CN117379597A - Hydrogel material with reactive oxygen species, antibacterial/anti-iron-death bone repair composite scaffold, preparation method and application thereof - Google Patents
Hydrogel material with reactive oxygen species, antibacterial/anti-iron-death bone repair composite scaffold, preparation method and application thereof Download PDFInfo
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- CN117379597A CN117379597A CN202311455023.7A CN202311455023A CN117379597A CN 117379597 A CN117379597 A CN 117379597A CN 202311455023 A CN202311455023 A CN 202311455023A CN 117379597 A CN117379597 A CN 117379597A
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- Prior art keywords
- hydrogel
- iron
- death
- quaternary ammonium
- ammonium salt
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Abstract
The invention belongs to the technical field of hydrogels, in particular to a hydrogel material with reactive oxygen and a preparation method thereof, and also relates to an antibacterial/anti-iron death bone repair composite stent comprising a tissue engineering porous stent material and the hydrogel material with reactive oxygen filled in the tissue engineering porous stent material, and in particular relates to an antibacterial/anti-iron death bone repair composite stent comprising a polycaprolactone/bioglass porous stent material with 3D printing and the hydrogel material with reactive oxygen filled in the porous stent material and a preparation method thereof; also relates to the application of the antibacterial/anti-iron death bone repair composite scaffold with active oxygen responsiveness in the treatment of infectious bone defect.
Description
Technical Field
The invention belongs to the technical field of hydrogels, in particular to a hydrogel material with reactive oxygen and a preparation method thereof, and also relates to an antibacterial/anti-iron death bone repair composite stent comprising a tissue engineering porous stent material and the hydrogel material with reactive oxygen filled in the tissue engineering porous stent material, and in particular relates to an antibacterial/anti-iron death bone repair composite stent comprising a polycaprolactone/bioglass porous stent material with 3D printing and the hydrogel material with reactive oxygen filled in the porous stent material and a preparation method thereof; also relates to the application of the antibacterial/anti-iron death bone repair composite scaffold with active oxygen responsiveness in the treatment of infectious bone defect.
Background
In orthopedic clinical treatment, infectious bone defects caused by bone and implant infections are one of the most challenging and persistent complications of orthopedic clinics. Infectious bone defects are mainly caused by open wounds combined with wound pollution, and are partially caused by perioperative infection of an intraosseous plant implantation operation, hematogenous osteomyelitis and other reasons. Bacteria and metabolites of the bacteria, which are implanted in bone tissues and bone implants, can cause local inflammatory reactions and tissue cell necrosis, destroy bone regeneration microenvironment and seriously affect fracture or bone defect healing. At present, the main treatment method for clinically treating infectious bone defects is to implant an antibiotic bone cement bead chain after thorough debridement, and then perform a secondary bone grafting operation after waiting for thorough control of infection. However, the problems of low healing capacity of bone regeneration of infected tissues, bacterial antibiotic resistance and the like exist, and serious physical and psychological and economic burdens are caused on patients. In addition to the removal of pathogenic bacteria from the foci of infection, how to rescue tissue cell damage caused by infection is also a key issue in the treatment of infectious bone defects. In the infected microenvironment, the secretion and the metabolite of pathogenic bacteria can induce the damage and death of the mesenchymal stem cells, inhibit the normal osteogenic differentiation and bone regeneration process of the mesenchymal stem cells, and finally lead to the failure of bone repair. It is desirable to promote repair of infected bone defects by rescuing bone marrow mesenchymal stem cell injury death and osteogenic differentiation disorders in the infected microenvironment.
Chinese literature (research on 3D printable high-strength and toughness dual-network polyethylene glycol diacrylate/chitosan hydrogel and bone property promotion thereof) (Deng Ziwei, university of North China university of technology, university of North China, university of great university of China, 2021.4) discloses a dual-network mechanism enhanced polyethylene glycol diacrylate (PEGDA)/Chitosan (CS) based composite hydrogel, chitosan is soaked in a citrate salt ion solution, and N-glucosamine units on the chitosan can be mixed with Cit in the citrate salt ion solution 3- And (3) forming an ionic bond, adding a small molecular weight PEGDA into the chitosan system, and activating double bonds at two ends of the PEGDA under ultraviolet stimulation to form covalent crosslinking points to obtain a covalent network, thereby forming the chitosan/PEGDA hydrogel of the synthetic ion-covalent double network. According to the technology, the formed chitosan/PEGDA hydrogel is directly subjected to 3D printing, the formed 3D printing support is still weak in mechanical strength and low in mechanical strength, the extruded material collapses on a platform when the formed chitosan/PEGDA hydrogel is directly subjected to 3D printing, and the aim of regenerating and repairing the bearing tissues can not be achieved even if the formed chitosan/PEDGA composite hydrogel is directly subjected to 3D printing.
As a new additive manufacturing technology that can be customized, 3D printing technology has been attracting attention in bone defect repair research in recent years. By using the 3D printing technology, the bone repair stent with the matched anatomical structure and controllable internal porosity and void structure can be prepared.
However, the existing 3D printing bracket for anti-infection bone repair lacks the function of rescuing tissue cell injury, and is difficult to rescue bone mesenchymal stem cell injury and osteogenic differentiation disorder caused by pathogenic bacteria of an infection focus, so that the bone repair of an infection part is slow or does not heal. An important reason for the lack of anti-infective bone repair scaffolds to rescue tissue damage is that specific ways and mechanisms of cell damage caused by infection are unknown, and targeted intervention on death of mesenchymal stem cell damage in the infected microenvironment is difficult. Moreover, the existing anti-infection bone repair 3D printing bracket can not intelligently adjust the release rate of antibacterial and bioactive components according to the pathogenic bacteria load and the inflammation degree of an infection focus, and the material is difficult to dynamically adapt to the change of the infection course. An important reason for the poor biocompatibility of materials is the lack of ability of the materials to respond to release of antimicrobial and bioactive components.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a hydrogel material having active oxygen responsiveness, and also to provide an antibacterial/anti-iron-death bone repair composite scaffold comprising a tissue engineering porous scaffold material and the hydrogel material having active oxygen responsiveness filled in the tissue engineering porous scaffold material, and an application in the treatment of infectious bone defect. Firstly, preparing ROS response hydrogel with antibacterial/anti-iron death activity by loading a certain amount of anti-iron death drugs into ROS response hydrogel based on chitosan quaternary ammonium salt; meanwhile, mesoporous bioglass loaded with an anti-iron death drug is mixed with polycaprolactone, a porous scaffold is prepared through a 3D printing technology, and finally, after the uncrosslinked antibacterial/anti-iron death hydrogel is assembled with the 3D printing porous scaffold, in-situ photocrosslinking is carried out, so that the required ROS-responded antibacterial/anti-iron death bone repair composite scaffold is prepared.
In order to achieve the above object, the solution adopted by the present invention is as follows:
in a first aspect, the invention provides a hydrogel material with reactive oxygen species, comprising a double-crosslinked hydrogel crosslinking system which is formed by taking phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (glycol) diacrylate (PEGDA-DTT) as precursors, and forming a boric acid ester bond between HACC-PBA and PEGDA-DTT molecules to form covalent crosslinking and PEGDA-DTT molecule unsaturated carbon-carbon double bond photocrosslinking.
Preferably, the hydrogel material having active oxygen responsiveness further comprises a drug loaded in the hydrogel cross-linking system.
Preferably, the medicament is an anti-iron death medicament, preferably, the anti-iron death medicament comprises one or more of Ferrostatin-1, liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine.
Preferably, the loading rate of the anti-iron death medicine in the hydrogel crosslinking system is 0.1% -10% (mass fraction).
Preferably, in the hydrogel material, the formed borate ester bond has an active oxygen response cleavage property, and chitosan quaternary ammonium salt having antibacterial activity and an anti-iron death drug can be simultaneously released in the presence of active oxygen (ROC) molecules.
