-dimensional printing, and a preparation method therefor. A free radical chain reaction between the double bonds of acrylamide at an end of a PIC side chain is initiated by means of ultraviolet light crosslinking, which effectively increases the rigidity of a PIC hydrogel. In a low-temperature liquid state, direct crosslinking can form a relatively weak non-fibrous structure; at room temperature or higher temperatures, crosslinking can form covalent bonds between polymer molecules on the basis of an already formed temperature-sensitive fibrous structure, which significantly improves the mechanical strength of the system and overcomes the problem of low rigidity of hydrogel.

Description

A three-dimensional printing photocrosslinked spiral polyisocyanide hydrogel matrix and its preparation method Technical Field

[0001] The present invention relates to the technical field of biomaterial preparation, and in particular to a temperature-sensitive photocrosslinked helical polyisocyanide hydrogel matrix (PIC) that can be used for ultraviolet light-cured three-dimensional printing and a preparation method thereof. Background Art

[0002] PIC hydrogel is a synthetic extracellular matrix (ECM) material that is highly similar to natural extracellular matrix structural protein gels. PIC polymers are composed of a semi-flexible polyisocyanide backbone with polypeptide-ethylene glycol side chains attached thereto. PIC hydrogels have extremely low gelling concentrations (the commonly used cell culture concentration is 0.1% w / v), excellent biocompatibility, temperature sensitivity for low-temperature liquid and high-temperature gelling, and stress-rigidifying mechanical characteristics similar to those of biological gels. These highly biomimetic and controllable physical and chemical properties have enabled them to be used in many biomedical applications, such as wound dressings, drug delivery, and three-dimensional cell culture.

[0003] Although PIC hydrogels have been widely used in biomedical engineering, their relatively low intrinsic stiffness, typically less than 1 kPa, and limited self-support have limited their application in bio-additive manufacturing. Therefore, effectively increasing the stiffness of PIC hydrogels and expanding their tissue engineering applications remains a pressing challenge.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to solve the problem raised in the above background art that the inherent stiffness of PIC hydrogel is usually less than 1 kPa and the self-supporting property is limited, which leads to very limited application of PIC hydrogel in bio-additive manufacturing.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention is to provide a method for preparing a three-dimensional printed photo-crosslinked helical polyisocyanide hydrogel matrix, comprising the following steps:

[0008] S1: Synthesis of a helical polyisocyanide peptide modified with an azide group on the side chain, namely PIC-N3;

[0009] S2: adding a cross-linking agent to the helical polyisocyanide polypeptide solution to obtain a double-bond modified helical polyisocyanide polypeptide;

[0010] S3: mixing the double-bond modified helical polyisocyanate polypeptide and a photoinitiator solution to obtain the three-dimensional printed photo-crosslinked helical polyisocyanate hydrogel matrix under ultraviolet light.

[0011] UV-light crosslinking can trigger a free radical chain reaction between the terminal acrylamide double bonds of the PIC side chains, effectively increasing the stiffness of the PIC hydrogel. At low temperatures, the liquid state can directly crosslink to form a weak, non-fibrous structure. Crosslinking at room temperature or higher can form covalent bonds between polymer molecules based on the already temperature-sensitive fibrous structure, significantly improving the mechanical strength of the system. The above preparation method is simple and easy to operate, and the prepared photocrosslinked helical polyisocyanide hydrogel matrix (helical polyisocyanide polypeptide, PIC) can be printed with a pre-defined shape before photocrosslinking. The ultimate mechanical strength of the hydrogel can be adjusted by controlling the PIC molecular weight and concentration. Furthermore, the entire crosslinking process is mild, the selected photoinitiator has good biocompatibility, and the minimal dosage does not affect subsequent cell survival. The synergistic effect of the physical gelation properties mediated by PIC hydrogen bonds and the chemical crosslinking of double bonds mediated by phenyl and lithium phosphonate (LAP) is fully utilized. The entire hydrogel system is very suitable as a UV-curable matrix for 3D printing of 3D materials. The above preparation method has a simple process and is suitable for a variety of biomedical applications. In addition, the materials used are all artificially synthesized, highly controllable, and repeatable, which can support the stable effect of biological three-dimensional printing.

[0012] Preferably, in step S1, a catalyst, such as nickel perchlorate hexahydrate, is added to the helical polyisocyanide polypeptide modified with an azide group on the side chain, thereby changing the average chain length of the helical polyisocyanide polypeptide and thus changing the final mechanical strength of the hydrogel.

