A hydrogel material with active oxygen responsiveness, an antibacterial / anti ferroptosis bone repair composite scaffold, a preparation method and application thereof

Abstract

The present application belongs to the technical field of hydrogel, and particularly relates to a hydrogel material with active oxygen responsiveness and a preparation method thereof, and further relates to an antibacterial / anti-ferric death bone repair composite scaffold comprising a tissue engineering porous scaffold material and the hydrogel material with active oxygen responsiveness filled in the tissue engineering porous scaffold material, in particular to an antibacterial / anti-ferric death bone repair composite scaffold comprising a 3D-printed polycaprolactone / bioglass porous scaffold material and the hydrogel material with active oxygen responsiveness filled in the porous scaffold material and a preparation method thereof, and further relates to application of the antibacterial / anti-ferric death bone repair composite scaffold with active oxygen responsiveness as described above to treatment of infectious bone defects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of hydrogel, and particularly relates to a hydrogel material with active oxygen responsiveness and a preparation method thereof, and further relates to an antibacterial / anti-ferroptosis bone repair composite scaffold comprising a tissue engineering porous scaffold material and the hydrogel material with active oxygen responsiveness filled in the tissue engineering porous scaffold material, and particularly relates to an antibacterial / anti-ferroptosis bone repair composite scaffold comprising a 3D-printed polycaprolactone / bioglass porous scaffold material and the hydrogel material with active oxygen responsiveness filled in the porous scaffold material and a preparation method thereof, and further relates to application of the antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness as described above in treatment of infectious bone defects. BACKGROUND

[0002] In orthopedic clinical treatment, infectious bone defects caused by bone and implant infection are one of the most challenging and persistent complications in orthopedic clinical treatment. Infectious bone defects are mainly caused by open trauma combined with wound contamination, and partly caused by perioperative infection of bone implantation surgery and hematogenous osteomyelitis. Bacteria and their metabolites colonized in bone tissue and bone implants can cause local inflammatory response and tissue cell necrosis, destroy the bone regeneration microenvironment, and seriously affect the healing of bone fractures or bone defects. At present, the main treatment method for clinical treatment of infectious bone defects is to implant antibiotic bone cement beads after thorough debridement, and then perform a second bone grafting surgery after the infection is completely controlled. However, there are problems such as low bone regeneration healing ability of infected tissue and bacterial antibiotic resistance, and it causes serious physical and economic burden to patients. In addition to removing pathogenic bacteria in the infection site, how to rescue the damage of tissue cells caused by infection is also a key problem in the treatment of infectious bone defects. In the infection microenvironment, pathogenic bacteria can induce bone marrow mesenchymal stem cells to damage and die, inhibit the normal osteogenic differentiation of bone marrow mesenchymal stem cells and the bone regeneration process, and ultimately lead to bone repair failure. It is hoped that by rescuing the damage and death of bone marrow mesenchymal stem cells and the osteogenic differentiation disorder in the infection microenvironment, the repair of infected bone defects can be promoted.

[0003] Chinese document "3D printable high-toughness double-network polyethylene glycol diacrylate / chitosan hydrogel and its osteogenic performance" (Deng Ziwei, Master's Degree Thesis of South China University of Technology, April 2021) discloses a composite hydrogel of polyethylene glycol diacrylate (PEGDA) / chitosan (CS) based on double-network mechanism enhancement. The N-glucosamine units on chitosan can react with Cit 3-Forming ionic bonds, small molecular weight PEGDA is also added in chitosan system, under ultraviolet stimulation, the double bond at both ends of PEGDA can be activated to form covalent crosslinking points, obtain covalent network, form the synthesis of ionic-covalent double network chitosan / PEGDA hydrogel. The document technology will form chitosan / PEGDA hydrogel directly 3D printing, the 3D printing scaffold formed in mechanical strength is still weak, the mechanical strength is low, the material will appear after extrusion on the platform collapse phenomenon, even if the direct 3D printing scaffold of the double network enhanced chitosan / PEDGA composite hydrogel still cannot achieve the purpose of weight-bearing tissue regeneration and repair.

[0004] 3D printing technology as a new type of additive manufacturing technology that can be personalized, in recent years, in the study of bone defect repair is concerned. Using 3D printing technology, the anatomical structure matching, internal porosity and void structure controllable bone repair scaffold can be prepared.

[0005] However, the existing anti-infection bone repair 3D printing scaffold lacks the function of rescuing tissue cell damage, and it is difficult to rescue the bone marrow mesenchymal stem cell damage and osteogenic differentiation disorder caused by the pathogenic bacteria of the infection focus, resulting in slow or non-healing of the bone repair at the infection site. The important reason why the anti-infection bone repair scaffold lacks the function of rescuing tissue damage is that the specific pathway and mechanism of cell damage caused by infection are unknown, and it is difficult to intervene in the death of mesenchymal stem cells in the infection microenvironment. Moreover, the existing anti-infection bone repair 3D printing scaffold cannot intelligently adjust the release rate of antibacterial and bioactive components according to the pathogenic bacteria load and inflammation degree of the infection focus, and the material is difficult to dynamically adapt to the changes of the infection course. The important reason why the material has poor biological adaptability is that the material lacks the ability to respond to the release of antibacterial components and bioactive components. SUMMARY

[0006] In view of this, the purpose of the present application is to provide a hydrogel material with active oxygen responsiveness, and to provide an antibacterial / anti-ferroptosis bone repair composite scaffold comprising a tissue engineering porous scaffold material and the hydrogel material with active oxygen responsiveness filled in the tissue engineering porous scaffold material, and an application in the treatment of infectious bone defects. First, a certain amount of anti-ferroptosis drug is loaded in the ROS-responsive hydrogel based on chitosan quaternary ammonium salt to prepare a ROS-responsive hydrogel with antibacterial / anti-ferroptosis activity; at the same time, the mesoporous bioglass loaded with anti-ferroptosis drug is mixed with polycaprolactone to prepare a porous scaffold by 3D printing technology, and finally the uncrosslinked antibacterial / anti-ferroptosis hydrogel and the 3D printed porous scaffold are assembled and subjected to in-situ photo-crosslinking to prepare the required ROS-responsive antibacterial / anti-ferroptosis bone repair composite scaffold.

[0007] To achieve the above-mentioned purpose, the solution adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a hydrogel material with active oxygen responsiveness, comprising a hydrogel cross-linking system formed by a double cross-linking network structure of a borate ester bond between HACC-PBA and PEGDA-DTT and a photo-cross-linking of an unsaturated carbon-carbon double bond of PEGDA-DTT molecules, with phenylboronic acid-modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol-modified polyethylene glycol (diol) diacrylate (PEGDA-DTT) as precursors.

[0009] Preferably, the hydrogel material with active oxygen responsiveness further comprises a drug loaded in the hydrogel cross-linking system.

[0010] Preferably, the drug is an anti-ferroptosis drug, and preferably the anti-ferroptosis drug comprises one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K, and N-acetylcysteine.

[0011] Preferably, the loading rate of the anti-ferroptosis drug in the hydrogel cross-linking system is 0.1%-10% (mass fraction).

[0012] Preferably, in the hydrogel material, the formed borate ester bond has an active oxygen response breaking property, and can release chitosan quaternary ammonium salt with antibacterial activity and anti-ferroptosis drugs simultaneously in the presence of active oxygen (ROC) molecules.

[0013] In a second aspect, the present application also provides a preparation method of the hydrogel material with active oxygen responsiveness as described above, comprising the following steps:

[0014] Preparation of phenylboronic acid-modified chitosan quaternary ammonium salt by normal temperature solution method;

[0015] Preparation of chitosan quaternary ammonium salt hydrogel cross-linking system by normal temperature solution method;

[0016] Preparation of chitosan quaternary ammonium salt hydrogel material loaded with anti-ferroptosis drugs by hydrogel encapsulation method.

[0017] Preferably, the step of preparing phenylboronic acid-modified chitosan quaternary ammonium salt by normal temperature solution method comprises: dissolving chitosan quaternary ammonium salt in a water-organic solvent dissolution system, then adding a phenylboronic acid substance, starting normal temperature stirring reaction after sufficient dissolution for 1-12h, then adding a reducing protective agent sodium cyanoborohydride, normal temperature stirring reaction for 3-96h after sufficient dissolution; dialysis treatment is performed on the obtained solution after reaction, and the dialysis time is 6-72h; finally, vacuum freeze-drying is performed to obtain phenylboronic acid-modified chitosan quaternary ammonium salt (HACC-PBA).