In a second aspect, the present invention also provides a method for preparing a hydrogel material having active oxygen responsiveness as described above, comprising the steps of:
preparing phenylboronic acid modified chitosan quaternary ammonium salt by a normal temperature solution method;
preparing a chitosan quaternary ammonium salt hydrogel crosslinking system by a normal temperature solution method;
the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death medicine is prepared by a hydrogel encapsulation method.
Preferably, the preparation method of the phenylboronic acid modified chitosan quaternary ammonium salt by the normal temperature solution method comprises the following steps: dissolving chitosan quaternary ammonium salt in a water and organic solvent dissolving system, adding phenylboronic acid substances, fully dissolving, starting stirring at normal temperature to react for 1-12h, adding a reducing protective agent sodium cyanoborohydride, fully dissolving, and stirring at normal temperature to react for 3-96h; dialyzing the obtained solution after the reaction for 6-72h; finally, the chitosan quaternary ammonium salt (HACC-PBA) modified by phenylboronic acid is obtained after vacuum freeze drying.
Preferably, the chitosan quaternary ammonium salt is hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and the substitution degree of the hydroxypropyl trimethyl ammonium chloride of the chitosan quaternary ammonium salt ranges from 5% to 60%; the phenylboronic acid material is at least one selected from 4-formylphenylboronic acid and 3-aminophenylborate.
Preferably, the step of preparing the chitosan quaternary ammonium salt hydrogel crosslinking system by a normal temperature solution method comprises the following steps: dissolving polyethylene glycol (glycol) diacrylate (PEGDA) and Dithiothreitol (DTT) in an aqueous solution, and stirring for reaction at normal temperature under dark condition for 0.5-6h after the polyethylene glycol (glycol) diacrylate and the Dithiothreitol (DTT) are fully dissolved; then adding phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA), fully dissolving, and stirring at normal temperature for reacting for 0.5-6h to obtain the chitosan quaternary ammonium salt hydrogel crosslinking system.
Preferably, the average molecular weight of the polyethylene glycol (glycol) diacrylate (PEGDA) is 250-10000, and the volume percentage is 5% -20%; the final concentration of Dithiothreitol (DTT) is 1-100mg/ml; the mass concentration of the phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) is 1-15mg/ml.
Preferably, the preparation method of the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death drug by the hydrogel encapsulation method comprises the following steps: adding the anti-iron death medicine into the chitosan quaternary ammonium salt hydrogel crosslinking system, and fully and uniformly stirring to obtain the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death medicine.
Preferably, the anti-iron death medicine comprises one or more of Ferrosistatin-1, liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, and the mass concentration of the anti-iron death medicine is 1-100mg/ml.
In a third aspect, the invention also provides an application of the hydrogel material with active oxygen responsiveness and the hydrogel material with active oxygen responsiveness prepared by the preparation method in preparation of bone repair materials, tissue engineering porous scaffold materials or 3D printing biological ink.
Preferably, the active oxygen responsive hydrogel material is used for coating of an implant of a bone repair material.
Preferably, the hydrogel material with active oxygen responsiveness is used for filling a tissue engineering porous scaffold material.
In a fourth aspect, the present invention also provides an antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness, comprising a tissue engineering porous scaffold material and the hydrogel material with active oxygen responsiveness as described above filled in the tissue engineering porous scaffold material.
Preferably, the tissue engineering porous scaffold material is a 3D printed polycaprolactone/bioglass porous scaffold material.
Preferably, the 3D printed polycaprolactone/bioglass porous scaffold material is loaded with an anti-iron death drug, wherein the anti-iron 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-iron death drug in the 3D printed polycaprolactone/bioglass porous scaffold material is 0.5% -10% (mass fraction).
Preferably, the filling rate of the hydrogel material with active oxygen responsiveness in the 3D printing polycaprolactone/bioglass porous scaffold material is 20% -300% (mass fraction).
In a fifth aspect, the present invention also provides a method for preparing the antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness as described above, comprising the steps of:
preparing a polycaprolactone/bioglass porous scaffold material loaded with an anti-iron death drug in a 3D printing mode;
and compounding the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death medicament with the polycaprolactone/bioglass porous scaffold material loaded with the anti-iron death medicament by a photo-crosslinking method to prepare the antibacterial/anti-iron death bone repair composite scaffold with active oxygen responsiveness.
Preferably, the step of preparing the polycaprolactone/bioglass porous scaffold material loaded with the anti-iron death drug by means of 3D printing comprises: dispersing the anti-iron death medicine in the water solution of the mesoporous bioglass for co-incubation, shaking and adsorbing for 6-8 hours on a shaking table, and then centrifugally collecting the bioglass adsorbed with the anti-iron death medicine, and obtaining the dried bioglass loaded with the anti-iron death medicine after vacuum freeze drying; then fully dissolving polycaprolactone in an organic solvent, adding bioglass powder loaded with an anti-iron death medicament, and uniformly mixing to obtain a polycaprolactone/bioglass raw material loaded with the anti-iron death medicament; and further adding the mixture raw materials into a 3D printing charging barrel, performing 3D printing according to the designed size information and the pore size by using a 3D printer, and completely drying to prepare the polycaprolactone/bioglass porous support loaded with the anti-iron death drug.
Preferably, the 3D printing method includes a normal temperature/low temperature printing method or a high temperature printing method.
Preferably, the step of compounding the active oxygen responsive hydrogel material with the anti-iron death drug loaded polycaprolactone/bioglass porous scaffold material by photo cross-linking method comprises: adding a photoinitiator into an uncrosslinked chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death drug to form a hydrogel system, and filling and wrapping the polycaprolactone/bioglass porous scaffold loaded with the anti-iron death drug by adopting a hydrogel filling method.
Preferably, the hydrogel filling method includes at least one of a tank drop method, a negative pressure suction method, and a soaking method.
Preferably, the hydrogel system is subjected to in-situ photocrosslinking reaction on the surface of the polycaprolactone/bioglass porous support loaded with the anti-iron death medicament by adopting light excitation irradiation; the photoinitiator is at least one selected from phenyl-2, 4, 6-trimethylbenzoyl lithium hypophosphite (LAP) and 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone (Irgacure 2959), the mass fraction of the photoinitiator is 0.1% -1%, and the light excitation irradiation time is 5-60s.
In a sixth aspect, the present invention also provides an antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness as described above and an application of the antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness prepared by the preparation method as described above in the treatment of infectious bone defect.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a hydrogel material with active oxygen responsiveness, an antibacterial/anti-iron death bone repair composite scaffold with active oxygen responsiveness and application thereof in treatment of infectious bone defects, and the prepared active oxygen responsiveness antibacterial/anti-iron death bone repair composite scaffold has intelligent response and antibacterial characteristics, has effects of saving mesenchymal stem cell iron death and promoting mesenchymal stem cell osteogenic differentiation, has an effect of obviously promoting healing of infectious bone defects, and has very important research and clinical application values.
Drawings
Fig. 1 is a schematic view of the formation of a hydrogel material having active oxygen responsiveness according to the present invention.
FIG. 2 is a schematic photograph of an antibacterial/anti-iron-death bone repair composite scaffold with active oxygen response in example 1 of the present invention; in the figure, (a) is a polycaprolactone/mesoporous bioglass scaffold, and (b) is an antibacterial/anti-iron death bone repair composite scaffold with active oxygen response.
FIG. 3 is a graph showing the antimicrobial properties of the antimicrobial/anti-iron-death bone repair composite scaffold with active oxygen response according to example 2 of the present invention; in the figure, (a) is a photograph of an antibacterial coated plate of a hydrogel composite stent loaded with different HACC-PBA contents, and (b) is a statistical graph of antibacterial efficacy.