[0013] Preferably, in step S1, the ratio of the added catalyst nickel perchlorate hexahydrate to the helical polyisocyanate polypeptide is in the range of 1:1000-1:5000. Within this ratio range, the chain length of the helical polyisocyanate polypeptide is appropriate, and the prepared hydrogel has a strong rigidity.

[0014] Preferably, the photoinitiator in step S3 includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959). Both are water-soluble and highly cytocompatible, and can be used to polymerize hydrogels or other polymeric materials. Due to their higher water solubility, faster polymerization rate at 365 nm, and absorbance at 400 nm (enabling visible light polymerization), these photoinitiators are superior to Irgacure 2959 and more suitable for biological applications.

[0015] Preferably, in step S3, the concentration of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) is in the range of 0.2-0.5% w / v. Within this concentration range, the photocrosslinking effect is better, so that the use effect of the photocrosslinked helical polyisocyanide hydrogel matrix is ​​better.

[0016] Preferably, the reaction temperature is adjusted after the mixing in step S3 to regulate the rigidity of the photo-crosslinked helical polyisocyanide hydrogel matrix.

[0017] Preferably, the cross-linking agent in step S2 includes a thiol reaction reagent containing acrylamide and dibenzocyclooctyne, such as DBCO-PEG4-Acrylamide; this type of cross-linking agent has good biocompatibility and good grafting effect. The entire cross-linking process is mild, the selected photoinitiator has good biocompatibility, and the amount used is very small and will not affect the survival of cells in the later stage. The synergistic effect of the physical gelation properties mediated by PIC hydrogen bonds and the double bond chemical cross-linking mediated by lithium phenyl (2,4,6-trimethylbenzoyl) phosphonate (LAP) is fully utilized. The entire hydrogel system is very suitable as a matrix for UV curing of three-dimensional material printing.

[0018] Preferably, the reaction time after adding the cross-linking agent in step S2 is 10-20 minutes. Within this time range, the cross-linking process is successfully completed and the efficiency of preparing the PIC hydrogel is optimal.

[0019] Preferably, in step S2, the ratio of double bonds to side chains in the double-bond modified helical polyisocyanide polypeptide is 0-3.3%, and this ratio can regulate the ultimate mechanical strength of the PIC hydrogel.

[0020] Preferably, the density of the azide groups in the helical polyisocyanide polypeptide is adjusted before step S1. By adjusting the density of the azide groups in PIC-N3, the final mechanical strength of the hydrogel can be adjusted.

[0021] The second aspect of the present invention is to provide a three-dimensional printing photo-crosslinked spiral polyisocyanate hydrogel matrix prepared by any of the above methods, wherein the photo-crosslinked spiral polyisocyanate hydrogel matrix has a fibrous structure.

[0022] The third aspect of the present invention is to provide the use of the above-mentioned three-dimensional printed photo-crosslinked helical polyisocyanide hydrogel matrix in a three-dimensional cell culture matrix model.