[0018] Preferably, the quaternary ammonium salt of chitosan is hydroxypropyl trimethyl ammonium chloride chitosan (HACC), the degree of substitution of hydroxypropyl trimethyl ammonium chloride of the quaternary ammonium salt of chitosan ranges from 5% to 60%; and the phenyl boronic acid substance is selected from at least one of 4-formyl phenyl boronic acid and 3-amino phenyl boronic acid hydrochloride.

[0019] Preferably, the step of preparing the quaternary ammonium salt of chitosan hydrogel cross-linking system by normal temperature solution method comprises: dissolving polyethylene glycol (diol) diacrylate (PEGDA) and dithiothreitol (DTT) in an aqueous solution, and after sufficient dissolution, starting to stir and react at room temperature in the dark for 0.5-6h; then adding phenyl boronic acid modified quaternary ammonium salt of chitosan (HACC-PBA), and after sufficient dissolution, stirring and reacting at room temperature for 0.5-6h to obtain the quaternary ammonium salt of chitosan hydrogel cross-linking system.

[0020] Preferably, the average molecular weight of the polyethylene glycol (diol) diacrylate (PEGDA) is 250-10000, and the volume percentage is 5%-20%; the final concentration of the dithiothreitol (DTT) is 1-100mg / ml; and the mass concentration of the phenyl boronic acid modified quaternary ammonium salt of chitosan (HACC-PBA) is 1-15mg / ml.

[0021] Preferably, the step of preparing the quaternary ammonium salt of chitosan hydrogel material loaded with anti-ferroptosis drugs by hydrogel encapsulation method comprises: adding anti-ferroptosis drugs into the quaternary ammonium salt of chitosan hydrogel cross-linking system, and stirring uniformly to obtain the quaternary ammonium salt of chitosan hydrogel material loaded with anti-ferroptosis drugs.

[0022] Preferably, the anti-ferroptosis drugs include one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, and the mass concentration of the anti-ferroptosis drugs is 1-100mg / ml.

[0023] In a third aspect, the present application also provides a use of the hydrogel material with active oxygen responsiveness as described above and the hydrogel material with active oxygen responsiveness prepared by the preparation method as described above in the preparation of bone repair materials, tissue engineering porous scaffold materials or 3D printing biological ink.

[0024] Preferably, the hydrogel material with active oxygen responsiveness is used for coating of implants of bone repair materials.

[0025] Preferably, the hydrogel material with active oxygen responsiveness is used for filling tissue engineering porous scaffold materials.

[0026] In a fourth aspect, the present application further provides an antibacterial / anti-ferric death bone repair composite scaffold with active oxygen responsiveness, comprising a tissue engineering porous scaffold material and a hydrogel material with active oxygen responsiveness as described above filled in the tissue engineering porous scaffold material.

[0027] Preferably, the tissue engineering porous scaffold material is a 3D-printed polycaprolactone / bioglass porous scaffold material.

[0028] Preferably, the 3D-printed polycaprolactone / bioglass porous scaffold material is loaded with an anti-ferric death drug, and the anti-ferric death drug comprises one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K, and N-acetylcysteine; preferably, the loading rate of the anti-ferric death drug in the 3D-printed polycaprolactone / bioglass porous scaffold material is 0.5%-10% (mass fraction).

[0029] Preferably, the filling rate of the hydrogel material with active oxygen responsiveness in the 3D-printed polycaprolactone / bioglass porous scaffold material is 20%-300% (mass fraction).

[0030] In a fifth aspect, the present application further provides a preparation method of the antibacterial / anti-ferric death bone repair composite scaffold with active oxygen responsiveness as described above, comprising the following steps:

[0031] Preparation of a polycaprolactone / bioglass porous scaffold material loaded with an anti-ferric death drug by 3D printing;

[0032] Preparation of an antibacterial / anti-ferric death bone repair composite scaffold with active oxygen responsiveness by compounding the chitosan quaternary ammonium salt hydrogel material loaded with an anti-ferric death drug and the polycaprolactone / bioglass porous scaffold material loaded with an anti-ferric death drug by photo-crosslinking.

[0033] Preferably, the step of preparing a polycaprolactone / bioglass porous scaffold material loaded with an anti-ferric death drug by 3D printing comprises: co-incubating an anti-ferric death drug in a mesoporous bioglass aqueous solution, shaking for 6-8 h on a shaking table, then centrifuging to collect the bioglass adsorbed with the anti-ferric death drug, and vacuum freeze-drying to obtain dried bioglass loaded with the anti-ferric death drug; then fully dissolving polycaprolactone in an organic solvent, adding the bioglass powder loaded with the anti-ferric death drug and mixing uniformly to obtain polycaprolactone / bioglass raw material loaded with the anti-ferric death drug; further adding the mixture raw material into a 3D printing cartridge, using a 3D printer to perform 3D printing according to the designed size information and pore size, and completely drying to prepare a polycaprolactone / bioglass porous scaffold loaded with an anti-ferric death drug.

[0034] Preferably, the 3D printing method comprises a normal / low temperature printing method or a high temperature printing method.

[0035] Preferably, the step of compounding the hydrogel material with active oxygen responsiveness and the poly (caprolactone) / bioglass porous scaffold material loaded with anti-ferric death drugs by the photo-crosslinking method comprises: adding a photo initiator into the uncrosslinked hydrogel material loaded with anti-ferric death drugs to form a hydrogel system, and then filling the poly (caprolactone) / bioglass porous scaffold wrapped with the anti-ferric death drug by a hydrogel filling method.

[0036] Preferably, the hydrogel filling method comprises at least one of a jar dripping method, a negative pressure suction method and a soaking method.

[0037] Preferably, the hydrogel system is subjected to in-situ photo-crosslinking reaction on the surface of the poly (caprolactone) / bioglass porous scaffold material loaded with anti-ferric death drugs by photo excitation irradiation; the photo initiator is at least one selected from the group consisting of lithium phenyl-2, 4, 6-trimethyl benzoyl phosphinate (LAP) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl propiophenone (Irgacure 2959), the mass fraction of the photo initiator is 0.1%-1%, and the photo excitation irradiation time is 5-60s.

[0038] In a sixth aspect, the present application also provides a use of the antibacterial / anti-ferric death bone repair composite scaffold with active oxygen responsiveness and the antibacterial / anti-ferric death bone repair composite scaffold with active oxygen responsiveness prepared by the preparation method in the treatment of infectious bone defects.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] The present application provides a hydrogel material with active oxygen responsiveness, and also provides an antibacterial / anti-ferric death bone repair composite scaffold with active oxygen responsiveness and a use thereof in the treatment of infectious bone defects. The prepared antibacterial / anti-ferric death bone repair composite scaffold with active oxygen responsiveness has intelligent response and antibacterial properties, and has the effects of rescuing mesenchymal stem cell ferric death and promoting mesenchymal stem cell osteogenic differentiation, and has obvious effect of promoting the healing of infectious bone defects, and has very important research and clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 A schematic diagram of the formation of the hydrogel material with active oxygen responsiveness involved in the present application.

[0042] Figure 2The schematic photo of the antibacterial / anti-ferruginous death bone repair composite scaffold with active oxygen response in Example 1 of the present application; in the figure, (a) is a polycaprolactone / mesoporous bioglass scaffold, (b) is an antibacterial / anti-ferruginous death bone repair composite scaffold with active oxygen response.

[0043] Figure 3 The antibacterial performance picture of the antibacterial / anti-ferruginous death bone repair composite scaffold with active oxygen response in Example 2 of the present application; in the figure, (a) is the antibacterial coating plate photo of the hydrogel composite scaffold loaded with different HACC-PBA contents, (b) is the antibacterial efficacy statistical chart.

[0044] Figure 4 The release performance picture of the chitosan quaternary ammonium salt hydrogel and the polycaprolactone / bioglass porous scaffold in Example 3 of the present application; in the figure, (a) is the FITC release curve of the HACC-DP hydrogel with different HACC-PBA contents in the simulated body fluid and the simulated body fluid containing hydrogen peroxide, (b) is the FITC release curve of the PCL / MBG scaffold loaded with FITC in the simulated body fluid and the simulated body fluid containing hydrogen peroxide.