FIG. 4 is a graph showing the release properties of chitosan quaternary ammonium salt hydrogel and polycaprolactone/bioglass porous scaffolds of example 3 of the present invention; in the figure, (a) is FITC release curve of HACC-DP hydrogel with different HACC-PBA content in simulated body fluid and hydrogen peroxide-containing simulated body fluid, and (b) is FITC release curve of FITC-loaded PCL/MBG bracket in simulated body fluid and hydrogen peroxide-containing simulated body fluid.
FIG. 5 is a photograph showing the death of mesenchymal stem cell iron caused by infection rescue by Ferrostatin-1 in example 4 of the present invention; in the figure, (a) is a fluorescent photograph of a rescue effect of escherichia coli and staphylococcus aureus induced mesenchymal stem cell lipid peroxidation and iron overload effect and an iron death inhibitor Ferrostatin-1, (b) is a flow cytometry live-dead staining analysis chart of escherichia coli and staphylococcus aureus induced mesenchymal stem cell death, and (c) is a quantitative analysis of a ratio of flow cytometry live-dead staining dead cells.
FIG. 6 is a photograph showing that Ferrositin-1 promotes osteoblast differentiation of mesenchymal stem cells infected with bacteria in example 5 of the present invention; in the figure, (a) is alkaline phosphatase staining and (b) is alizarin red S staining.
FIG. 7 is a graph showing the in vivo antibacterial effect of the composite scaffolds for antibacterial/anti-iron-death bone repair with an active oxygen response according to example 6 of the present invention; in the figure, (a) is in vivo bacterial in-vivo fluorescence detection of a femoral condyle infectious bone defect model of a rat, and (b) is in vivo bacterial in-vivo fluorescence signal quantitative analysis.
FIG. 8 is a photograph showing the promotion of infectious bone loss repair by an antibacterial/anti-iron death bone repair composite scaffold having an active oxygen response in example 7 of the present invention; in the figure, (a) is micro-CT analysis of a rat femoral condyle infectious bone defect model, and (b) is quantitative analysis of micro-CT bone mass and bone trabecular number.
Detailed Description
The invention provides a hydrogel material with active oxygen responsiveness and capable of delivering and continuously releasing therapeutic drugs, which comprises a three-dimensional network structure hydrogel crosslinking system and drugs loaded in the hydrogel crosslinking system.
< double crosslinked hydrogel System >
As one embodiment of the invention, the hydrogel crosslinking system is a double-crosslinked hydrogel crosslinking system which takes phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (glycol) diacrylate (PEGDA-DTT) as precursors, and forms a boric acid ester bond covalent crosslinking between HACC-PBA and PEGDA-DTT molecules and a PEGDA-DTT molecule unsaturated carbon-carbon double bond photocrosslinking.
Chitosan is an N-deacetylated product of chitin, and has a structural formula shown in the following formula (1). Chitosan is the only basic aminopolysaccharide existing in a large amount in natural saccharides, has many special physical and chemical properties and physiological functions, such as good adsorptivity, film forming property, fiber forming property, moisture absorption and moisture retention property, and has very good biocompatibility and degradation property, but the application of chitosan is limited to a certain extent due to the influence of the solubility of chitosan.
The quaternization modification of chitosan is one of methods for modifying chitosan, and is a chitosan derivative obtained by introducing a quaternary ammonium group into amino groups of chitosan or connecting a low-molecular quaternary ammonium salt to the amino groups. The quaternized chitosan has better water solubility than the chitosan and the chitin, so that the quaternized chitosan can better exert the effect of the chitosan. The modified chitosan quaternary ammonium salt not only has the typical properties of quaternary ammonium salt, such as antibacterial and bacteriostatic properties and moisture absorption and retention properties, but also keeps the original good properties of film forming property, flocculation property, biocompatibility, biodegradation and the like of chitosan.
The chitosan quaternary ammonium salt is taken as a commercial antibacterial agent, and is a natural polysaccharide derivative with good biocompatibility and antibacterial activity. Depending on the quaternization modification method of chitosan, common chitosan quaternary ammonium salts include, but are not limited to, N-trimethyl chitosan quaternary ammonium salt, N-methyl-N, N-double long chain alkylated chitosan quaternary ammonium salt, O-acrylamide-N-chitosan quaternary ammonium salt (NMA-HTCC), hydroxypropyl trimethyl chitosan quaternary ammonium salt (HACC), and the like. According to the reported antibacterial property experiments, quaternary ammonium groups with positive electricity in quaternized chitosan molecules can obviously promote the mutual combination between chitosan molecules and cell wall anions of thalli, so that the thalli die. Meanwhile, the chitosan quaternary ammonium salt has no obvious toxicity to the bone marrow mesenchymal stem cells within a certain substitution degree range (1% -60%), can promote the bone marrow mesenchymal stem cells to differentiate into bone, simultaneously plays a good antibacterial effect, and inhibits the formation of biological membranes.
Among them, hydroxypropyl trimethyl chitosan quaternary ammonium salt (HACC) is the most excellent in bacteriostatic activity among these chitosan quaternary ammonium salts, and its bacteriostatic activity is enhanced with the increase of positive charges. 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 covalent cross-linked high molecular polymer in the hydrogel material.
As a preferred embodiment of the present invention, the hydroxypropyl trimethyl ammonium chloride chitosan (HACC) used in the present invention is also modified with phenylboronic acid, and the chemical structural formula of the phenyl boronic acid modified hydroxypropyl trimethyl ammonium chloride chitosan (HACC-PBA) is shown in the following formula (3).
The hydrogel crosslinking system provided by the invention is a double-crosslinked-network-structure hydrogel crosslinking system, the phenyl boric acid-modified hydroxypropyl trimethyl ammonium chloride chitosan (HACC-PBA) is a precursor of one of the double crosslinked networks, and the matrix of the other high polymer crosslinked network is dithiothreitol-modified polyethylene glycol (diol) diacrylate (PEGDA-DTT) shown in the following formula (4). Polyethylene glycol (glycol) diacrylate (PEGDA) is a polymer material with good biocompatibility and degradability, can form a hydrogel crosslinked network under the excitation of a photoinitiator and blue light, can be used as a good drug loading and releasing system, and is widely studied by photo-crosslinking water-carrying hydrogel systems based on PEGDA at present.
In the hydrogel material provided by the invention, phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (glycol) diacrylate (PEGDA-DTT) are used as precursors to form a hydrogel crosslinking system with a double-crosslinking network structure, a boric acid ester bond is formed between the HACC-PBA and the PEGDA-DTT molecules to carry out covalent crosslinking, the two networks are mutually connected through the boric acid ester bond (shown in figure 1), the formed boric acid ester bond is a chemical group with Reactive Oxygen (ROS) response activity and has ROS response fracture characteristics, and meanwhile, the boric acid ester bond can also increase the adsorption of a medicament loaded in the hydrogel material with the double-crosslinking network structure hydrogel crosslinking system so as to increase the loading rate of the medicament, and the boric acid ester bond can also play a role of delivering the medicament through connecting a target molecule and continuously releasing the medicament in a Reactive Oxygen (ROS) response.
< drug loaded in hydrogel Cross-Linked System >
As one embodiment of the invention, in the hydrogel material, the drug loaded in a uniformly dispersed form in a double-crosslinked hydrogel crosslinking system formed by phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (glycol) diacrylate (PEGDA-DTT) serving as precursors is an anti-iron death drug. Iron death is a novel programmed mode of cell death characterized by intracellular iron overload and lipid peroxidation accumulation, and the role of iron death in infection-related cell injury and death is of increasing concern. It has been reported in the literature that inhibiting iron death can alleviate the extent of damage to infected cells and accelerate pathogen clearance.
The anti-iron death medicine selected by the invention is one or more selected from Ferrosistatin-1, liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, wherein the loading rate of the anti-iron death medicine in the hydrogel crosslinking system is 0.1-10% (mass fraction). In the embodiment of the invention, the Ferrostatin-1 is taken as an example for explaining the technical scheme of the invention, but the Ferrostatin-1 is not limited to the explanation.