[0023] The thermosensitive nature of helical polyisocyanate hydrogels allows them to gel at room temperature, enabling solid-state UV crosslinking. After localized photocrosslinking, the uncrosslinked portions can be converted to a liquid state by cooling, facilitating removal. By grafting acrylamide groups onto helical polyisocyanate polypeptides of varying molecular weights, a UV-curable hydrogel matrix for 3D printing / tissue engineering is obtained. Helical polyisocyanate polypeptides modified with double bonds in their side chains exhibit thermosensitive properties, enabling the hydrogel matrix to form a basic structure at room temperature and above, enabling photocrosslinking and printing in the solid state. This photocrosslinked helical polyisocyanate hydrogel matrix exhibits excellent cytocompatibility and mimics the fibrous hierarchical structure and nonlinear mechanical properties of the natural extracellular matrix, providing a sufficient support environment for cell growth, promoting cell adhesion, proliferation, and targeted functional expression.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This application proposes a three-dimensional printing photocrosslinked helical polyisocyanide hydrogel matrix and its preparation method. By grafting acrylamide groups onto PIC molecules of different molecular weights, a three-dimensional printing / tissue engineering hydrogel matrix that can be cured by ultraviolet light is obtained. Ultraviolet light crosslinking can trigger a free radical chain reaction between the double bonds of acrylamide at the end of the PIC side chains, effectively improving the rigidity of the hydrogel. At low temperatures, the liquid state can directly crosslink to form a weaker non-fibrous structure; at room temperature or higher temperatures, crosslinking can form covalent bonds between polymer molecules based on the already temperature-sensitive fibrous structure, significantly improving the mechanical strength of the system. The ultimate mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of the PIC. The entire crosslinking process is mild, the selected photoinitiator has good biocompatibility, and the extremely small amount used will not affect the survival of cells in the later stage. The synergistic effect of the physical gelation properties mediated by the hydrogen bonds of the PIC and the double bond chemical crosslinking mediated by phenyl and lithium phosphonate (LAP) is fully utilized. The entire hydrogel system is very suitable as a matrix for UV curing of three-dimensional materials. The crosslinking agent used in the preparation method of the present invention is a macromolecule with excellent biocompatibility and gel-forming properties. This results in a hydrogel scaffold with low toxicity and good cell compatibility. While providing the three-dimensional environment required for cell growth, it also promotes cell adhesion, growth, and proliferation. Furthermore, the preparation method is simple and suitable for a variety of biomedical applications. Furthermore, the materials used are all artificially synthesized, resulting in high controllability and repeatability. This preparation method is unique and highly innovative compared to existing photocuring systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0027] Figure 1 is a molecular structure diagram of a PIC polymer (a) and a small molecule cross-linking agent (b);

[0028] FIG2 is a method for preparing a three-dimensional printed photo-crosslinked helical polyisocyanide hydrogel matrix;

[0029] FIG3 is a photo-crosslinked rheological curve of a 3D-printed photo-crosslinked helical polyisocyanide hydrogel matrix at low temperature (5° C.);

[0030] FIG4 is a photo-crosslinked rheological curve of a 3D-printed photo-crosslinked helical polyisocyanide hydrogel matrix at room temperature (25° C.);

[0031] Figure 5 shows the photocrosslinking rheological curve of the 3D printed photocrosslinked helical polyisocyanide hydrogel matrix at body temperature (37°C). DETAILED DESCRIPTION

[0032] The technical solution of this patent will be further described in detail below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.

[0033] PIC hydrogels have been widely used in biomedical engineering. However, their relatively low intrinsic stiffness, typically less than 1 kPa, and limited self-supporting properties have limited their application in bio-additive manufacturing. Therefore, effectively increasing the stiffness of PIC hydrogels and expanding their tissue engineering applications remains an urgent challenge.

[0034] In order to solve the above problems, the embodiments of the present application provide a three-dimensional printing photocross-linked spiral polyisocyanate hydrogel matrix and a preparation method thereof. By grafting acrylamide groups on spiral PIC molecules of different molecular weights, UV-curable three-dimensional printing hydrogel matrices of different concentrations are prepared. The hydrogel matrix makes full use of the inherent temperature-sensitive properties of PIC hydrogel. It is liquid at low temperatures (5°C) and changes into a gel state after rising to room temperature or higher temperatures (25°C). PIC inks in different states can be covalently cross-linked by UV light at different temperatures. Not only does it solve the problem of low inherent rigidity of the hydrogel, but the obtained hydrogel matrix can provide cells with a biomimetic fibrous microenvironment, and can modify bioactive polypeptides through click chemistry, thereby promoting cell adhesion, proliferation and other functional expressions.

[0035] The technical solution of this patent will be further described in detail below in conjunction with specific embodiments. Wherein, the methods used in the examples are all conventional methods unless otherwise specified.

[0036] Example 1: A 3D printed photocrosslinked helical polyisocyanide hydrogel matrix

[0037] This embodiment introduces a three-dimensional printed photo-crosslinked helical polyisocyanide hydrogel matrix, including a helical polyisocyanide polypeptide (PIC) with double bond modifications on the side chain.

[0038] The thermosensitive nature of the PIC hydrogel itself allows it to gel at room temperature, making solid-state UV crosslinking possible. After photocrosslinking, the uncrosslinked portion can be converted to a liquid state by cooling, making it easy to remove. By grafting acrylamide groups onto PIC molecules of different molecular weights, a hydrogel matrix for 3D printing / tissue engineering that can be cured by UV light is obtained. The helical polyisocyanate polypeptide with double bond modifications on the side chain has thermosensitive properties, which can enable the hydrogel matrix to have a basic structure at room temperature and higher temperatures, and realize photocrosslinking printing in the solid state. This photocrosslinked helical polyisocyanate hydrogel matrix has good cell compatibility and has a fibrous hierarchical structure and nonlinear mechanical characteristics that mimic the natural extracellular matrix. It can provide a sufficient support environment for cell growth, which is conducive to cell adhesion, proliferation and targeted function expression.