[0045] Figure 5 The picture of Ferrostatin-1 saving mesenchymal stem cell ferroptosis caused by infection in Example 4 of the present application; in the figure, (a) is the fluorescence photo of the effect of E. coli and S. aureus on inducing lipid peroxidation and iron overload of bone marrow mesenchymal stem cells and the rescue effect of the ferroptosis inhibitor Ferrostatin-1, (b) is the flow cytometry live / dead staining analysis chart of E. coli and S. aureus inducing mesenchymal stem cell death, (c) is the flow cytometry live / dead staining quantitative analysis of the proportion of dead cells.

[0046] Figure 6 The picture of Ferrostatin-1 promoting osteogenic differentiation of mesenchymal stem cells infected with bacteria in Example 5 of the present application; in the figure, (a) is alkaline phosphatase staining, (b) is alizarin red S staining.

[0047] Figure 7 The picture of the antibacterial effect of the antibacterial / anti-ferruginous death bone repair composite scaffold with active oxygen response in Example 6 of the present application in vivo; in the figure, (a) is the in vivo bacterial live fluorescence detection of the rat femoral condyle infectious bone defect model, (b) is the quantitative analysis of the in vivo bacterial live fluorescence signal.

[0048] Figure 8 The picture of the antibacterial / anti-ferruginous death bone repair composite scaffold with active oxygen response in Example 7 of the present application promoting the repair of infectious bone defects; in the figure, (a) is the micro-CT analysis of the rat femoral condyle infectious bone defect model, (b) is the quantitative analysis of the bone mass and trabecular number of micro-CT. DETAILED DESCRIPTION

[0049] The present application provides a hydrogel material with active oxygen responsiveness, which can deliver and continuously release therapeutic drugs, comprising a three-dimensional network structure hydrogel crosslinking system and drugs loaded in the hydrogel crosslinking system.

[0050] <Three-crosslinking network structure hydrogel crosslinking system>

[0051] As an embodiment of the present application, the hydrogel crosslinking system is a three-crosslinking network structure hydrogel crosslinking system formed by HACC-PBA and PEGDA-DTT intermolecularly forming a borate ester bond covalent crosslinking and PEGDA-DTT molecularly forming a photo-crosslinking carbon-carbon double bond.

[0052] Chitosan is an N-deacetylation product of chitin, and its structural formula is shown in the following formula (1). Chitosan is the only basic amino polysaccharide in natural sugars, and has many special physical and chemical properties and physiological functions, such as good adsorption, film forming, fiber forming, moisture absorption and retention, biocompatibility and degradation performance. However, due to the influence of its solubility, its application is limited to a certain extent.

[0053]

[0054] Quaternization modification of chitosan is one of the methods for modifying chitosan, which introduces quaternary ammonium groups on the amino group of chitosan or connects a low molecular quaternary ammonium salt to the amino group to obtain a kind of chitosan derivative. Due to the better water solubility of quaternized chitosan than chitin and chitosan, it can better exert the efficacy of chitosan. The modified chitosan quaternary ammonium salt not only has the properties of typical quaternary ammonium salt, such as antibacterial and antifungal properties and moisture absorption and retention, but also maintains the original good film forming, flocculation, biocompatibility and biodegradation properties of chitosan.

[0055] Chitosan quaternary ammonium salt as a commercial antibacterial agent is a kind of natural polysaccharide derivatives with good biocompatibility and antibacterial activity. According to the different modification methods of chitosan quaternization, the common chitosan quaternary ammonium salt includes but is not limited to N-trimethyl chitosan quaternary ammonium salt, N-methyl-N,N-bis long chain alkyl chitosan quaternary ammonium salt, O-acrylamide-N-chitosan quaternary ammonium salt (NMA-HTCC), hydroxypropyl trimethyl chitosan quaternary ammonium salt (HACC) and the like. Through the reported antibacterial performance experiment, it is found that the positively charged quaternary ammonium group in the quaternary ammonium chitosan molecule can obviously promote the mutual combination between the chitosan molecule and the cell wall negative ions of bacteria, so as to cause the death of bacteria. At the same time, the chitosan quaternary ammonium salt has no obvious toxicity to bone marrow mesenchymal stem cells within a certain degree of substitution (1%-60%), can promote the osteogenic differentiation of bone marrow mesenchymal stem cells and at the same time play a good antibacterial effect, and inhibit the formation of biofilm.

[0056] And the hydroxypropyl trimethyl chitosan quaternary ammonium salt (HACC) is the best among these chitosan quaternary ammonium salts in antibacterial activity, and the antibacterial activity is enhanced with the increase of positive charge. Therefore, the hydroxypropyl trimethyl ammonium chloride chitosan (HACC) with the structure shown in the following formula (2) is selected as the three-dimensional structure matrix of the covalently crosslinked polymer in the hydrogel material provided by the present application.

[0057]

[0058] As a preferred embodiment of the present application, the hydroxypropyl trimethyl ammonium chloride chitosan (HACC) used in the present application is also modified by phenylboronic acid. The chemical structure of the hydroxypropyl trimethyl ammonium chloride chitosan (HACC-PBA) modified by phenylboronic acid is shown in the following formula (3).

[0059]

[0060] The hydrogel crosslinking system provided by the present application is a double crosslinking network structure hydrogel crosslinking system. The above hydroxypropyl trimethyl ammonium chloride chitosan (HACC-PBA) modified by phenylboronic acid is a precursor of one of the crosslinking networks of the double crosslinking network. The matrix of the other polymer crosslinking network is a dithiothreitol modified polyethylene glycol (diol) diacrylate (PEGDA-DTT) shown in the following formula (4). Polyethylene glycol (diol) diacrylate (PEGDA) is a kind of high molecular material with good biocompatibility and degradability. Under the action of photoinitiator and blue light excitation, it can form a hydrogel crosslinking network, and can be used as a good drug loading and release system. At present, there are more biological researches on the light crosslinking drug-loaded hydrogel system based on PEGDA.

[0061]

[0062] In the hydrogel material provided by the present application, the benzene boronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and the dithiothreitol modified polyethylene glycol (diol) diacrylate (PEGDA-DTT) are precursors to form a double crosslinking network structure hydrogel crosslinking system. The HACC-PBA and the PEGDA-DTT molecules are covalently crosslinked by forming borate ester bonds, and the two networks are connected to each other through the borate ester bonds (as shown in Figure 1 The borate ester bond formed is a chemical group with ROS response activity, has ROS responsive breaking characteristics, and can increase the adsorption of the drug loaded in the double crosslinking network structure hydrogel crosslinking system in the hydrogel material, thereby increasing the drug loading rate. In addition, the borate ester bond can also play a role in delivering drugs and ROS-responsive sustained release of drugs by connecting target molecules.

[0063] <Drug loaded in the hydrogel crosslinking system>

[0064] As an embodiment of the present application, the drug loaded in the double crosslinking network structure hydrogel crosslinking system in the hydrogel material provided by the present application is an anti-ferroptosis drug. Ferroptosis is a new type of programmed cell death characterized by intracellular iron overload and lipid peroxidation accumulation. The role of ferroptosis in infection-related cell damage and death is increasingly concerned. Some documents report that inhibiting ferroptosis can alleviate the degree of infection cell damage and accelerate the clearance of pathogenic bacteria.

[0065] The anti-ferroptosis drug selected by the present application is selected from one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine. The loading rate of the anti-ferroptosis drug in the hydrogel crosslinking system is 0.1%-10% (mass fraction). In the embodiments of the present application, Ferrostatin-1 is used as an example to illustrate the technical solutions of the present application, but the present application is not limited thereto.

[0066] <Hydrogel material>

[0067] The hydrogel material provided by the present application comprises the double crosslinking network structure hydrogel crosslinking system described above and the anti-ferroptosis drug loaded in the hydrogel crosslinking system described above.

[0068] In the hydrogel material provided by the application, the benzene boronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and the dithiothreitol modified polyethylene glycol (diol) diacrylate (PEGDA-DTT) are precursors to form a double crosslinking network structure hydrogel crosslinking system. The HACC-PBA and the PEGDA-DTT molecules are covalently crosslinked by forming borate ester bonds, and the two networks are connected to each other through the borate ester bonds (as shown in Figure 1 The borate ester bond formed is a chemical group with ROS response activity, has ROS responsive breaking characteristics, and can increase the adsorption of the anti-ferroptosis drug loaded in the double crosslinking network structure hydrogel crosslinking system in the hydrogel material, thereby increasing the loading rate of the anti-ferroptosis drug. In addition, the borate ester bond can also play a role in delivering the anti-ferroptosis drug and ROS-responsive sustained release of the drug by connecting target molecules.