< hydrogel Material >
The hydrogel material provided by the invention comprises the double-crosslinked hydrogel crosslinking system and the anti-iron death drug loaded in the hydrogel crosslinking system.
In the hydrogel material provided by the invention, phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (glycol) diacrylate (PEGDA-DTT) are used as precursors to form a hydrogel crosslinking system with a double-crosslinking network structure, boric acid ester bonds are formed between HACC-PBA and PEGDA-DTT molecules to carry out covalent crosslinking, the two networks are mutually connected through the boric acid ester bonds (shown in figure 1), the formed boric acid ester bonds are chemical groups with Reactive Oxygen (ROS) response activity and have ROS response fracture characteristics, and simultaneously, the boric acid ester bonds can also increase the adsorption of an anti-iron death drug loaded in the hydrogel material with the double-crosslinking network structure, so that the loading rate of the anti-iron death drug is increased, and the boric acid ester bonds can also play the roles of delivering the anti-iron death drug through connecting target molecules and continuously releasing the drug with Reactive Oxygen (ROS) response.
In the hydrogel material provided by the invention, the characteristic of reactive oxygen breakage of the borate ester bond can enable the hydrogel material to simultaneously release chitosan quaternary ammonium salt with antibacterial activity and an anti-iron death drug in the presence of active oxygen molecules. Therefore, the chitosan quaternary ammonium salt hydrogel can flexibly adjust the release rate of the antibacterial and antibody death molecules according to the inflammation degree of the infection part, and has better biocompatibility.
< preparation of hydrogel Material >
The invention also provides a preparation method of the hydrogel material with active oxygen responsiveness, which comprises the following steps:
step (1), preparing phenylboronic acid modified chitosan quaternary ammonium salt by a normal temperature solution method:
dissolving chitosan quaternary ammonium salt in a water and organic solvent dissolving system, adding phenylboronic acid substances, fully dissolving, starting stirring at normal temperature to react for 1-12h (4-12 h, 6-12h, 4-10h and 6-10h, for example), adding a reducing protective agent sodium cyanoborohydride, fully dissolving, and stirring at normal temperature to react for 3-96h (3-80 h, 3-60h, 3-40h, 3-20h, 3-10h, 15-80h, 15-60h, 15-40h and 15-20h, for example); after the reaction, the obtained solution is subjected to dialysis treatment for 6-72 hours (which may be, for example, 6-60 hours, 6-40 hours, 6-20 hours, 6-10 hours, 15-60 hours, 15-40 hours, 15-20 hours); finally, the chitosan quaternary ammonium salt (HACC-PBA) modified by phenylboronic acid is obtained after vacuum freeze drying.
In step (1), the chitosan quaternary ammonium salt is hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and 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, from 5% to 50%, from 5% to 40%, from 5% to 30%, from 5% to 20%, from 5% to 10%, from 10% to 60%, from 10% to 50%, from 10% to 40%, from 10% to 30%, from 10% to 20%, from 15% to 60%, from 15% to 50%, from 15% to 40%, from 15% to 30%, from 20% to 60%, from 20% to 40%, from 20% to 30%, from 30% to 60%, from 30% to 50%, from 40% to 50%; the phenylboronic acid material is at least one selected from 4-formylphenylboronic acid and 3-aminophenylborate.
Step (2), preparing a chitosan quaternary ammonium salt hydrogel crosslinking system by a normal temperature solution method:
dissolving polyethylene glycol (glycol) diacrylate (PEGDA) shown in the following formula (5) and Dithiothreitol (DTT) shown in the following formula (6) in an aqueous solution, and stirring and reacting for 0.5-6 hours at normal temperature in a dark place after the polyethylene glycol (glycol) diacrylate and the Dithiothreitol (DTT) are fully dissolved; then adding phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA), fully dissolving, and stirring at normal temperature for reacting for 0.5-6h to obtain the chitosan quaternary ammonium salt hydrogel crosslinking system.
In step (2), the polyethylene glycol (diol) diacrylate (PEGDA) has an average molecular weight of 250-10000, a volume percentage of 5-20% (which may for example be 5-15%, 5-10%, 8-20%, 8-15%, 8-10%, 10-20%, 10-15%, 15-20%); the final concentration of Dithiothreitol (DTT) is 1-100mg/ml (which may be, for example, 10-100mg/ml, 10-90mg/ml, 10-80mg/ml, 10-70mg/ml, 10-60mg/ml, 10-50mg/ml, 10-40mg/ml, 10-30mg/ml, 10-20mg/ml, 20-100mg/ml, 20-90mg/ml, 20-80mg/ml, 20-70mg/ml, 20-60mg/ml, 20-50mg/ml, 20-40mg/ml, 20-30mg/ml, 30-100mg/ml, 30-90mg/ml, 30-80mg/ml, 30-70 mg/ml) 30-60mg/ml, 30-50mg/ml, 30-40mg/ml, 40-100mg/ml, 40-90mg/ml, 40-80mg/ml, 40-70mg/ml, 40-60mg/ml, 40-50mg/ml, 50-100mg/ml, 50-90mg/ml, 50-80mg/ml, 50-70mg/ml, 50-60mg/ml, 60-100mg/ml, 60-90mg/ml, 60-80mg/ml, 60-70mg/ml, 70-100mg/ml, 70-90mg/ml, 70-80mg/ml, 80-100mg/ml, 80-90mg/ml, 90-100 mg/ml; the mass concentration of the phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) is 1-15mg/ml (which can be 1-12mg/ml,1-10mg/ml,1-8mg/ml,1-6mg/ml,1-4mg/ml,3-15mg/ml,3-12mg/ml,3-10mg/ml,3-8mg/ml,3-6mg/ml,5-15mg/ml,5-12mg/ml,5-10mg/ml,5-8mg/ml,7-15mg/ml,7-12mg/ml,7-10mg/ml,9-15mg/ml,9-12mg/ml,11-15mg/ml,11-13mg/ml and 13-15 mg/ml).
Step (3), preparing the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death drug by a hydrogel encapsulation method:
adding the anti-iron death medicine into the chitosan quaternary ammonium salt hydrogel crosslinking system, and fully and uniformly stirring to obtain the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death medicine.
In the step (3), the anti-iron death medicine comprises one or more of Ferrostatin-1, liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, the mass concentration of the anti-iron death medicine is 1-100mg/ml (can be, for example, 10-100mg/ml, 10-90mg/ml, 10-80mg/ml, 10-70mg/ml, 10-60mg/ml, 10-50mg/ml, 10-40mg/ml, 10-30mg/ml, 10-20mg/ml, 20-100mg/ml, 20-90mg/ml, 20-80mg/ml, 20-70mg/ml, 20-60mg/ml, 20-50mg/ml, 20-40mg/ml, 20-30mg/ml, 30-100mg/ml, 30-90mg/ml 30-80mg/ml, 30-70mg/ml, 30-60mg/ml, 30-50mg/ml, 30-40mg/ml, 40-100mg/ml, 40-90mg/ml, 40-80mg/ml, 40-70mg/ml, 40-60mg/ml, 40-50mg/ml, 50-100mg/ml, 50-90mg/ml, 50-80mg/ml, 50-70mg/ml, 50-60mg/ml, 60-100mg/ml, 60-90mg/ml, 60-80mg/ml, 60-70mg/ml, 70-100mg/ml, 70-90mg/ml, 70-80mg/ml, 80-100mg/ml, 80-90mg/ml, 90-100 mg/ml).
