Three-dimensional printing photo-crosslinking spiral polyisocyanide hydrogel matrix and preparation method thereof

Abstract

The invention provides a three-dimensional printing photo-crosslinking spiral polyisocyanide hydrogel matrix and a preparation method thereof. Ultraviolet crosslinking can initiate free radical chain reaction between acrylamide double bonds at the tail end of a PIC side chain, and the rigidity of the PIC hydrogel is effectively improved. At a low temperature, a liquid state can be directly crosslinked to form a relatively weak non-fibrous structure; covalent bonds among polymer molecules can be formed on the basis of a fibrous structure formed in a temperature-sensitive mode through crosslinking at room temperature or higher temperature, the mechanical strength of a system is remarkably improved, and the problem that in the prior art, hydrogel is low in rigidity is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial preparation, and in particular to a temperature-sensitive photo-crosslinked helical polyisocyanurate 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 the natural extracellular matrix structural protein gel. PIC polymers are composed of a semi-flexible polyisocyanate main chain and polypeptide-ethylene glycol side chains thereon. PIC hydrogel has an extremely low gelation concentration (the commonly used cell culture concentration is 0.1% w / v), excellent biocompatibility, low-temperature liquid and high-temperature gelation temperature sensitivity, and stress-rigidified mechanical characteristics similar to biological gels. The above highly biomimetic and controllable physical and chemical properties have enabled it to be used in many biomedical fields, such as wound dressings, drug delivery, and three-dimensional cell culture.

[0003] Although PIC hydrogels have been widely used in the field of biomedical engineering, the inherent stiffness of PIC hydrogels is relatively low, usually less than 1 kPa, and the self-supporting capacity is limited, which leads to very limited application of PIC hydrogels in bio-additive manufacturing. Therefore, how to effectively improve the stiffness of PIC hydrogels and expand their application range in tissue engineering is a problem that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to solve the problem mentioned in the above background technology 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.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention is to provide a method for preparing a three-dimensional printed photo-crosslinked helical polyisocyanate hydrogel matrix, comprising the following steps:

[0007] S1: Synthesis of a helical polyisocyanide peptide modified with azide groups on the side chains, namely PIC-N3;

[0008] S2: adding a cross-linking agent to the helical polyisocyanate polypeptide solution to obtain a double-bond modified helical polyisocyanate polypeptide;

[0009] 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.

[0010] UV crosslinking can trigger a free radical chain reaction between the double bonds of acrylamide at the end of the PIC side chain, effectively improving the stiffness of the PIC 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 on the basis of 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 photo-crosslinked helical polyisocyanate hydrogel matrix (helical polyisocyanate polypeptide, PIC) can achieve three-dimensional printing with a pre-shaped shape before photocrosslinking. The final mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of PIC. In addition, the entire crosslinking 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. Give full play to the synergistic effect of the physical gelation properties mediated by PIC hydrogen bonds and the double bond chemical crosslinking mediated by phenyl and lithium phosphonate (LAP). The entire hydrogel system is very suitable as a matrix for UV curing of three-dimensional material printing. 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 highly repeatable, and can support the stable effect of biological three-dimensional printing.

[0011] Preferably, in step S1, a catalyst, such as nickel perchlorate hexahydrate, is added to the helical polyisocyanate polypeptide modified with an azide group on the side chain, thereby changing the average chain length of the helical polyisocyanate polypeptide and further changing the final mechanical strength of the hydrogel.

[0012] 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 rigidity of the prepared hydrogel is relatively strong.

[0013] 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 of which have good water solubility and high cell compatibility and can be used for the polymerization of hydrogels or other polymer materials. Because of its higher water solubility, faster polymerization rate under 365nm light, and absorbance at 400nm (visible light polymerization can be achieved), this photoinitiator is superior to Irgacure 2959 and more suitable for biological applications.

[0014] Preferably, in step S3, the concentration range of the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP) 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.