[0039] The photocrosslinked helical polyisocyanide hydrogel matrix leverages the inherent temperature-sensitive properties of PIC (liquid at low temperatures, solidified at high temperatures) to allow photocrosslinking in both liquid and gel states. The hydrogel's stiffness can be controlled by adjusting the polymer concentration, crosslinking density, and polymerization temperature.

[0040] Furthermore, the chain length of PIC is adjustable, the azide density is adjustable, the photocrosslinking density is adjustable, and the concentration is adjustable, thereby adjusting the stiffness of the photocrosslinked helical polyisonitrile hydrogel matrix, so that the prepared photocrosslinked helical polyisonitrile hydrogel matrix can be suitable for photocrosslinking application scenarios with different requirements.

[0041] Furthermore, a photoinitiator is used to react with the photocrosslinked helical polyisonitrile hydrogel matrix to produce photocrosslinking for three-dimensional shaping. The photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure2959). The concentrations of the two are adjustable, the biocompatibility is good, and the damage to cells is less, which is conducive to the application of the photocrosslinked helical polyisonitrile hydrogel matrix.

[0042] By grafting acrylamide groups onto PIC molecules of varying molecular weights, a hydrogel matrix for 3D printing / tissue engineering that can be cured with UV light is obtained. By fully utilizing the thermosensitive properties of PIC, the hydrogel matrix can have a basic structure at room temperature and above, enabling photocrosslinking and printing in the solid state. The resulting hydrogel 3D scaffold has excellent cell compatibility and possesses a fibrous hierarchical structure and nonlinear mechanical characteristics that mimic the natural extracellular matrix. This can provide a sufficient support environment for cell growth, facilitating cell adhesion, proliferation, and targeted functional expression. The entire PIC hydrogel system is very suitable as a matrix for UV-curing 3D material printing.

[0043] The above-mentioned 3D-printed photocrosslinked helical polyisocyanate hydrogel matrix has changed its original linear structure to a fibrous structure, overcoming the free radical chain reaction between the double bonds of acrylamide at the end of the PIC side chains that can be triggered by UV crosslinking, effectively improving the rigidity of the hydrogel. At low temperatures, the liquid state can directly crosslink to form a weaker non-fibrous structure; crosslinking at room temperature or higher temperatures can form covalent bonds between polymer molecules based on the already temperature-sensitive fibrous structure, significantly improving the mechanical strength of the system and overcoming the low rigidity of hydrogels existing in the prior art. The 3D-printed photocrosslinked helical polyisocyanate hydrogel matrix has extremely high application value in the preparation of 3D culture matrix models.

[0044] Example 2: Preparation method of three-dimensional printing photocrosslinked helical polyisocyanide hydrogel matrix

[0045] This embodiment provides a method for preparing the three-dimensional printing photo-crosslinked spiral polyisonitrile hydrogel matrix described in Example 1. The photo-crosslinked spiral polyisonitrile hydrogel matrix is ​​an ultraviolet light-cured nano-photo-crosslinked spiral polyisonitrile hydrogel matrix that can be used for three-dimensional printing.

[0046] Please refer to FIG2 , which shows a three-dimensional printing photo-crosslinked helical polyisocyanide hydrogel matrix and its preparation method, which specifically includes the following steps:

[0047] S1: Synthesis of a helical polyisocyanide peptide modified with an azide group on the side chain, namely PIC-N3;

[0048] S2: adding a catalyst of nickel perchlorate hexahydrate to the helical polyisocyanide polypeptide modified with azide groups on the side chains, thereby changing the average chain length of PIC-N3 and thus changing the final mechanical strength of the hydrogel;

[0049] S3: dissolving PIC-N3 in MilliQ to obtain a PIC-N3 solution, wherein the concentration of PIC-N3 is 10 mg / ml;

[0050] S4: Add a crosslinker to the PIC-N3 solution. For example, dissolve the crosslinker DBCO-PEG4-acrylamide in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / ml and add the crosslinker dropwise to the PIC solution in an appropriate proportion. After stirring evenly, let the mixture stand on ice for at least 15 minutes. The double bond is modified on the PIC side chain through a ring strain-promoted azide-alkyne cycloaddition (SPAAC) reaction to obtain a double-bond-modified helical polyisocyanide polypeptide. Please refer to Figure 1 for the molecular formula of the double bond modified on the PIC side chain.