[0069] In the hydrogel material provided by the application, the borate ester bond has the characteristic of ROS-responsive breaking, which can enable the hydrogel material of the application to release the chitosan quaternary ammonium salt with antibacterial activity and the anti-ferroptosis drug at the same time in the presence of ROS molecules. Therefore, the chitosan quaternary ammonium salt hydrogel of the application can flexibly adjust the release rate of antibacterial and anti-ferroptosis molecules according to the degree of inflammation at the infection site, and has better biological adaptation performance.

[0070] <Preparation of the hydrogel material>

[0071] The application also provides a preparation method of the hydrogel material with ROS responsiveness as described above, comprising the following steps:

[0072] Step (1), preparing the benzene boronic acid modified chitosan quaternary ammonium salt by a normal temperature solution method:

[0073] The chitosan quaternary ammonium salt is dissolved in a water and organic solvent dissolution system, then a benzene boronic acid substance is added, and after being fully dissolved, normal temperature stirring reaction is started for 1-12 h (for example, 4-12 h, 6-12 h, 4-10 h, 6-10 h), then a reducing protective agent sodium cyanoborohydride is added, and after being fully dissolved, normal temperature stirring reaction is started for 3-96 h (for example, 3-80 h, 3-60 h, 3-40 h, 3-20 h, 3-10 h, 15-80 h, 15-60 h, 15-40 h, 15-20 h); after the reaction, the obtained solution is subjected to dialysis treatment for 6-72 h (for example, 6-60 h, 6-40 h, 6-20 h, 6-10 h, 15-60 h, 15-40 h, 15-20 h); and finally, vacuum freeze-drying is performed to obtain the benzene boronic acid modified chitosan quaternary ammonium salt (HACC-PBA).

[0074] In step (1), the chitosan quaternary ammonium salt is hydroxypropyl trimethyl ammonium chloride chitosan (HACC), the degree of substitution of the hydroxypropyl trimethyl ammonium chloride of the chitosan quaternary ammonium salt ranges from 5% to 60% (may be, for example, 5% to 50%, 5% to 40%, 5% to 30%, 5% to 20%, 5% to 10%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 60%, 15% to 50%, 15% to 40%, 15% to 30%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 60%, 40% to 50%, 50% to 60%); the phenyl boronic acid substance is selected from at least one of 4-formyl phenyl boronic acid and 3-amino phenyl boronic acid hydrochloride.

[0075] Step (2), preparation of chitosan quaternary ammonium salt hydrogel crosslinking system by normal temperature solution method:

[0076] Polyethylene glycol (diol) diacrylate (PEGDA) shown in the following formula (5) and dithiothreitol (DTT) shown in the following formula (6) are dissolved in an aqueous solution, after sufficient dissolution, start to react under light protection at normal temperature with stirring for 0.5-6h; then add phenyl boronic acid modified chitosan quaternary ammonium salt (HACC-PBA), after sufficient dissolution, react at normal temperature with stirring for 0.5-6h, to obtain chitosan quaternary ammonium salt hydrogel crosslinking system.

[0077]

[0078] In step (2), the average molecular weight of the polyethylene glycol (diol) diacrylate (PEGDA) is 250-10000, and the volume percentage is 5%-20% (which can be, for example, 5%-15%, 5%-10%, 8%-20%, 8%-15%, 8%-10%, 10%-20%, 10%-15%, 15%-20%); the final concentration of the dithiothreitol (DTT) is 1-100 mg / ml (which can be, for example, 10-100 mg / ml, 10-90 mg / ml, 10-80 mg / ml, 10-70 mg / ml, 10-60 mg / ml, 10-50 mg / ml, 10-40 mg / ml, 10-30 mg / ml, 10-20 mg / ml, 20-100 mg / ml, 20-90 mg / ml, 20-80 mg / ml, 20-70 mg / ml, 20-60 mg / ml, 20-50 mg / ml, 20-40 mg / ml, 20-30 mg / ml, 30-100 mg / ml, 30-90 mg / ml, 30-80 mg / ml, 30-70 mg / ml, 30-60 mg / ml, 30-50 mg / ml, 30-40 mg / ml, 40-100 mg / ml, 40-90 mg / ml, 40-80 mg / ml, 40-70 mg / ml, 40-60 mg / ml, 40-50 mg / ml, 50-100 mg / ml, 50-90 mg / ml, 50-80 mg / ml, 50-70 mg / ml, 50-60 mg / ml, 60-100 mg / ml, 60-90 mg / ml, 60-80 mg / ml, 60-70 mg / ml, 70-100 mg / ml, 70-90 mg / ml, 70-80 mg / ml, 80-100 mg / ml, 80-90 mg / ml, 90-100 mg / ml); the mass concentration of the phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) is 1-15 mg / ml (which can be, for example, 1-12 mg / ml, 1-10 mg / ml, 1-8 mg / ml, 1-6 mg / ml, 1-4 mg / ml, 3-15 mg / ml, 3-12 mg / ml, 3-10 mg / ml, 3-8 mg / ml, 3-6 mg / ml, 5-15 mg / ml, 5-12 mg / ml, 5-10 mg / ml, 5-8 mg / ml, 7-15 mg / ml, 7-12 mg / ml, 7-10 mg / ml, 9-15 mg / ml, 9-12 mg / ml, 11-15 mg / ml, 11-13 mg / ml, 13-15 mg / ml).

[0079] Step (3), preparation of chitosan quaternary ammonium salt hydrogel material loaded with anti-iron death drugs by hydrogel encapsulation method:

[0080] The anti-ferroptosis drug is added into the chitosan quaternary ammonium salt hydrogel cross-linking system, and fully stirred to obtain a chitosan quaternary ammonium salt hydrogel material loaded with the anti-ferroptosis drug.

[0081] In step (3), the anti-ferroptosis drug includes one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K, and N-acetylcysteine, and the mass concentration of the anti-ferroptosis drug is 1-100 mg / ml (which may be, for example, 10-100 mg / ml, 10-90 mg / ml, 10-80 mg / ml, 10-70 mg / ml, 10-60 mg / ml, 10-50 mg / ml, 10-40 mg / ml, 10-30 mg / ml, 10-20 mg / ml, 20-100 mg / ml, 20-90 mg / ml, 20-80 mg / ml, 20-70 mg / ml, 20-60 mg / ml, 20-50 mg / ml, 20-40 mg / ml, 20-30 mg / ml, 30-100 mg / ml, 30-90 mg / ml, 30-80 mg / ml, 30-70 mg / ml, 30-60 mg / ml, 30-50 mg / ml, 30-40 mg / ml, 40-100 mg / ml, 40-90 mg / ml, 40-80 mg / ml, 40-70 mg / ml, 40-60 mg / ml, 40-50 mg / ml, 50-100 mg / ml, 50-90 mg / ml, 50-80 mg / ml, 50-70 mg / ml, 50-60 mg / ml, 60-100 mg / ml, 60-90 mg / ml, 60-80 mg / ml, 60-70 mg / ml, 70-100 mg / ml, 70-90 mg / ml, 70-80 mg / ml, 80-100 mg / ml, 80-90 mg / ml, 90-100 mg / ml).

[0082] <Applications of the hydrogel material>

[0083] The hydrogel material provided by this invention is a three-dimensional network structure material with high water content and excellent biocompatibility, making it widely applicable in the field of biomedical engineering, especially in medical applications such as drug delivery and cartilage repair. Hydrogels are widely studied for drug delivery due to their injectability, ease of chemical modification, easy degradation, and high permeability. As the hydrogel continuously degrades, the drug is also continuously released. Simultaneously, due to the better permeability of the hydrogel, tissues and cells can better absorb the drug contained within it. In orthopedic applications, the hydrogel can be used alone as a coating for implants or as a filler for porous materials to provide greater coverage in the gap between the implant and bone, thereby better stimulating bone growth.

[0084] In a preferred embodiment of the present invention, the chitosan quaternary ammonium saline hydrogel material loaded with anti-ferroptosis drug of the present invention is filled into a 3D printed porous scaffold material. The filling method of the hydrogel material is selected from at least one of drip irrigation, negative pressure suction and soaking. In the embodiment of the present invention, the negative pressure suction method is used for filling.