[0015] Preferably, the reaction temperature is adjusted after the mixing in step S3 to regulate the rigidity of the photo-crosslinked helical polyisocyanide hydrogel matrix.

[0016] Preferably, the cross-linking agent in step S2 includes a thiol reaction reagent containing acrylamide and dibenzocyclooctyne, such as DBCO-PEG4-Acrylamide; such cross-linking agents have 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.

[0017] 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 PIC hydrogel is optimal.

[0018] Preferably, in step S2, the ratio of double bonds to side chains in the double-bond modified helical polyisocyanurate polypeptide is 0-3.3%, and this ratio can regulate the final mechanical strength of the PIC hydrogel.

[0019] Preferably, the density of the azide groups in the helical polyisocyanurate polypeptide is adjusted before step S1, and the final mechanical strength of the hydrogel can be adjusted by adjusting the density of the azide groups in PIC-N3.

[0020] The second aspect of the present invention is to provide a three-dimensional printed photo-crosslinked helical polyisocyanate hydrogel matrix prepared by any of the above methods, wherein the photo-crosslinked helical polyisocyanate hydrogel matrix has a fibrous structure.

[0021] The third aspect of the present invention is to provide the use of the above-mentioned three-dimensional printed photo-cross-linked helical polyisocyanide hydrogel matrix in a three-dimensional cell culture matrix model.

[0022] The thermosensitive nature of the helical polyisocyanate hydrogel itself enables it to gel at room temperature, making solid-state UV crosslinking possible. After local photocrosslinking, the temperature can be lowered to convert the uncrosslinked part into a liquid state, which is convenient for removal. By grafting acrylamide groups on helical polyisocyanate polypeptide 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 modification on the side chain has thermosensitive properties, which can make the hydrogel matrix 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.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present application proposes a three-dimensional printing photo-crosslinked spiral polyisocyanate hydrogel matrix and a preparation method thereof. By grafting acrylamide groups on 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 chain, effectively improving the rigidity of the hydrogel. At low temperatures, the liquid state can be directly crosslinked to form a weaker non-fibrous structure; crosslinking at room temperature or higher temperatures can form covalent bonds between polymer molecules on the basis of the already temperature-sensitively formed fibrous structure, significantly improving the mechanical strength of the system. The final mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of PIC. The entire crosslinking 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. Give full play to the synergistic effect of the physical gelation properties mediated by PIC hydrogen bonds and the double bond chemical crosslinking mediated by phenyl and lithium phosphonate (LAP). The entire hydrogel system is very suitable as a matrix for ultraviolet light curing of three-dimensional material printing. 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, and promotes the adhesion, growth and proliferation of cells while providing the three-dimensional environment required for cell growth. In addition, the preparation method has a simple process and is suitable for a variety of biomedical applications. At the same time, the materials used are all artificially synthesized, with high controllability and strong repeatability. This preparation method is unique and highly innovative compared to the existing photocuring system. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. 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 paying any creative work.

[0026] Figure 1 The molecular structure diagram of a PIC polymer (a) and a small molecule cross-linking agent (b);

[0027] Figure 2 A method for preparing a three-dimensionally printed photo-crosslinked helical polyisocyanide hydrogel matrix;

[0028] FIG3 is a photo-crosslinked rheological curve of a three-dimensionally printed photo-crosslinked helical polyisocyanide hydrogel matrix at low temperature (5° C.);

[0029] FIG4 is a photo-crosslinked rheological curve of a three-dimensionally printed photo-crosslinked helical polyisocyanide hydrogel matrix at room temperature (25° C.);

[0030] FIG5 is a photo-crosslinked rheological curve of a three-dimensionally printed photo-crosslinked helical polyisocyanate hydrogel matrix at body temperature (37° C.). DETAILED DESCRIPTION

[0031] The technical solution of this patent will be further described in detail below in conjunction with specific implementation methods. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the technical field to which this application belongs.