< use of hydrogel Material >
The hydrogel material provided by the invention is a three-dimensional network structure material, has high water content and excellent biocompatibility, so that the hydrogel can be widely applied to the field of biomedical engineering, and has great value in medical applications such as drug delivery, cartilage repair and the like. Hydrogels are widely studied for drug delivery due to their injectability, chemical modification susceptibility, degradability and high permeability. The drug is released continuously while the hydrogel is degraded continuously. Meanwhile, the hydrogel has better permeability, so that tissues and cells can absorb the medicines in the hydrogel better. In orthopedic applications, hydrogels may be filled alone, used as a coating for implants, or used to fill porous materials to provide a greater degree of coverage in the gap between the implant and the bone, thereby better stimulating bone growth.
As a preferred embodiment of the present invention, the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death drug of the present invention is filled in the 3D printed porous scaffold material, and the filling method of the hydrogel material is at least one selected from a drip irrigation method, a negative pressure suction method and a soaking method, and in the embodiment of the present invention, the filling is performed by adopting the negative pressure suction method.
In the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death drug, as shown in the figure 1, phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (glycol) diacrylate (PEGDA-DTT) are used as precursors to form a hydrogel crosslinking system with a double-crosslinking network structure, boric acid ester bonds are formed between HACC-PBA and PEGDA-DTT molecules to carry out covalent crosslinking, the two networks are connected with each other through the boric acid ester bonds, and double bonds at two ends of the PEGDA-DTT shown in the formula (4) can carry out crosslinking reaction under the light excitation in the presence of a photoinitiator. Therefore, after a certain amount of photoinitiator is added into the uncrosslinked chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death drug, the hydrogel material is filled and wrapped in the 3D printed porous support material by adopting a negative pressure suction method, and then the hydrogel material is subjected to in-situ photocrosslinking reaction on the surface of the 3D printed porous support material by using light excitation irradiation, so that the composite support is obtained. The composite scaffold is implanted into a human body or an animal body as a bone implant, boric acid ester bonds with Reactive Oxygen Species (ROS) response rupture characteristics in a hydrogel system are ruptured, chitosan quaternary ammonium salt with antibacterial activity and an anti-iron death medicament are continuously released in the presence of active oxygen molecules, and the release rate of the antibacterial and antibody death molecules can be flexibly adjusted according to the inflammation degree of an infection part.
<3D printed porous bone repair scaffold Material >
As a new additive manufacturing technology that can be customized, 3D printing technology has been attracting attention in bone defect repair research in recent years. By using the 3D printing technology, the bone repair stent with the matched anatomical structure and controllable internal porosity and void structure can be prepared. At present, a plurality of researches report the application of a composite 3D printing bracket formed by a macromolecule degradable material and bioglass in bone repair treatment, and the bracket has better biodegradability and mechanical property. Meanwhile, the bioglass component has stronger bone promoting capability and drug slow release capability. According to the invention, the anti-iron death medicine is innovatively loaded in the bioglass with a pore canal structure, and the bone repair stent with the long-acting slow-release function of the anti-iron death medicine is prepared through the 3D printing stent.
The polycaprolactone/bioglass porous scaffold loaded with the anti-iron death medicament provided by the invention takes a mixture of mesoporous bioglass loaded with a certain amount of the anti-iron death medicament and polycaprolactone as raw materials, and the porous scaffold with a certain gap size and geometry is prepared by a 3D printing mode. Polycaprolactone and bioglass are degradable materials approved by FDA for clinic, so the scaffold prepared by the invention has higher biological safety, and the anti-iron death drug loaded in the mesoporous bioglass can be slowly released in a local microenvironment along with the biodegradation of the scaffold, thereby providing a long-term protective effect for bone tissue regeneration and repair in an infected microenvironment.
As an embodiment of the present invention, the step of preparing the polycaprolactone/bioglass porous scaffold loaded with the anti-iron death drug by means of 3D printing comprises: dispersing the anti-iron death medicine in the water solution of the mesoporous bioglass for co-incubation, shaking and adsorbing for 6-8 hours on a shaking table, then centrifugally collecting the bioglass adsorbed with the anti-iron death medicine, and preparing the dried bioglass loaded with the anti-iron death medicine by using a vacuum freeze drying method; further, fully dissolving the polycaprolactone in an organic solvent, adding bioglass powder loaded with the anti-iron death medicament, and uniformly mixing to prepare a polycaprolactone/bioglass raw material loaded with the anti-iron death medicament; further, adding the mixture raw materials into a 3D printing charging barrel, performing 3D printing according to the designed size information and the pore size by using a 3D printer, and completely drying to prepare the polycaprolactone/bioglass porous support loaded with the anti-iron death drug.
As a preferred embodiment of the present invention, the mass fraction of the anti-iron death drug in the mesoporous bioglass adsorption system is 5% -40% (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%), and the mass fraction of the anti-iron death drug-loaded mesoporous bioglass in the polycaprolactone/bioglass raw material system is 10% -50% (may be, for example, 10% -45%, 10% -40%, 10% -35%, 10% -30%, 10% -25%, 10% -20%, 10% -15% -50%, 15% -45%, 15% -40%, 15% -35%, 15% -30%, 15% -25%, 20% -25% -30%, 25% -25%, 25% -30%, 20% -30%, 25% -40%, 25% -25%, 25% -25% and 20% -25% and 25% -40% 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 mode includes a normal temperature/low temperature printing or a high temperature printing mode.
As one embodiment of the invention, the anti-iron death medicine selected by the invention is selected from one or more of Ferrosistatin-1, liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, wherein the loading rate of the anti-iron death medicine in the 3D printed polycaprolactone/bioglass porous scaffold material is 0.5% -10% (mass fraction). In the embodiment of the invention, the Ferrostatin-1 is taken as an example for explaining the technical scheme of the invention, but the Ferrostatin-1 is not limited to the explanation.
< antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness >
The invention also provides an antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness, which comprises a tissue engineering porous scaffold material and the hydrogel material with active oxygen responsiveness, which is filled in the tissue engineering porous scaffold material.
As one embodiment of the invention, the tissue engineering porous scaffold material is the 3D-printed porous bone repair scaffold material, and is especially the polycaprolactone/bioglass porous scaffold loaded with the anti-iron death drug.
As a preferred embodiment of the invention, the filling rate of the polycaprolactone/bioglass porous support material loaded with the anti-iron death medicine provided by the invention is 20-300% (mass fraction) by filling the hydrogel material with the active oxygen responsiveness.
The invention compounds the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death medicament with the polycaprolactone/bioglass porous scaffold material loaded with the anti-iron death medicament by a photo-crosslinking method to prepare the antibacterial/anti-iron death bone repair composite scaffold with active oxygen responsiveness, and the preparation method comprises the following specific steps: adding a photoinitiator into an uncrosslinked chitosan quaternary ammonium salt hydrogel material loaded with an anti-iron death drug to form a hydrogel system, filling and wrapping the polycaprolactone/bioglass porous scaffold loaded with the anti-iron death drug by a hydrogel filling method, and carrying out in-situ photocrosslinking reaction on the surface of the polycaprolactone/bioglass porous scaffold loaded with the anti-iron death drug by light excitation irradiation. As an embodiment of the present invention, the hydrogel filling method includes at least one of a drip irrigation method, a negative pressure suction method and a soaking method, the photoinitiator is selected from at least one of phenyl-2, 4, 6-trimethylbenzoyl lithium hypophosphite (LAP) and 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylbenzophenone (Irgacure 2959), the mass fraction of the photoinitiator is 0.1% -1%, and the light excitation irradiation time is 5-60s.