[0085] In the chitosan quaternary ammonium salt gel material loaded with anti-ferroptosis drugs provided by this invention, such as Figure 1 As shown, a double-crosslinked network structure hydrogel crosslinking system is formed using phenylboronic acid-modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol-modified polyethylene glycol (PEGDA-DTT) as precursors. HACC-PBA and PEGDA-DTT molecules form borate ester bonds for covalent crosslinking, and the two networks are interconnected through these bonds. As shown in formula (4), the double bonds at both ends of PEGDA-DTT can undergo crosslinking under photoexcitation in the presence of a photoinitiator. Therefore, in this invention, after adding a certain amount of photoinitiator to the uncrosslinked chitosan quaternary ammonium salt hydrogel material loaded with anti-ferroptosis drugs, the hydrogel material is filled and encapsulated in a 3D-printed porous scaffold material using a negative pressure suction method. Then, photoexcitation is used to irradiate the hydrogel material on the surface of the 3D-printed porous scaffold material to induce an in-situ photocrosslinking reaction, thus obtaining a composite scaffold. When this composite scaffold is implanted into a human or animal as a bone implant, the borate ester bonds in the hydrogel system, which have reactive oxygen species (ROS) responsive cleavage characteristics, break. In the presence of ROS molecules, chitosan quaternary ammonium salts with antibacterial activity and anti-ferroptosis drugs are continuously released. Furthermore, the release rate of antibacterial and antibody death molecules can be flexibly adjusted according to the degree of inflammation at the site of infection.

[0086] <3D Printed Porous Bone Repair Scaffold Materials>

[0087] 3D printing technology as a new type of additive manufacturing technology of personalized customization, in recent years in the bone defect repair research is concerned. Using 3D printing technology, the anatomical structure matching, internal porosity and gap structure controllable bone repair scaffold can be prepared. At present, a number of studies have reported that the composite 3D printing scaffold composed of high molecular weight degradable material and bioglass has good biodegradability and mechanical properties. At the same time, the bioglass composition has strong osteogenesis ability and drug release ability. The application innovatively loads the anti-ferroptosis drug in the bioglass with pore structure, and prepares a bone repair scaffold with long-acting sustained release function of anti-ferroptosis drug through 3D printing scaffold.

[0088] The polycaprolactone / bioglass porous scaffold loaded with anti-ferroptosis drug provided by the application is prepared by mixing a certain amount of mesoporous bioglass loaded with anti-ferroptosis drug and polycaprolactone, and then using a 3D printing method to prepare a porous scaffold with a certain gap size and geometric shape. Polycaprolactone and bioglass are degradable materials approved by FDA for clinical use, so the scaffold prepared by the application has high biological safety. The anti-ferroptosis drug loaded in the mesoporous bioglass can be released in the local microenvironment with the biodegradation of the scaffold, providing long-term protection effect for bone tissue regeneration and repair in the infected microenvironment.

[0089] As an embodiment of the application, the steps of preparing the polycaprolactone / bioglass porous scaffold loaded with anti-ferroptosis drug by 3D printing method include: dispersing the anti-ferroptosis drug in the aqueous solution of mesoporous bioglass for co-incubation, shaking for 6-8h on a shaking table, then centrifuging to collect the bioglass loaded with anti-ferroptosis drug, and using vacuum freeze-drying method to prepare dry bioglass loaded with anti-ferroptosis drug; further, dissolving polycaprolactone in an organic solvent, adding bioglass powder loaded with anti-ferroptosis drug and mixing uniformly to prepare polycaprolactone / bioglass raw material loaded with anti-ferroptosis drug; further, adding the mixture raw material into the 3D printing cartridge, using the 3D printer to 3D print according to the designed size information and pore size, and preparing the polycaprolactone / bioglass porous scaffold loaded with anti-ferroptosis drug after complete drying.

[0090] As a preferred embodiment of the present application, the mass fraction of the anti-ferroptosis drug in the mesoporous bioglass adsorption system is 5%-40% (which may be, for example, 5%-35%, 5%-30%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, 10%-15%, 15%-40%, 15%-35%, 15%-30%, 15%-25%, 15%-20%, 20%-40%, 20%-35%, 20%-30%, 20%-25%, 25%-40%, 25%-35%, 25%-30%, 30%-40%, 30%-35%, 35%-40%). The mass fraction of the mesoporous bioglass loaded with the anti-ferroptosis drug in the polycaprolactone / bioglass raw material system is 10%-50% (which may be, for example, 10%-45%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, 10%-15%, 15%-50%, 15%-45%, 15%-40%, 15%-35%, 15%-30%, 15%-25%, 15%-20%, 20%-50%, 20%-45%, 20%-40%, 20%-35%, 20%-30%, 20%-25%, 25%-50%, 25%-45%, 25%-40%, 25%-35%, 25%-30%, 30%-50%, 30%-45%, 30%-40%, 30%-35%, 35%-50%, 35%-45%, 35%-40%, 40%-50%, 40%-45%, 45%-50%). The 3D printing method includes normal / low-temperature printing or high-temperature printing.

[0091] As an embodiment of the present application, the anti-ferroptosis drug selected by the present application is selected from one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, wherein the loading rate of the anti-ferroptosis drug in the 3D-printed polycaprolactone / bioglass porous scaffold material is 0.5%-10% (mass fraction). In the embodiment of the present application, Ferrostatin-1 is taken as an example to illustrate the technical solutions of the present application, but the present application is not limited thereto.

[0092] <Antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness>

[0093] The present application also provides an antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness, which comprises a tissue engineering porous scaffold material and a hydrogel material with active oxygen responsiveness as described above filled in the tissue engineering porous scaffold material.

[0094] As an embodiment of the present application, the tissue engineering porous scaffold material is a 3D-printed porous bone repair scaffold material as described above, in particular a polycaprolactone / bioglass porous scaffold loaded with anti-ferroptosis drugs provided by the present application.

[0095] As a preferred embodiment of the present application, the filling rate of the hydrogel material with active oxygen responsiveness provided by the present application in the polycaprolactone / bioglass porous scaffold material loaded with anti-ferroptosis drugs provided by the present application is 20%-300%(mass fraction).

[0096] The present application prepares an antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness by compounding the above-mentioned chitosan quaternary ammonium salt hydrogel material loaded with anti-ferroptosis drugs provided by the present application and the above-mentioned polycaprolactone / bioglass porous scaffold material loaded with anti-ferroptosis drugs provided by the present application through photo-crosslinking method, and the specific steps include: adding a photo initiator to the uncrosslinked chitosan quaternary ammonium salt hydrogel material loaded with anti-ferroptosis drugs to form a hydrogel system, then filling the polycaprolactone / bioglass porous scaffold wrapped with the anti-ferroptosis drugs by using a hydrogel filling method, and irradiating the hydrogel system with light excitation to cause in-situ photo-crosslinking reaction on the surface of the polycaprolactone / bioglass porous scaffold loaded with anti-ferroptosis drugs. As an embodiment of the present application, the hydrogel filling method includes at least one of the drip filling method, the negative pressure suction method and the soaking method, the photo initiator is selected from at least one of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), the mass fraction of the photo initiator is 0.1%-1%, and the light excitation irradiation time is 5-60s.

[0097] In the antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness provided by the present application, as Figure 1As shown, the benzene boronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (diol) diacrylate (PEGDA-DTT) are used as precursors to form a double cross-linking network structure hydrogel cross-linking system, and the HACC-PBA and PEGDA-DTT molecules are covalently cross-linked by forming borate ester bonds, and the double bonds at both ends of the PEGDA-DTT shown in formula (4) can be cross-linked under light excitation in the presence of a photoinitiator. Therefore, after adding a certain amount of photoinitiator into the uncross-linked chitosan quaternary ammonium salt hydrogel material loaded with an anti-ferroptosis drug, the hydrogel material is filled and wrapped in the above-mentioned poly (caprolactone) / bioglass porous scaffold loaded with an anti-ferroptosis drug by using, for example, a negative pressure suction method, and then the hydrogel material is subjected to in-situ photocrosslinking reaction on the surface of the poly (caprolactone) / bioglass porous scaffold loaded with an anti-ferroptosis drug by using light excitation irradiation to obtain a composite scaffold. The composite scaffold is implanted into the human or animal body as a bone implant, and in the hydrogel system, the borate ester bonds with the active oxygen (ROS) responsive breaking characteristics are broken, and the chitosan quaternary ammonium salt with antibacterial activity and the anti-ferroptosis drug are continuously released in the presence of active oxygen molecules, and the release rate of the antibacterial and anti-ferroptosis molecules can be flexibly adjusted according to the degree of inflammation at the infection site. In the composite scaffold, the porous scaffold used is a poly (caprolactone) / bioglass porous scaffold with high biological safety, and the anti-ferroptosis drug loaded in the mesoporous bioglass can be released in the local microenvironment with the biodegradation of the porous scaffold, thereby further providing a long-term protection effect for bone tissue regeneration and repair in the infection microenvironment.