[0051] S5: Mixing the double-bond modified PIC-N3 and the photoinitiator solution to obtain a three-dimensional printed photo-crosslinked helical polyisocyanide hydrogel matrix under ultraviolet light; wherein the final concentration of each component is:

[0052] The ratio of the catalyst nickel perchlorate hexahydrate to the helical polyisocyanide polypeptide is 1:1000 or 1:5000;

[0053] PIC concentration was 1 mg / ml or 8 mg / ml;

[0054] The proportion of double bonds in the side chain is 3.3%;

[0055] The concentration of the photoinitiator was 0.5% w / v.

[0056] In a further embodiment, the density of the azide groups of PIC-N3 in step S1 can be adjusted, and the final mechanical strength of the hydrogel can be adjusted by adjusting the density of the azide groups in PIC-N3.

[0057] In a further embodiment, the photoinitiator in step S5 includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959). Both have good water solubility and high cell compatibility and can be used to polymerize hydrogels or other polymeric materials. Due to their higher water solubility, faster polymerization rate under 365nm light, and absorbance at 400nm (enabling visible light polymerization), these photoinitiators are superior to Irgacure 2959 and more suitable for biological applications.

[0058] In a further embodiment, the concentration range of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) in step S3 is 0.2-0.5% w / v. Within this concentration range, the photocrosslinking effect is better, so that the use effect of the photocrosslinked helical polyisonitrile hydrogel matrix is ​​better.

[0059] In a further embodiment, the cross-linking agent in step S4 includes a thiol reaction reagent containing acrylamide and dibenzocyclooctyne, such as DBCO-PEG4-Acrylamide. This type of cross-linking agent has good biocompatibility and good gelling effect, so that the prepared hydrogel three-dimensional scaffold has low toxicity and good cell compatibility. While providing the three-dimensional environment required for cell growth, it can promote the adhesion, growth and proliferation of cells. The entire cross-linking process is mild, the selected photoinitiator has good biocompatibility, and the amount used is very small and will not affect the survival of cells in the later stage. The synergistic effect of the physical gelling properties mediated by PIC hydrogen bonds and the double bond chemical cross-linking mediated by lithium phenyl (2,4,6-trimethylbenzoyl) phosphonate (LAP) is fully utilized. The entire hydrogel system is very suitable as a matrix for UV-curing of three-dimensional materials.

[0060] In a further embodiment, in step S1, the ratio of the added catalyst nickel perchlorate hexahydrate to PIC-N3 is in the range of 1:1000-1:5000. Within this ratio range, the chain length of PIC-N3 is appropriate, and the prepared hydrogel has stronger rigidity.

[0061] In a further embodiment, the reaction time after adding the cross-linking agent in step S4 is 10-20 minutes. Within this time range, the cross-linking process is successfully completed, and the efficiency of preparing the PIC hydrogel is optimal.

[0062] In a further embodiment, the ratio of double bonds in the side chains of the double-bond modified PIC-N3 in step S4 is 0-3.3%. The PIC hydrogel with this double bond ratio has better rigidity and is more convenient for its application.

[0063] In a further embodiment, the reaction temperature is adjusted after the mixing in step S5 to control the rigidity of the photo-crosslinked helical polyisocyanide hydrogel matrix.

[0064] This method fully utilizes the thermosensitive properties of PIC, allowing the hydrogel matrix to have a basic structure at room temperature and above, enabling photocrosslinking printing in the solid state. The resulting hydrogel three-dimensional scaffold has good cell compatibility and possesses a fibrous hierarchical structure and nonlinear mechanical characteristics that mimic the natural extracellular matrix. It can provide a sufficient support environment for cell growth, facilitating cell adhesion, proliferation, and targeted functional expression. The thermosensitive properties of the helical polyisocyanate hydrogel allow it to gel at room temperature, making solid-state UV crosslinking possible. After photocrosslinking, the uncrosslinked portion can be converted to a liquid state by cooling, facilitating removal.