In the antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness, as shown in figure 1, phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (glycol) diacrylate (PEGDA-DTT) are used as precursors to form a double-crosslinked network structure hydrogel crosslinking system, borate ester bonds are formed between HACC-PBA and PEGDA-DTT molecules to carry out covalent crosslinking, and double bonds at two ends of the PEGDA-DTT shown in formula (4) can carry out crosslinking reaction under the light excitation in the presence of a photoinitiator. Therefore, after a certain amount of photoinitiator is added into the uncrosslinked chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death drug, the hydrogel material is filled and wrapped in the polycaprolactone/bioglass porous support loaded with the anti-iron death drug by adopting a negative pressure suction method, and then the hydrogel material is subjected to in-situ photocrosslinking reaction on the surface of the polycaprolactone/bioglass porous support loaded with the anti-iron death drug by using light excitation irradiation, so that the composite support is obtained. The composite scaffold is implanted into a human body or an animal body as a bone implant, boric acid ester bonds with Reactive Oxygen Species (ROS) response rupture characteristics are ruptured in a hydrogel system, chitosan quaternary ammonium salt with antibacterial activity and an anti-iron death medicament are continuously released in the presence of active oxygen molecules, and the release rate of the antibacterial and antibody death molecules can be flexibly adjusted according to the inflammation degree of an infection part. In the composite scaffold, the adopted porous scaffold is a polycaprolactone/bioglass porous scaffold with higher biosafety, and the anti-iron death drug loaded in the mesoporous bioglass can be slowly released in a local microenvironment along with biodegradation of the porous scaffold, so that a long-term protection effect is further provided for bone tissue regeneration and repair in an infected microenvironment.
Therefore, the composite scaffold provided by the invention can respond to active oxygen to release antibacterial components and anti-iron death components through a hydrogel system in an acute infection period, protect the activity of mesenchymal stem cells while killing bacteria, and promote the osteogenic differentiation of the mesenchymal stem cells through the slow release of the anti-iron death drugs in mesoporous bioglass in a middle-long period bone repair process, so that the bimodal release of early antibacterial and anti-iron death and the promotion of bone tissue regeneration repair are realized.
According to the invention, the boric acid ester bond is used for connecting chitosan quaternary ammonium salt and PEGDA for the first time, so that the photo-crosslinking ROS responsive antibacterial hydrogel is prepared, and the photo-crosslinking ROS responsive hydrogel loaded with the antibacterial/anti-iron death activity of the anti-iron death medicine is used as a responsive component of the composite 3D printing bone repair stent, so that the antibacterial/anti-iron death bone repair composite stent with reactive oxygen responsiveness is prepared. The antibacterial/anti-iron death bone repair composite scaffold with active oxygen responsiveness can realize that the release rate of an antibacterial component and an anti-iron death component can be adjusted in response to the active oxygen signal dynamic state in an infection microenvironment at early infection, and the activity of mesenchymal stem cells can be saved by inhibiting iron death at the same time of antibacterial, and the osteogenic differentiation and bone repair of the mesenchymal stem cells can be promoted by long-term slow release of the anti-iron death component in treatment.
The technical scheme of the present invention will be further described with reference to specific examples, but the scope of the present invention is not limited to these examples. All changes and equivalents that do not depart from the gist of the invention are intended to be within the scope of the invention.
Example 1 preparation of an antibacterial/anti-iron death composite scaffold with active oxygen response:
the chitosan quaternary ammonium salt with the substitution degree of about 26 percent is dissolved in a mixed solution system of 50ml of deionized water and 25ml of absolute ethyl alcohol, 100mg of 4-formylphenyl boric acid is added, and the mixture is stirred and reacted for 1h at the temperature of 25 ℃. Then 150mg of sodium cyanoborohydride was added, and after complete dissolution, the reaction was stirred at 25℃for 18 hours. The obtained solution was dialyzed in pure water for 48 hours using 3500Da dialysis bag, and then phenylboronic acid modified chitosan quaternary ammonium salt was obtained using a vacuum freeze-drying method.
The polyethylene glycol (glycol) diacrylate and dithiothreitol are dissolved in PBS aqueous solution, so that the final concentration of the polyethylene glycol (glycol) diacrylate is 150mg/ml, the final concentration of the dithiothreitol is 100mg/ml, and after the polyethylene glycol (glycol) diacrylate and the dithiothreitol are fully dissolved, the reaction is started to be carried out under the condition of light shielding and normal temperature stirring, and the reaction time is 2 hours. Then adding phenylboronic acid modified chitosan quaternary ammonium salt to enable the mass fraction of the phenylboronic acid modified chitosan quaternary ammonium salt to be 0-1.5%, and stirring and reacting for 1h at normal temperature in a dark place. Then adding Ferrostatin-1 and photoinitiator LAP to make the final concentration of the Ferrostatin-1 and the photoinitiator be 20mg/ml and 1mg/ml respectively, so as to obtain the chitosan quaternary ammonium salt hydrogel system loaded with Ferrostatin-1.
The Ferrostatin-1-loaded mesoporous bioglass is obtained by incubating the Ferrostatin-1 which is an anti-iron death drug prepared in advance and the mesoporous bioglass in a mass ratio of 1:7 in an aqueous solution, shaking and adsorbing for 6 hours on a shaking table, centrifuging and then performing freeze drying treatment. After the polycaprolactone is dissolved by using dichloromethane, mesoporous bioglass loaded with Ferrostatin-1 is added, and the mixture is fully and uniformly stirred to prepare the mixed paste suitable for 3D printing. Wherein the mass ratio of the mesoporous bioglass powder loaded with the Ferrositin-1 to the polycaprolactone is 3:7. After re-volatilisation to the appropriate viscosity, the paste is loaded into a 3D printing cartridge and then into a 3D printer. A borderless grid circular model with a diameter of 3mm, a height of 4mm and a gap spacing of 0.5mm is arranged on an extrusion type 3D printer, the printing speed is 1.8mm/s, and the extrusion pressure is 3.0bar. 3D printing is carried out to obtain the porous bracket loaded with Ferrostatin-1.
The porous scaffold loaded with the Ferrostatin-1 is soaked in an uncrosslinked chitosan quaternary ammonium salt hydrogel system loaded with the Ferrostatin-1. The hydrogel system was allowed to fully fill the porous scaffold voids using a negative pressure venting method. The hydrogel system was then crosslinked by irradiating the porous scaffold supporting the hydrogel system with blue light for 30 s. An antibacterial/anti-iron death hydrogel composite 3D printed bone repair scaffold with ROS response was prepared. FIG. 2 shows the composite scaffold (Fer-1@HACC-DP-PCL/MBG scaffold) without hydrogel support (HACC-DP-PCL/MBG scaffold) loaded with hydrogel.
Example 2 antimicrobial properties of an antimicrobial/anti-iron death composite scaffold with active oxygen response:
antibacterial/anti-iron death composite scaffolds (about 20 g) loaded with different amounts of phenylboronic acid modified chitosan quaternary ammonium salt were soaked in 200 μl containing 5.5X10 5 CFU/ml of Staphylococcus aureus (ATCC 25923) in the CAMHB medium, incubated in a shaker for 18 hours at 37℃and 200rpm. After incubation, the scaffolds were removed and after 5min of ultrasonic agitation with 1ml of sterile PBS, the antibacterial rate of the scaffolds was calculated using plating colony counting. As can be seen from fig. 3, the antibacterial efficiency of the composite scaffold containing 1% phenylboronic acid modified chitosan quaternary ammonium salt in the hydrogel system is highest when the antibacterial rate of the scaffold is greater than 99% when the phenylboronic acid modified chitosan quaternary ammonium salt content in the hydrogel is 0.5-1.5% compared with the case that the phenylboronic acid modified chitosan quaternary ammonium salt group is not added in the hydrogel.