[0098] Therefore, the composite scaffold provided by the present application can release antibacterial components and anti-ferroptosis components in response to active oxygen through the hydrogel system in the acute phase of infection, kill bacteria while protecting the activity of mesenchymal stem cells, promote the osteogenic differentiation of mesenchymal stem cells in the medium and long-term bone repair process through the slow release of the anti-ferroptosis drug in the mesoporous bioglass, and achieve early antibacterial and anti-ferroptosis dual-mode release and promotion of bone tissue regeneration and repair.

[0099] The present application first uses borate ester bonds to connect chitosan quaternary ammonium salt and PEGDA to prepare a light cross-linking ROS responsive antibacterial hydrogel, and uses the antibacterial / anti-ferroptosis active light cross-linking ROS responsive hydrogel loaded with an anti-ferroptosis drug as a responsive component of a composite 3D printed bone repair scaffold to prepare an antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness. The antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness provided by the present application can adjust the release rate of antibacterial components and anti-ferroptosis components in response to the active oxygen signal in the infection microenvironment in the early stage of infection, kill bacteria while inhibiting ferroptosis to save the activity of mesenchymal stem cells, and promote the osteogenic differentiation of mesenchymal stem cells and bone repair in the medium and long-term through the long-acting slow release of the anti-ferroptosis components.

[0100] The technical solutions of the present application are further described below in combination with specific embodiments, but the protection scope of the present application is not limited to these embodiments. Any changes or equivalent replacements without departing from the concept of the present application are included in the protection scope of the present application.

[0101] Example 1, preparation of an antibacterial / anti-ferric death composite scaffold with active oxygen response:

[0102] Dissolve the chitosan quaternary ammonium salt with a substitution degree of about 26% in a mixed solution system of 50 ml deionized water and 25 ml anhydrous ethanol, add 100 mg 4-formylphenylboronic acid, and stir at 25°C for 1 h. Then add 150 mg sodium cyanoborohydride, fully dissolve, and then stir at 25°C for 18 h. Use a 3500 Da dialysis bag to dialyze the obtained solution in pure water for 48 h, and then use a vacuum freeze-drying method to obtain the phenylboronic acid modified chitosan quaternary ammonium salt.

[0103] Dissolve polyethylene glycol (diol) diacrylate and dithiothreitol in an aqueous PBS solution, so that the final concentration of polyethylene glycol (diol) diacrylate is 150 mg / ml and the final concentration of dithiothreitol is 100 mg / ml. After fully dissolving, start the reaction under dark and normal temperature stirring, and the reaction time is 2 h. Then add the phenylboronic acid modified chitosan quaternary ammonium salt so that its mass fraction is 0-1.5%, and stir at room temperature under dark for 1 h. Then add the anti-ferric death drug Ferrostatin-1 and the photoinitiator LAP so that their final concentrations are 20 mg / ml and 1 mg / ml respectively, to prepare the chitosan quaternary ammonium salt hydrogel system loaded with Ferrostatin-1.

[0104] The anti-ferric death drug Ferrostatin-1 prepared in advance is co-incubated with mesoporous bioglass in an aqueous solution at a mass ratio of 1:7. After adsorption for 6 h on a shaking bed, centrifugation is performed and freeze-drying treatment is carried out to obtain mesoporous bioglass loaded with Ferrostatin-1. After dissolving polycaprolactone in dichloromethane, the mesoporous bioglass loaded with Ferrostatin-1 is added and fully stirred to prepare a mixed paste suitable for 3D printing. The mass ratio of mesoporous bioglass loaded with Ferrostatin-1 to polycaprolactone is 3:7. After evaporation to a suitable viscosity, the paste is loaded into a 3D printing cartridge and then into a 3D printer. On the extrusion type 3D printer, a borderless grid circular model with a diameter of 3 mm, a height of 4 mm, and a void spacing of 0.5 mm is set, the printing speed is 1.8 mm / s, and the extrusion pressure is 3.0 bar. The 3D printing is carried out to obtain a porous scaffold loaded with Ferrostatin-1.

[0105] The Ferrostatin-1 loaded porous scaffold as described above was immersed in the uncrosslinked Ferrostatin-1 loaded chitosan quaternary ammonium salt hydrogel system. The hydrogel system was used to fill the porous scaffold voids sufficiently using the negative pressure exhaust method. Then, the porous scaffold loaded with the hydrogel system was irradiated with blue light for 30 s to crosslink the hydrogel system. The ROS-responsive antibacterial / anti-ferroptosis hydrogel composite 3D printed bone repair scaffold was prepared. As shown in Figure 2 The loaded hydrogel composite scaffold (Fer-1@HACC-DP-PCL / MBG scaffold) and the unloaded hydrogel scaffold (HACC-DP-PCL / MBG scaffold) are shown.

[0106] Example 2, antibacterial performance of the ROS-responsive antibacterial / anti-ferroptosis composite scaffold:

[0107] The antibacterial / anti-ferroptosis composite scaffold loaded with different contents of phenylboronic acid modified chitosan quaternary ammonium salt (about 20 g) was immersed in 200 μl of CAMHB medium containing 5.5 x 10 5 CFU / ml of Staphylococcus aureus (ATCC25923) and incubated in a shaker for 18 hours at 37°C, 200 rpm. After incubation, the scaffold was removed, 1 ml of sterile PBS was added and ultrasonically shaken for 5 min, and then the plate colony counting method was used to calculate the antibacterial rate of the scaffold. From Figure 3 It can be seen that compared with the group without adding phenylboronic acid modified chitosan quaternary ammonium salt in the hydrogel, the antibacterial rate of the scaffold is greater than 99% when the content of phenylboronic acid modified chitosan quaternary ammonium salt in the hydrogel is 0.5-1.5%, and the antibacterial efficiency of the composite scaffold containing 1% phenylboronic acid modified chitosan quaternary ammonium salt in the hydrogel system is the highest.

[0108] Example 3, release performance of chitosan quaternary ammonium salt hydrogel and polycaprolactone / bioglass porous scaffold:

[0109] Ferrostatin-1 was replaced by fluorescein FITC with similar molecular weight, added into chitosan quaternary ammonium salt hydrogel system and 3D printed porous scaffold, the release pattern of Ferrostatin-1 was simulated by measuring the release of FITC. Chitosan quaternary ammonium salt hydrogels loaded with FITC but different content of phenylboronic acid modified chitosan quaternary ammonium salt (0.5%-1.5%) were immersed in simulated body fluid with hydrogen peroxide (100 μM) and without hydrogen peroxide, volume ratio 1:100, incubated in a constant temperature shaker at 37°C, 200 rpm. The cumulative release amount of FITC was calculated by measuring the concentration of FITC in SBF at 0, 0.5, 1, 2, 3, 4, 5, 6 days. 3D printed porous scaffolds loaded with FITC were immersed in simulated body fluid with hydrogen peroxide (100 μM) and without hydrogen peroxide, mass volume ratio 1 g: 100 ml, incubated in a constant temperature shaker at 37°C, 200 rpm. The cumulative release amount of FITC was calculated by measuring the concentration of FITC in SBF at 0, 1, 4, 7, 14, 21, 28 days. From Figure 4 It can be seen that the release rate of FITC of chitosan quaternary ammonium salt hydrogel system with 0.5%, 1%, 1.5% content of phenylboronic acid modified chitosan quaternary ammonium salt is significantly accelerated in the presence of hydrogen peroxide, and ROS-responsive release performance can be achieved within 0-6 days. The FITC in polycaprolactone / bioglass can achieve long-acting slow release of FITC within 0-28 days. This indicates that the composite scaffold composed of chitosan quaternary ammonium salt hydrogel and polycaprolactone / bioglass scaffold can achieve ROS-responsive release in the short term, and slow release in the long term.