[0032] PIC hydrogels have been widely used in the field of biomedical engineering. However, the inherent stiffness of PIC hydrogels is relatively low, usually less than 1 kPa, and the self-supporting property is limited, which leads to very limited application of PIC hydrogels in bio-additive manufacturing. Therefore, how to effectively improve the stiffness of PIC hydrogels and expand their application range in tissue engineering is a problem that needs to be solved urgently.

[0033] In order to solve the above problems, the embodiments of the present application provide a three-dimensional printing photo-cross-linked spiral polyisocyanate hydrogel matrix and a preparation method thereof. By grafting acrylamide groups on spiral PIC molecules of different molecular weights, hydrogel matrices of different concentrations for UV-curable three-dimensional printing are prepared. The hydrogel matrix makes full use of the inherent temperature-sensitive properties of PIC hydrogels. 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 bionic fibrous microenvironment, and can modify bioactive polypeptides through click chemistry, thereby promoting cell adhesion, proliferation and other functional expressions.

[0034] The technical solution of this patent will be further described in detail below in conjunction with specific implementation methods. Among them, the methods used in the embodiments are all conventional methods unless otherwise specified.

[0035] Example 1: A three-dimensional printed photo-crosslinked helical polyisocyanate hydrogel matrix

[0036] This embodiment introduces a three-dimensional printed photo-crosslinked helical polyisocyanate hydrogel matrix, including a helical polyisocyanate polypeptide (PIC) with double bonds modified on the side chain.

[0037] The thermosensitive nature of PIC hydrogel itself enables it to gel at room temperature, making solid-state UV crosslinking possible. After photocrosslinking, the temperature can be lowered to convert the uncrosslinked part into a liquid state for easy removal. By grafting acrylamide groups on 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 modification on the side chain has thermosensitive properties, which can make the hydrogel matrix 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 sufficient support environment for cell growth, which is conducive to cell adhesion, proliferation and targeted function expression.

[0038] The photo-crosslinked helical polyisocyanate hydrogel matrix can be photo-crosslinked in both liquid and gel states by virtue of the inherent temperature-sensitive properties of PIC (liquid at low temperature, solidified at high temperature). The rigidity of the hydrogel can be regulated by adjusting the concentration of the polymer, the crosslinking density and the polymerization temperature.

[0039] 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.

[0040] Furthermore, a photoinitiator is used to react with a photo-crosslinked helical polyisonitrile hydrogel matrix to produce photo-crosslinking for three-dimensional shaping. The photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphite (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure2959). Both have adjustable concentrations, good biocompatibility, and less damage to cells, which is conducive to the application of the photo-crosslinked helical polyisonitrile hydrogel matrix.

[0041] 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. By making full use of the thermosensitive properties of PIC, the hydrogel matrix can have a basic structure at room temperature and higher temperatures, and achieve photocrosslinking printing in the solid state. The obtained hydrogel 3D scaffold has good cell compatibility and has a fibrous hierarchical structure and nonlinear mechanical characteristics that mimic the natural extracellular matrix. It can provide sufficient support environment for cell growth and is conducive to cell adhesion, proliferation and targeted function expression. The entire PIC hydrogel system is very suitable as a matrix for UV curing of 3D materials.

[0042] The above-mentioned 3D printed photo-crosslinked helical polyisocyanate hydrogel matrix has changed its original linear structure and has a fibrous structure, which overcomes the free radical chain reaction between the double bonds of acrylamide at the end of the PIC side chain that can be induced by ultraviolet light crosslinking, and effectively improves 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 on the basis of the already temperature-sensitive fibrous structure, significantly improving the mechanical strength of the system and overcoming the problem of low rigidity of the hydrogel in the prior art. The 3D printed photo-crosslinked helical polyisocyanate hydrogel matrix has extremely high application value in the preparation of 3D culture matrix models.

[0043] Example 2: Preparation method of three-dimensional printed photo-crosslinked helical polyisocyanate hydrogel matrix

[0044] 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.