Example 3 release properties of chitosan quaternary ammonium salt hydrogel and polycaprolactone/bioglass porous scaffold:
and replacing the Ferrostatin-1 with fluorescein FITC with a molecular weight similar to that of the Ferrostatin-1, adding the fluorescein FITC into a chitosan quaternary ammonium salt hydrogel system and a 3D printing porous bracket, and simulating the release mode of the Ferrostatin-1 by measuring the release of the FITC. Chitosan quaternary ammonium salt hydrogels loaded with FITC but with different phenylboronic acid modified chitosan quaternary ammonium salt contents (0.5% -1.5%) were immersed in simulated body fluid containing hydrogen peroxide (100 μm) and no hydrogen peroxide at a volume ratio of 1:100 and incubated in a constant temperature shaker at 37 ℃ at 200rpm. The FITC cumulative release was calculated by measuring FITC concentration in SBF at 0,0.5,1,2,3,4,5,6 days. FITC-loaded 3D printed porous scaffolds were immersed in simulated body fluids containing hydrogen peroxide (100. Mu.M) and no hydrogen peroxide at a mass to volume ratio of 1g:100ml, incubated at 37℃and 200rpm in a thermostatted shaker. The FITC cumulative release was calculated by measuring FITC concentration in SBF at 0,1,4,7,14,21,28 days. From fig. 4, it can be seen that the phenylboronic acid modified chitosan quaternary ammonium salt content is 0.5%,1% and 1.5% of the chitosan quaternary ammonium salt hydrogel system has significantly accelerated FITC release rate in the presence of hydrogen peroxide, and can realize ROS responsive release performance in 0-6 days. FITC in polycaprolactone/bioglass can realize long-acting slow release of FITC within 0-28 days. The composite scaffold composed of chitosan quaternary ammonium salt hydrogel and polycaprolactone/bioglass scaffold can realize ROS response release in a short term and slow release in a long term.
Example 4, anti-iron death drug Ferrostatin-1 rescue infection resulted in mesenchymal stem cell iron death:
the salvage effect of Ferrostatin-1 on mesenchymal stem cell death under the infection condition is detected by lipid peroxidation staining and dead living staining methods. Use of bone marrow derived mesenchymal stem cells (BMSCs) at 20×10 4 Individual cells/well density were seeded in 6-well plates overnight to allow cell attachment. Cells were pretreated with 10. Mu.M Ferrostatin-1 for 12h. Thereafter, staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were used to co-culture with BMSCs at a multiplicity of infection (MOI) of 10 for 12h, and during co-culture, the rescue group was continued to be treated with 10. Mu.M Ferrostatin-1. Using a C11-BODIPY and Liperfluo lipid peroxidation fluorescent probe, using a Ferroorange ferrous iron fluorescent probe to dye cells, using a confocal microscope to shoot, and evaluating the regulation effect of Ferrostatin-1 on BMSCs lipid peroxidation and ferrous iron ion accumulation in a sensory microenvironment; or using live-dead staining flow assays after the end of co-culture to evaluate the rescue effect of Ferrostatin-1 on BMSCs cell death in the infected microenvironment. The result shows that the use of 10 mu M Ferrostatin-1 to treat cells can obviously reduce lipid peroxidation level of BMSCs in cells when staphylococcus aureus and escherichia coli are infected, inhibit ferrous ion accumulation in cells and reduce cell death rate. From FIG. 5, it can be seen that the damage and iron death of the infected micro-environmental mesenchymal stem cells can be saved by releasing Ferrostatin-1 in the composite scaffold.
Example 5 anti-iron death drug Ferrostatin-1 promotes osteoblastic differentiation of mesenchymal Stem cells after infection
The regulation of mesenchymal stem cell osteogenic differentiation after infection by Ferrostatin-1 treatment was examined 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 the bacteria was aspirated and washed twice with sterile PBS, and osteogenic induction medium containing 10. Mu.M Ferrostatin-1 with 20. Mu.g/ml gentamicin was added. ALP staining was performed 7 days after induction and ARS staining was performed 14 days after induction. As can be seen from FIG. 6, treatment with 10. Mu.M Ferrostatin-1 promotes ALP expression and mineralization nodule formation in mesenchymal stem cells, and promotes osteoblast differentiation of mesenchymal stem cells.
Example 6 antimicrobial efficacy of antimicrobial/anti-iron death 3D printed bone repair composite scaffolds with reactive oxygen species in an infectious bone defect model:
antibacterial performance of the antibacterial/anti-iron death 3D printed bone repair composite scaffold with active oxygen response was evaluated in a rat femoral condyle infectious bone defect model by in vivo fluorescence imaging of small animals. A cylindrical bone defect with a diameter of 3mm and a depth of 4mm was produced at the distal end of the femoral condyle of a rat by trephine, and 50. Mu.L of a bone defect containing 5X 10 was dropped 8 CFUs/mL bioluminescent Staphylococcus aureus (Xen 29), after stent implantation, the skin and muscle were sutured. Bacterial load was assessed 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks, 12 weeks after molding by measuring bacterial fluorescence signals at the site of molding using in vivo imaging. As can be seen from FIG. 7, bacterial fluorescence signals can be continuously detected at the animal molding position of a non-antibacterial component group (Bone wax, PCL/MBG scaffold) after 12 weeks of operation, and bacterial signal intensity of the animal molding position of a chitosan quaternary ammonium salt hydrogel group (HACC-DP-PCL/MBG scaffold) and a Fer-1@HACC-DP-PCL/MBG scaffold) is obviously reduced after 3 days of molding, and the bacterial signal intensity is reduced to below a detection limit after one week, so that the bracket loaded with the chitosan quaternary ammonium salt hydrogel has a strong antibacterial effect and can effectively remove pathogenic bacteria at the infectious Bone defect position.
Example 7, antibacterial/anti-iron death bone repair composite scaffold with reactive oxygen species response promotes infectious bone defect repair:
the bone repair effect of the antibacterial/anti-iron-death bone repair composite scaffold in the rat femoral condyle infectious bone defect model in example 6 was evaluated by microscopic CT bone measurement. The model rats were euthanized 6 and 12 weeks after surgery, and rat femoral tissue was fixed using paraformaldehyde solution. The distal femur of the rat was then scanned using micro-CT (Scanco Medical μCT 80). And calculating bone morphometric parameters such as BV/TV, tb.N and the like. As can be seen from FIG. 8, at the time points of 6 weeks and 12 weeks, no antibacterial component group (Bone wax, PCL/MBG scaffold) occurred in the femur of the animals, severe osteolysis and pathological fracture occurred, no pathological fracture occurred in the femur of the animals loaded with chitosan quaternary ammonium salt hydrogel group (HACC-DP-PCL/MBG scaffold and Fer-1@HACC-DP-PCL/MBG scaffold), and new Bone formation occurred in the Bone defect site. The antibacterial/anti-iron death bone repair composite stent loaded with the anti-iron death drug Ferrostatin-1 has more bone mass at the femoral condyle defect part compared with a pure antibacterial stent group, and BV/TV and Tb.N are obviously higher than those of the pure antibacterial stent group. This suggests that intervention for iron death promotes osteogenesis and bone repair after infection, and that the antibacterial/anti-iron death scaffold is effective in promoting repair of infectious bone defects.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (24)
1. A hydrogel material with active oxygen responsiveness is characterized by comprising a double-crosslinked hydrogel crosslinking system which is formed by taking phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) and dithiothreitol modified polyethylene glycol (glycol) diacrylate (PEGDA-DTT) as precursors, and forming boric acid ester bond covalent crosslinking between HACC-PBA and PEGDA-DTT molecules and photocrosslinking with PEGDA-DTT molecular unsaturated carbon-carbon double bonds.
2. The reactive oxygen species responsive hydrogel material of claim 1, wherein the reactive oxygen species responsive hydrogel material further comprises a drug loaded in the hydrogel cross-linking system.
3. The hydrogel material with reactive oxygen species of claim 2, wherein the drug is an anti-iron death drug, preferably the anti-iron death drug comprises one or more of Ferrostatin-1, liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine.
4. A hydrogel material according to claim 2 or 3, wherein the loading rate of the anti-iron death drug in the hydrogel cross-linking system is 0.1% to 10% (mass fraction).