[0065] The cross-linking agent used in the preparation method of the present invention is a macromolecule with good biocompatibility and good gelling effect, so that the prepared hydrogel three-dimensional scaffold has low toxicity and good cell compatibility. While providing the three-dimensional environment required for cell growth, it promotes the adhesion, growth and proliferation of cells. The preparation method of the present invention has a simple process and is suitable for a variety of biomedical applications. The materials used in the above preparation method are all artificially synthesized, with high controllability and strong repeatability. The preparation method of the present invention is simple and fast, the selected materials are novel, and the properties are biomimetic and controllable, and it is suitable for promotion to a variety of biomedical medical uses.

[0066] The photo-crosslinked helical polyisocyanate hydrogel matrix prepared by this preparation method can achieve three-dimensional printing with a pre-shaped shape before photo-crosslinking; the final mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of PIC. In addition, the conditions of the entire cross-linking process are mild, the selected photoinitiator has good biocompatibility, and the extremely small amount used will not affect the survival of cells in the later stage. The synergistic effect of the physical gelation properties mediated by PIC hydrogen bonds and the double bond chemical cross-linking mediated by phenyl and lithium phosphonate (LAP) is fully utilized. The entire hydrogel system is very suitable as a matrix for UV-curing of three-dimensional materials. This preparation method is suitable for a variety of biomedical applications; in addition, the materials used are all artificially synthesized, with high controllability and strong repeatability, which is conducive to reducing the production costs of enterprises.

[0067] Example 3: Performance testing of 3D printed photocrosslinked helical polyisocyanide hydrogel matrix

[0068] In this example, a series of experiments were conducted to test the performance of the three-dimensional printed photo-crosslinked helical polyisocyanide hydrogel matrix constructed in this scheme.

[0069] Mix well on ice to obtain a UV-curable hydrogel matrix precursor that can be used for 3D printing. Temperature sweep test and light curing test were performed on the rheometer. For the UV point light curing system, set the light intensity to 50%.

[0070] Low-temperature crosslinking: UV irradiation at 5°C for 100 seconds, followed by a temperature sweep from 5 to 37°C, a heating rate of 1.0°C / min, a strain of 1%, and a frequency of 1.0 Hz. The results are shown in Figure 3. Figures 3A-D show the rheological results of PIC at low temperature (5°C) photocrosslinking. The abscissa shows temperature, the ordinate shows storage modulus G', and the black squares show the change in storage modulus with temperature. Blue light crosslinking was performed at 5°C, followed by heating to 37°C. The PIC chain length (controlled by the catalyst nickel perchlorate hexahydrate:monomer ratio, 1:1000 or 1:5000, abbreviated as 1k or 5k) and concentration (1 or 8 mg / ml) both affected the storage modulus G' before and after crosslinking.

[0071] Room-temperature crosslinking: A temperature sweep was first performed from 5°C to 25°C at a heating rate of 1.0°C / min, a strain of 1%, and a frequency of 1.0 Hz. The material was then UV-irradiated at 25°C for 100 s, followed by heating to 37°C using the same parameters. The results are shown in Figure 4. Figures 4A-D show the rheological properties of the PIC during room-temperature (25°C) photocrosslinking. The abscissa shows temperature, and the ordinate shows the storage modulus G'. The black squares show the change in storage modulus with temperature, and the arrows indicate the direction of temperature change: upward arrows indicate heating, and downward arrows indicate cooling. The material was first heated from 5°C to 25°C, then crosslinked with blue light at 25°C, then heated to 37°C, and finally cooled to 5°C. Both the PIC chain length (controlled by the catalyst nickel perchlorate hexahydrate:monomer ratio, 1:1000 or 1:5000, abbreviated as 1k or 5k) and concentration (1 or 8 mg / ml) affected the storage modulus G' before and after crosslinking.