[0110] Example 4, Anti-ferroptosis drug Ferrostatin-1 rescues mesenchymal stem cells from ferroptosis caused by infection:

[0111] The rescue effect of Ferrostatin-1 on the death of mesenchymal stem cells under infection was detected by lipid peroxidation staining and live and dead staining methods. Bone marrow-derived mesenchymal stem cells (BMSCs) were used at 20x10 4Cells were seeded in 6-well plates at a density of 1 cell / well and allowed to adhere overnight. Cells were pre-treated with 10 mM Ferrostatin-1 for 12 h. After that, S. aureus (ATCC 25923) and E. coli (ATCC 25922) were used to co-culture with BMSCs at a multiplicity of infection (MOI) of 10 for 12 h. During the co-culture, the rescue group continued to be treated with 10 mM Ferrostatin-1. Cells were stained with C11-BODIPY and Liperfluo lipid peroxidation fluorescent probes, FerroOrange ferrous ion fluorescent probe, and imaged using a confocal microscope to evaluate the regulatory effect of Ferrostatin-1 on lipid peroxidation and ferrous ion accumulation in the infected microenvironment; or flow cytometry analysis was used to evaluate the rescue effect of Ferrostatin-1 on cell death in BMSCs in the infected microenvironment after co-culture. The results showed that treatment of cells with 10 mM Ferrostatin-1 can significantly reduce the level of lipid peroxidation in BMSCs infected with S. aureus and E. coli, inhibit the accumulation of ferrous ions in cells, and reduce cell death rate. From Figure 5 It can be seen that Ferrostatin-1 can be released in the composite scaffold to rescue mesenchymal stem cells from damage and ferroptosis in the infected microenvironment.

[0112] Example 5, Anti-ferroptosis drug Ferrostatin-1 promotes osteogenic differentiation of mesenchymal stem cells after infection

[0113] The regulatory effect of Ferrostatin-1 treatment on osteogenic differentiation of mesenchymal stem cells after infection was detected by alkaline phosphatase (ALP) staining and alizarin red (ARS) staining methods. Using the co-culture model as in Example 4, after the end of co-culture, the medium containing bacteria was aspirated, and washed twice with sterile PBS, and osteogenic induction medium containing 10 mM Ferrostatin-1 and 20 pg / ml gentamicin was added. ALP staining was performed 7 days after induction, and ARS staining was performed 14 days after induction. From Figure 6 It can be seen that treatment with 10 mM Ferrostatin-1 can promote ALP expression and mineralized nodule formation in mesenchymal stem cells, and promote osteogenic differentiation of mesenchymal stem cells.

[0114] Example 6, Antibacterial / anti-ferroptosis 3D-printed bone repair composite scaffold with active oxygen response in an infected bone defect model:

[0115] The antibacterial performance of the antibacterial / antiferruginous 3D-printed bone repair composite scaffold with active oxygen response was evaluated in the rat femoral condyle infectious bone defect model by small animal live body fluorescence imaging. A cylindrical bone defect with a diameter of 3 mm and a depth of 4 mm was made at the distal end of the rat femoral condyle using a trephine, 50 μL of 5×10 8 CFUs / mL bioluminescent Staphylococcus aureus (Xen 29) was dripped, and the skin and muscle were sutured after the scaffold was implanted. The bacterial fluorescence signal at the modeling site was measured using live imaging at 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks, and 12 weeks after modeling to evaluate the bacterial load. The bone volume / total volume (BV / TV) and trabecular number (Tb.N) of the femur were calculated from the micro-CT images. Figure 7 It can be seen that the bacterial fluorescence signal at the animal modeling site of the group without antibacterial components (Bonewax, PCL / MBG scaffold) can be detected continuously at 12 weeks after the operation, while the bacterial signal intensity at the animal modeling site of the group loaded with chitosan quaternary ammonium salt hydrogel (HACC-DP-PCL / MBG scaffold and Fer-1@HACC-DP-PCL / MBG scaffold) decreased significantly at 3 days after modeling and decreased to below the detection limit after one week, indicating that the scaffold loaded with chitosan quaternary ammonium salt hydrogel has a strong antibacterial effect and can effectively eliminate pathogenic bacteria at the infectious bone defect site.

[0116] Example 7: Antibacterial / antiferruginous bone repair composite scaffold with active oxygen response promotes the repair of infectious bone defects:

[0117] The bone repair effect of the antibacterial / antiferruginous bone repair composite scaffold in the rat femoral condyle infectious bone defect model as in Example 6 was evaluated by micro-CT bone measurement. At 6 weeks and 12 weeks after the operation, the modeling rats were euthanized, and the rat femur tissue was fixed using a paraformaldehyde solution. Then, the distal end of the rat femur was scanned using a micro-CT (Scanco Medical μCT80). The bone morphometric parameters such as BV / TV and Tb.N were calculated. From Figure 8 It can be seen that at the 6-week and 12-week time points, the femur of the animal in the group without antibacterial components (Bonewax, PCL / MBG scaffold) had severe osteolysis and pathological fractures, the femur of the animal in the group loaded with chitosan quaternary ammonium salt hydrogel (HACC-DP-PCL / MBG scaffold and Fer-1@HACC-DP-PCL / MBG scaffold) did not have pathological fractures, and new bone formation occurred at the bone defect site. However, the antibacterial / antiferruginous bone repair composite scaffold loaded with the antiferruginous drug Ferrostatin-1 had more bone mass at the femoral condyle defect site than the simple antibacterial scaffold group, and the BV / TV and Tb.N were significantly higher than those of the simple antibacterial scaffold group. This indicates that intervention in ferruginous death can promote post-infection osteogenesis and bone repair, and the antibacterial / antiferruginous scaffold can effectively promote the repair of infectious bone defects.

[0118] The above merely preferred embodiments of the present application, it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. A hydrogel material having a reactive oxygen species responsiveness, characterized by, The application relates to a hydrogel cross-linking system with a double cross-linking network structure, which comprises a benzene boronic acid modified chitosan quaternary ammonium salt HACC-PBA and a dithiothreitol modified polyethylene glycol (diol) diacrylate PEGDA-DTT as precursors, a borate ester bond formed between the HACC-PBA and the PEGDA-DTT molecules for covalent cross-linking, and a photo-cross-linking formed between the PEGDA-DTT molecules and unsaturated carbon-carbon double bonds; and an anti-ferroptosis drug loaded in the hydrogel cross-linking system. The chemical structure of the HACC-PBA is shown in formula (3): Formula (3); The chemical structure of the PEGDA-DTT is shown in formula (4): Formula (4); The active oxygen responsive hydrogel material is prepared by the following process: dissolving polyethylene glycol (diol) diacrylate PEGDA and dithiothreitol DTT in an aqueous solution, starting to stir at room temperature in the dark for 0.5-6 hours after sufficient dissolution; then adding benzene boronic acid modified chitosan quaternary ammonium salt HACC-PBA, stirring at room temperature for 0.5-6 hours after sufficient dissolution, to obtain a chitosan quaternary ammonium salt hydrogel cross-linking system; adding an anti-ferroptosis drug in the chitosan quaternary ammonium salt hydrogel cross-linking system, and stirring uniformly to obtain a chitosan quaternary ammonium salt hydrogel material loaded with the anti-ferroptosis drug.

2. The hydrogel material having a reactive oxygen species responsiveness according to claim 1, wherein, The anti-ferroptosis drug comprises one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine.

3. The hydrogel material having a reactive oxygen species responsiveness according to claim 1, wherein, The loading rate of the anti-ferroptosis drug in the hydrogel cross-linking system is 0.1%-10% in terms of mass fraction.

4. The hydrogel material having a reactive oxygen species responsiveness according to any one of claims 1 to 3, characterized in that, In the hydrogel material, the formed borate ester bond has active oxygen response breaking characteristics, and can release the chitosan quaternary ammonium salt with antibacterial activity and the anti-ferroptosis drug simultaneously in the presence of active oxygen ROC molecules.