5. The active oxygen responsive hydrogel material of any one of claims 1-4, wherein the borate ester linkage formed in the hydrogel material has active oxygen responsive cleavage properties that allow simultaneous release of the chitosan quaternary ammonium salt having antibacterial activity and the anti-iron death agent in the presence of active oxygen (ROC) molecules.
6. A method for preparing a hydrogel material having active oxygen responsiveness according to any one of claims 1 to 5, comprising the steps of:
preparing phenylboronic acid modified chitosan quaternary ammonium salt by a normal temperature solution method;
preparing a chitosan quaternary ammonium salt hydrogel crosslinking system by a normal temperature solution method;
the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death medicine is prepared by a hydrogel encapsulation method.
7. The method of preparing the phenylboronic acid modified chitosan quaternary ammonium salt according to claim 6, wherein the step of preparing the phenylboronic acid modified chitosan quaternary ammonium salt by a normal temperature solution method comprises the following steps: dissolving chitosan quaternary ammonium salt in a water and organic solvent dissolving system, adding phenylboronic acid substances, fully dissolving, starting stirring at normal temperature to react for 1-12h, adding a reducing protective agent sodium cyanoborohydride, fully dissolving, and stirring at normal temperature to react for 3-96h; dialyzing the obtained solution after the reaction for 6-72h; finally, the chitosan quaternary ammonium salt (HACC-PBA) modified by phenylboronic acid is obtained after vacuum freeze drying.
8. The preparation method according to claim 7, wherein the chitosan quaternary ammonium salt is hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and the degree of substitution of the hydroxypropyl trimethyl ammonium chloride of the chitosan quaternary ammonium salt is in the range of 5% -60%; the phenylboronic acid material is at least one selected from 4-formylphenylboronic acid and 3-aminophenylborate.
9. The method of preparing a chitosan quaternary ammonium salt hydrogel cross-linking system according to claim 6, wherein the step of preparing the chitosan quaternary ammonium salt hydrogel cross-linking system by a normal temperature solution method comprises: dissolving polyethylene glycol (glycol) diacrylate (PEGDA) and Dithiothreitol (DTT) in an aqueous solution, and stirring for reaction at normal temperature under dark condition for 0.5-6h after the polyethylene glycol (glycol) diacrylate and the Dithiothreitol (DTT) are fully dissolved; then adding phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA), fully dissolving, and stirring at normal temperature for reacting for 0.5-6h to obtain the chitosan quaternary ammonium salt hydrogel crosslinking system.
10. The method of claim 9, wherein the polyethylene glycol (diol) diacrylate (PEGDA) has an average molecular weight of 250-10000, in the range of 5-20% by volume; the final concentration of Dithiothreitol (DTT) is 1-100mg/ml; the mass concentration of the phenylboronic acid modified chitosan quaternary ammonium salt (HACC-PBA) is 1-15mg/ml.
11. The method of preparing a chitosan quaternary ammonium salt hydrogel material loaded with an anti-iron-death drug according to claim 6, wherein the step of preparing the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron-death drug by a hydrogel encapsulation method comprises: adding the anti-iron death medicine into the chitosan quaternary ammonium salt hydrogel crosslinking system, and fully and uniformly stirring to obtain the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death medicine.
12. The preparation method of claim 11, wherein the anti-iron death agent comprises one or more of Ferrostatin-1, liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine, and the mass concentration of the anti-iron death agent is 1-100mg/ml.
13. Use of the active oxygen responsive hydrogel material according to any one of claims 1 to 5 and the active oxygen responsive hydrogel material prepared by the preparation method according to any one of claims 6 to 12 for preparing bone repair materials, tissue engineering porous scaffold materials or 3D printing bio-ink.
14. The use according to claim 13, wherein the active oxygen responsive hydrogel material is used for coating of an implant of a bone repair material.
15. The use according to claim 14, wherein the active oxygen responsive hydrogel material is used to fill a tissue engineering porous scaffold material.
16. An antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness, which is characterized by comprising a tissue engineering porous scaffold material and the hydrogel material with active oxygen responsiveness, which is filled in the tissue engineering porous scaffold material.
17. The active oxygen responsive antibacterial/anti-iron death bone repair composite scaffold according to claim 16, wherein the tissue engineering porous scaffold material is a 3D printed polycaprolactone/bioglass porous scaffold material.
18. The antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness according to claim 17, wherein the 3D printed polycaprolactone/bioglass porous scaffold material is loaded with an anti-iron-death drug comprising one or more of Ferrostatin-1, liproxstatin-1, deferoxamine, vitamin E, vitamin K and N-acetylcysteine; preferably, the loading rate of the anti-iron death drug in the 3D printed polycaprolactone/bioglass porous scaffold material is 0.5% -10% (mass fraction).
19. The antimicrobial/anti-iron-death bone repair composite scaffold according to claim 18, wherein the filling rate of the active oxygen-responsive hydrogel material filled in the 3D printed polycaprolactone/bioglass porous scaffold material is 20% -300% (mass fraction).
20. A method of preparing an antibacterial/anti-iron-death bone repair composite scaffold according to any one of claims 16 to 19, comprising the steps of:
preparing a polycaprolactone/bioglass porous scaffold material loaded with an anti-iron death drug in a 3D printing mode;
and compounding the chitosan quaternary ammonium salt hydrogel material loaded with the anti-iron death medicament with the polycaprolactone/bioglass porous scaffold material loaded with the anti-iron death medicament by a photo-crosslinking method to prepare the antibacterial/anti-iron death bone repair composite scaffold with active oxygen responsiveness.
21. The method of preparing a polycaprolactone/bioglass porous scaffold material loaded with anti-iron death drug according to claim 20, wherein the step of preparing the polycaprolactone/bioglass porous scaffold material loaded with anti-iron death drug by 3D printing comprises: dispersing the anti-iron death medicine in the water solution of the mesoporous bioglass for co-incubation, shaking and adsorbing for 6-8 hours on a shaking table, and then centrifugally collecting the bioglass adsorbed with the anti-iron death medicine, and obtaining the dried bioglass loaded with the anti-iron death medicine after vacuum freeze drying; then fully dissolving polycaprolactone in an organic solvent, adding bioglass powder loaded with an anti-iron death medicament, and uniformly mixing to obtain a polycaprolactone/bioglass raw material loaded with the anti-iron death medicament; adding the mixture raw materials into a 3D printing charging barrel, performing 3D printing according to the designed size information and the pore size by using a 3D printer, and completely drying to prepare the polycaprolactone/bioglass porous support loaded with the anti-iron death drug; preferably, the 3D printing method includes a normal temperature/low temperature printing method or a high temperature printing method.
22. The method of claim 20, wherein the step of compounding the active oxygen responsive hydrogel material with the anti-iron death drug loaded polycaprolactone/bioglass porous scaffold material by photo cross-linking comprises: adding a photoinitiator into an uncrosslinked chitosan quaternary ammonium salt hydrogel material loaded with an anti-iron death drug to form a hydrogel system, and filling and wrapping the polycaprolactone/bioglass porous scaffold loaded with the anti-iron death drug by adopting a hydrogel filling method; preferably, the hydrogel filling method includes at least one of a tank drop method, a negative pressure suction method, and a soaking method.
23. The method of claim 22, wherein the hydrogel system undergoes an in situ photocrosslinking reaction on the surface of the anti-iron death drug loaded polycaprolactone/bioglass porous scaffold using light-activated irradiation; the photoinitiator is at least one selected from phenyl-2, 4, 6-trimethylbenzoyl lithium hypophosphite (LAP) and 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone (Irgacure 2959), the mass fraction of the photoinitiator is 0.1% -1%, and the light excitation irradiation time is 5-60s.
24. Use of the antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness according to any one of claims 16 to 19 and the antibacterial/anti-iron-death bone repair composite scaffold with active oxygen responsiveness prepared by the preparation method according to any one of claims 20 to 23 in the treatment of infectious bone defects.
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