[0045] Please see attached Figure 2 , is a three-dimensional printing photo-crosslinked helical polyisocyanate hydrogel matrix and a preparation method thereof, which specifically comprises the following steps:

[0046] S1: Synthesis of a helical polyisocyanide peptide modified with azide groups on the side chains, namely PIC-N3;

[0047] S2: adding a catalyst of nickel perchlorate hexahydrate to the helical polyisocyanurate polypeptide modified with an azide group on the side chain, thereby changing the average chain length of PIC-N3 and further changing the final mechanical strength of the hydrogel;

[0048] S3: dissolving PIC-N3 in MilliQ to obtain a PIC-N3 solution, wherein the concentration of PIC-N3 is 10 mg / ml;

[0049] S4: Add a cross-linking agent to the PIC-N3 solution, for example, dissolve the cross-linking agent DBCO-PEG4-acrylamide in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / ml, and add the cross-linking agent dropwise to the PIC solution in an appropriate proportion; stir evenly and place the solution on ice for at least 15 minutes to react, and modify the double bond 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 the attached Figure 1 , which is the molecular formula of the PIC side chain modified with a double bond.

[0050] S5: Mixing the double-bond modified PIC-N3 and the photoinitiator solution to obtain a three-dimensional printed photo-crosslinked helical polyisocyanurate hydrogel matrix under ultraviolet light; wherein the final concentration of each component is:

[0051] The ratio of the catalyst nickel perchlorate hexahydrate to the helical polyisocyanide polypeptide is 1:1000 or 1:5000;

[0052] PIC concentration was 1 mg / ml or 8 mg / ml;

[0053] The proportion of double bonds in the side chain is 3.3%;

[0054] The concentration of the photoinitiator was 0.5% w / v.

[0055] In a further embodiment, the density of the azide groups of PIC-N3 in step S1 is adjustable, and the final mechanical strength of the hydrogel can be adjusted by adjusting the density of the azide groups in PIC-N3.

[0056] 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 of which have good water solubility and high cell compatibility and can be used for polymerization of hydrogels or other polymer materials. Because of its higher water solubility, faster polymerization rate under 365nm light, and absorbance at 400nm (visible light polymerization can be achieved), this photoinitiator is superior to Irgacure 2959 and more suitable for biological applications.

[0057] In a further embodiment, in step S3, the concentration range of the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP) is 0.2-0.5% w / v. Within this concentration range, the photo-crosslinking effect is better, so that the use effect of the photo-crosslinked helical polyisonitrile hydrogel matrix is ​​better.

[0058] 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. Such cross-linking agents have 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. Give full play to 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). The entire hydrogel system is very suitable as a matrix for UV-curing of three-dimensional materials.

[0059] 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 rigidity of the prepared hydrogel is relatively strong.

[0060] 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 PIC hydrogel is optimal.

[0061] In a further embodiment, in the double-bond modified PIC-N3 in step S4, the ratio of double bonds to side chains is 0-3.3%. The PIC hydrogel with this double bond ratio has better rigidity and is more convenient for its application.

[0062] 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.

[0063] This method makes full use of the thermosensitive properties of PIC, which can make the hydrogel matrix have a basic structure at room temperature and higher temperatures, and realize photo-crosslinking printing in the solid state. The obtained hydrogel three-dimensional scaffold 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 and is conducive to cell adhesion, proliferation and targeted functional expression. The thermosensitive properties of the helical polyisocyanate hydrogel itself enable it to gel at room temperature, making solid-state ultraviolet light crosslinking possible. After photo-crosslinking, the temperature can be lowered to convert the uncrosslinked part into a liquid state, which is convenient for removal.

[0064] 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, and 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 bionic and controllable, which is suitable for promotion to a variety of biomedical medical uses.

[0065] The photo-crosslinked helical polyisocyanate hydrogel matrix prepared by this preparation method can realize 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 crosslinking process are 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 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 material printing. 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 cost of the enterprise.

[0066] Example 3: Performance test of 3D printed photo-crosslinked helical polyisocyanurate hydrogel matrix

[0067] In this example, a series of experiments were performed to test the performance of the three-dimensional printed photo-cross-linked helical polyisocyanurate hydrogel matrix constructed in this scheme.