5. A method for producing the hydrogel material having activity oxygen responsiveness according to any one of claims 1 to 4, characterized by, The method comprises the following steps: The benzene boronic acid modified chitosan quaternary ammonium salt is prepared by a normal-temperature solution method: dissolving chitosan quaternary ammonium salt in a water and organic solvent dissolving system, then adding benzene boronic acid substances, starting to stir at room temperature for 1-12 hours after sufficient dissolution, then adding a reduction protective agent sodium cyanoborohydride, stirring at room temperature for 3-96 hours after sufficient dissolution; after the reaction, the obtained solution is subjected to dialysis treatment, and the dialysis time is 6-72 hours; finally, vacuum freeze-drying is carried out to obtain the benzene boronic acid modified chitosan quaternary ammonium salt HACC-PBA; The chitosan quaternary ammonium salt hydrogel cross-linking system is prepared by a normal-temperature solution method: dissolving polyethylene glycol (diol) diacrylate PEGDA and dithiothreitol DTT in an aqueous solution, starting to stir at room temperature in the dark for 0.5-6 hours after sufficient dissolution; then adding benzene boronic acid modified chitosan quaternary ammonium salt HACC-PBA, stirring at room temperature for 0.5-6 hours after sufficient dissolution, to obtain a chitosan quaternary ammonium salt hydrogel cross-linking system; The chitosan quaternary ammonium salt hydrogel material loaded with the anti-ferroptosis drug is prepared by a hydrogel encapsulation method: adding an anti-ferroptosis drug in the chitosan quaternary ammonium salt hydrogel cross-linking system, and stirring uniformly to obtain a chitosan quaternary ammonium salt hydrogel material loaded with the anti-ferroptosis drug.

6. The preparation method according to claim 5, characterized in that, The quaternary ammonium salt of chitosan is hydroxypropyl trimethyl ammonium chloride chitosan HACC, the degree of substitution of hydroxypropyl trimethyl ammonium chloride of the quaternary ammonium salt of chitosan ranges from 5% to 60%; the phenyl boronic acid substance is selected from at least one of 4-formyl phenyl boronic acid and 3-amino phenyl boronic acid hydrochloride.

7. The preparation method according to claim 5, characterized in that, The average molecular weight of the polyethylene glycol (diol) diacrylate PEGDA ranges from 250 to 10,000, the volume percentage ranges from 5% to 20%; the final concentration of the dithiothreitol DTT ranges from 1 to 100 mg / ml; the mass concentration of the phenyl boronic acid modified quaternary ammonium salt of chitosan HACC-PBA ranges from 1 to 15 mg / ml.

8. The preparation method according to claim 5, characterized in that, The anti-ferroptosis drug includes one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, and the mass concentration of the anti-ferroptosis drug ranges from 1 to 100 mg / ml.

9. Use of the hydrogel material with active oxygen responsiveness according to any one of claims 1-4 or the hydrogel material with active oxygen responsiveness prepared by the preparation method according to any one of claims 5-8 in the preparation of a bone repair material.

10. Use according to claim 9, characterized in that, The hydrogel material with active oxygen responsiveness is used for coating of an implant of a bone repair material.

11. Use of the hydrogel material with active oxygen responsiveness according to any one of claims 1-4 or the hydrogel material with active oxygen responsiveness prepared by the preparation method according to any one of claims 5-8 for filling a tissue engineering porous scaffold material.

12. An antibacterial / anti-ferrideath bone repair composite scaffold with active oxygen responsiveness, characterized in that, The tissue engineering porous scaffold material is a 3D printed polycaprolactone / bioglass porous scaffold material loaded with an anti-ferroptosis drug.

13. The antibacterial / anti-ferrideath bone repair composite scaffold with active oxygen responsiveness according to claim 12, characterized in that, The anti-ferroptosis drug includes one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine.

14. The antibacterial / anti-ferrideath bone repair composite scaffold with active oxygen responsiveness according to claim 13, characterized in that, The filling rate of the hydrogel material with active oxygen responsiveness in the 3D printed polycaprolactone / bioglass porous scaffold material ranges from 20% to 300% in mass fraction.

15. A method for preparing the antibacterial / anti-ferrideath bone repair composite scaffold with active oxygen responsiveness according to any one of claims 12-14, characterized in that, The method comprises the following steps: Preparation of a polycaprolactone / bioglass porous scaffold material loaded with an anti-ferroptosis drug by 3D printing; Preparation of an antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness by compounding the chitosan quaternary ammonium salt hydrogel material loaded with an anti-ferroptosis drug and the polycaprolactone / bioglass porous scaffold material loaded with an anti-ferroptosis drug by a photo-crosslinking method. The quaternary ammonium salt of chitosan is hydroxypropyl trimethyl ammonium chloride chitosan HACC, the degree of substitution of hydroxypropyl trimethyl ammonium chloride of the quaternary ammonium salt of chitosan ranges from 5% to 60%; the phenyl boronic acid substance is selected from at least one of 4-formyl phenyl boronic acid and 3-amino phenyl boronic acid hydrochloride. The average molecular weight of the polyethylene glycol (diol) diacrylate PEGDA ranges from 250 to 10,000, the volume percentage ranges from 5% to 20%; the final concentration of the dithiothreitol DTT ranges from 1 to 100 mg / ml; the mass concentration of the phenyl boronic acid modified quaternary ammonium salt of chitosan HACC-PBA ranges from 1 to 15 mg / ml. The anti-ferroptosis drug includes one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, and the mass concentration of the anti-ferroptosis drug ranges from 1 to 100 mg / ml.

9. Use of the hydrogel material with active oxygen responsiveness according to any one of claims 1-4 or the hydrogel material with active oxygen responsiveness prepared by the preparation method according to any one of claims 5-8 in the preparation of a bone repair material. The hydrogel material with active oxygen responsiveness is used for coating of an implant of a bone repair material.

11. Use of the hydrogel material with active oxygen responsiveness according to any one of claims 1-4 or the hydrogel material with active oxygen responsiveness prepared by the preparation method according to any one of claims 5-8 for filling a tissue engineering porous scaffold material. The tissue engineering porous scaffold material is a 3D printed polycaprolactone / bioglass porous scaffold material loaded with an anti-ferroptosis drug. The anti-ferroptosis drug includes one or more of Ferrostatin-1, Liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine. The filling rate of the hydrogel material with active oxygen responsiveness in the 3D printed polycaprolactone / bioglass porous scaffold material ranges from 20% to 300% in mass fraction. The method comprises the following steps: Preparation of a polycaprolactone / bioglass porous scaffold material loaded with an anti-ferroptosis drug by 3D printing; Preparation of an antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness by compounding the chitosan quaternary ammonium salt hydrogel material loaded with an anti-ferroptosis drug and the polycaprolactone / bioglass porous scaffold material loaded with an anti-ferroptosis drug by a photo-crosslinking method.

16. The method of claim 15, wherein, The steps of preparing the poly-caprolactone / bio-glass porous scaffold material loaded with anti-ferroptosis drugs by 3D printing include: dispersing the anti-ferroptosis drugs in the aqueous solution of mesoporous bio-glass for co-incubation, shaking for 6-8 hours on a shaking table, then centrifuging to collect the bio-glass loaded with the anti-ferroptosis drugs, and vacuum freeze-drying to obtain dried bio-glass loaded with the anti-ferroptosis drugs; then dissolving the poly-caprolactone in an organic solvent, adding the bio-glass powder loaded with the anti-ferroptosis drugs and mixing uniformly to obtain the poly-caprolactone / bio-glass raw material loaded with the anti-ferroptosis drugs; further adding the mixture raw material into a 3D printing cartridge, using a 3D printer to perform 3D printing according to the designed size information and pore size, and preparing the poly-caprolactone / bio-glass porous scaffold loaded with the anti-ferroptosis drugs after complete drying.

17. The preparation method according to claim 15, characterized in that, The steps of compounding the hydrogel material with active oxygen responsiveness and the poly-caprolactone / bio-glass porous scaffold material loaded with the anti-ferroptosis drugs by a photo-crosslinking method include: adding a photo-initiator in the uncrosslinked chitosan quaternary ammonium salt hydrogel material loaded with the anti-ferroptosis drugs to form a hydrogel system, and then filling the poly-caprolactone / bio-glass porous scaffold loaded with the anti-ferroptosis drugs by a hydrogel filling method.

18. The method of claim 17, wherein, The hydrogel system is subjected to in-situ photo-crosslinking reaction on the surface of the poly-caprolactone / bio-glass porous scaffold loaded with the anti-ferroptosis drugs by photo-excitation irradiation; the photo-initiator is at least one selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), the mass fraction of the photo-initiator is 0.1%-1%, and the photo-excitation irradiation time is 5-60s.

19. Use of the antibacterial / anti-ferroptosis bone repair composite scaffold with active oxygen responsiveness according to any one of claims 12-14 or prepared by the preparation method according to any one of claims 15-18 in the preparation of an infectious bone defect treatment material.

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