[0072] Body-temperature cross-linking: A temperature sweep was first performed from 5°C to 37°C at a heating rate of 1.0°C / min, a strain of 1%, and a frequency of 1.0 Hz. The material was then UV-irradiated at 37°C for 100 s, followed by cooling to 5°C using the same parameters. The results are shown in Figure 5. Figures 5A-D show the rheological results of PIC photocross-linking at body temperature (37°C). The abscissa shows temperature, and the ordinate shows the storage modulus G'. The black squares show the change in storage modulus with temperature, and the arrows indicate the direction of temperature change: upward arrows indicate heating, and downward arrows indicate cooling. The material was first heated from 5°C to 37°C, then UV-cross-linked at 37°C, and then cooled to 5°C. The PIC chain length (controlled by the catalyst nickel perchlorate hexahydrate:monomer ratio, 1:1000 or 1:5000, abbreviated as 1k or 5k) and concentration (1 or 8 mg / ml) both affected the storage modulus G' before and after cross-linking.

[0073] This application proposes a photocrosslinked helical polyisocyanide hydrogel matrix for 3D printing and a preparation method thereof. By grafting acrylamide groups onto PIC molecules of varying molecular weights, a UV-curable hydrogel matrix for 3D printing / tissue engineering is obtained. UV-light crosslinking can trigger a free radical chain reaction between the terminal acrylamide double bonds of the PIC side chains, effectively increasing the hydrogel's stiffness. At low temperatures, the liquid state can directly crosslink to form a weak, non-fibrous structure. At room temperature or higher, crosslinking can form covalent bonds between polymer molecules within the already temperature-sensitive fibrous structure, significantly enhancing the system's mechanical strength. The ultimate mechanical strength of the hydrogel can be adjusted by controlling the PIC molecular weight and concentration. The entire crosslinking process is mild, and the selected photoinitiator has excellent biocompatibility, requiring minimal dosage and will not affect subsequent cell survival. The synergistic effect of the physical gelation properties of the PIC mediated by hydrogen bonds and the chemical crosslinking of double bonds mediated by phenyl (2,4,6-trimethylbenzoyl) and lithium phosphonate (LAP) is fully utilized. The entire hydrogel system is well-suited as a UV-curable matrix for 3D printing. The crosslinking agent used in the preparation method of this invention is a macromolecule with excellent biocompatibility and gel-forming properties. This results in a hydrogel scaffold with low toxicity and excellent cell compatibility. While providing a three-dimensional environment for cell growth, it also promotes cell adhesion, growth, and proliferation. This preparation method is simple and suitable for a variety of biomedical applications. Furthermore, the materials used are all synthetic, offering high controllability and reproducibility. This unique preparation method is highly innovative compared to existing photocuring systems.

[0074] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A method for preparing a three-dimensional printed photo-crosslinked helical polyisocyanate hydrogel matrix, characterized in that: The following steps are involved: S1: Synthesis of helical polyisocyanide peptides modified with azide groups on the side chains; S2: adding a cross-linking agent to the helical polyisocyanate polypeptide solution to obtain a double-bond modified helical polyisocyanate polypeptide; S3: Mixing the double-bond modified helical polyisocyanate polypeptide and the photoinitiator solution to obtain the three-dimensional printed photo-cross-linked helical polyisocyanate hydrogel matrix under ultraviolet light.

2. The method according to claim 1, characterized in that In the step S1, a catalyst is added to the helical polyisocyanate polypeptide whose side chain is modified with an azide group, thereby changing the average chain length of the helical polyisocyanate polypeptide.

3. The method according to claim 2, characterized in that In the step S1, the ratio of the added catalyst nickel perchlorate hexahydrate to the helical polyisocyanate polypeptide is in the range of 1:1000-1:5000.

4. The method according to claim 1, characterized in that The photoinitiator in step S3 includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

5. The method according to claim 4, characterized in that The concentration of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate in step S3 ranges from 0.2 to 0.5% w / v.

6. The method according to claim 1, characterized in that In the double-bond modified helical polyisocyanurate polypeptide in step S2, the ratio of double bonds to side chains is 0-3.3%.

7. The method according to claim 1, characterized in that After the mixing in step S3, the reaction temperature is adjusted to regulate the rigidity of the photo-crosslinked helical polyisocyanide hydrogel matrix.

8. A three-dimensional printed photo-crosslinked helical polyisocyanate hydrogel matrix, characterized in that: Prepared by the method according to any one of claims 1 to 7.

9. The three-dimensional printing photo-crosslinked helical polyisocyanide hydrogel matrix according to claim 8, characterized in that: It has a fibrous structure.

10. Use of the three-dimensional printed photo-crosslinked helical polyisocyanide hydrogel matrix as claimed in claim 8 in a three-dimensional cell culture matrix model.

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