[0068] The mixture was mixed evenly on ice to obtain a UV-curable hydrogel matrix precursor that can be used for 3D printing. The precursor was subjected to temperature scanning test and light curing test on a rheometer. S1500 UV point light curing system, set the light intensity to 50%.

[0069] Low temperature crosslinking: UV irradiation at 5°C for 100s, followed by temperature scanning in the range of 5-37°C, heating rate of 1.0°C / min, strain of 1%, and frequency of 1.0Hz. The results are shown in Figure 3. Figure 3A -D is the rheological result of PIC photocrosslinking at low temperature (5°C). The horizontal axis shows the temperature, the vertical axis shows the storage modulus G', and the black squares show the change of storage modulus with temperature. Blue light crosslinking was performed at 5°C and then heated to 37°C. The PIC chain length (regulated 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 affect the storage modulus G' before and after crosslinking.

[0070] Room temperature crosslinking: First, a temperature scan was performed in the range of 5-25°C, a heating rate of 1.0°C / min, a strain of 1%, and a frequency of 1.0 Hz, followed by UV irradiation at 25°C for 100 s, and then the temperature was raised to 37°C with the same parameters. The results are shown in Figure 4. Figure 4A -D is the rheological result of PIC photocrosslinking at room temperature (25°C). The horizontal axis shows the temperature, the vertical axis shows the storage modulus G', the black square shows the change of storage modulus with temperature, and the arrow indicates the direction of temperature change. The upward arrow indicates heating, and the downward arrow indicates cooling. First, the material is heated from 5°C to 25°C, then blue light crosslinked at 25°C, then heated to 37°C, and finally cooled to 5°C. The PIC chain length (regulated by the catalyst nickel perchlorate hexahydrate: monomer ratio, 1:1000 or 1:5000, abbreviated as 1k or 5k) and concentration 1 (or 8mg / ml) both affect the storage modulus G' before and after crosslinking.

[0071] Body temperature cross-linking: First, a temperature scan was performed in the range of 5-37°C, a heating rate of 1.0°C / min, a strain of 1%, and a frequency of 1.0Hz, followed by UV irradiation at 37°C for 100s, followed by cooling to 5°C with the same parameters. The results are shown in Figure 5. Figure 5A -D is the rheological result of PIC photocrosslinking at body temperature (37°C). The horizontal axis shows the temperature, the vertical axis shows the storage modulus G', the black square shows the change of storage modulus with temperature, and the arrow indicates the direction of temperature change. The upward arrow indicates heating, and the downward arrow indicates cooling. First, the material is heated from 5°C to 37°C, then UV crosslinked at 37°C, and then cooled to 5°C. The PIC chain length (regulated 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 affect the storage modulus G' before and after crosslinking.

[0072] The present application proposes a three-dimensional printing photo-crosslinked spiral polyisocyanide hydrogel matrix and a preparation method thereof. By grafting acrylamide groups on 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 chain, 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 on the basis of the already temperature-sensitively formed fibrous structure, significantly improving the mechanical strength of the system. The final mechanical strength of the hydrogel can be adjusted by controlling the molecular weight and concentration of PIC. The entire crosslinking 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. Give full play to the synergistic effect of the physical gelation properties mediated by PIC hydrogen bonds and the double bond chemical crosslinking mediated by phenyl (2,4,6-trimethylbenzoyl) and lithium phosphonate (LAP). The entire hydrogel system is very suitable as a matrix for ultraviolet light curing of three-dimensional material printing. The crosslinking 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, and promotes the adhesion, growth and proliferation of cells while providing the three-dimensional environment required for cell growth. This preparation method has a simple process and is suitable for a variety of biomedical applications; at the same time, the materials used are all artificially synthesized, with high controllability and strong repeatability; this preparation method is unique and highly innovative compared to the existing photocuring system.

[0073] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

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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