CN109265691A - Hydrogel and the preparation method and application thereof - Google Patents
Hydrogel and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention relates to technical field of biological materials, more particularly to a kind of hydrogel and preparation method thereof, alpha-beta unsaturated acids structure is grafted in chitosan molecule chain respectively, mercapto groups are grafted in tropocollagen molecule chain, it is modified by the active structure to two kinds of biomaterials, utilize Michael addition reaction, the rapid curing of hydrogel is realized with chemical crosslinking mechanism, to realize the 3D printability of material, the mechanical strength and elasticity modulus of hydrogel are adjusted by changing the concentration of the material after modifying.The hydrogel curing rate is fast, and good biocompatibility, mechanical strength are adjustable, stability is good, and biodegradation rate is adjustable.The general formula of the hydrogel are as follows: WithAny one of；Wherein, R1The residue moiety of amino is removed for chitosan；R2For hydrogen atom, alkyl or alkylidene；R3For hydroxyl, hydroxy derivatives, amino or aminoderivative；R4The residue moiety of amino and carboxyl is removed for collagen；R5For alkylidene or substituted alkylene.
Description
Technical field
The present invention relates to technical field of biological materials, in particular to a kind of hydrogel and the preparation method and application thereof.
Background technique
3D printing, also referred to as increasing material manufacturing cause dramatic change in many fields, as engineering science, process industry, education,
Medicine.3D biometric print makes for biocompatible materials, cell and complementary ingredient to be assembled into active with three-dimensional function
Human organ or tissue are possibly realized.Compared with abiotic printing, 3D biometric print is increasingly complex, is related to biocompatible materials
Selection, cell type, growth, differentiation factor the considerations of and the problems such as tissue construction, wherein the selection of material is most important.
Hydrogel is a kind of with hydrophilic radical, can absorb large quantity of moisture swelling but three-dimensional netted polymerization not soluble in water
Object is suitble to adherency, growth, proliferation and the differentiation of various different cells because of its Nomenclature Composition and Structure of Complexes similar with extracellular matrix, at
The important materials of tissue and organ are constructed for 3D biometric print.There is solidification speed in current major part 3D biometric print hydrogel
Degree is relatively slow, colloid mechanical strength and toughness are smaller or the problems such as can not regulate and control, and greatly reduces its printability and application range.
Summary of the invention
The first purpose of this invention is to provide a kind of preparation method of hydrogel, and preparation process is simple, Neng Gouyou
Effect ground be prepared curing rate fastly, good biocompatibility, mechanical strength is adjustable, stability in the medium is good and biological
The adjustable hydrogel of degradation speed.
Second object of the present invention is to provide a kind of hydrogel, and curing rate is fast, good biocompatibility, mechanical strong
Spend that stability adjustable, in the medium is good, and biodegradation rate is adjustable, and application range is big.
Third object of the present invention is to provide above-mentioned hydrogel in production biologic medical material or tissue engineering material
Or the application on 3D printing material.
The present invention solves its technical problem and adopts the following technical solutions to realize.
A kind of preparation method of hydrogel comprising: maleic anhydride modification chitosan and sulfhydrylation collagen are carried out
Michael addition reaction；The general formula of maleic anhydride modification chitosan are as follows:Sulfhydrylation collagen leads to
Formula are as follows:
Wherein, R1The residue moiety of amino is removed for shitosan macromolecule；R2For hydrogen atom, alkyl or alkylidene；R3For hydroxyl
Base, hydroxy derivatives, amino or aminoderivative；R4The residue moiety of amino and carboxyl is removed for collagenous polymer；R5For alkylene
Base or substituted alkylene.
A kind of hydrogel is prepared, general formula by the preparation method of above-mentioned hydrogel are as follows:
Any one of；
Wherein, R1The residue moiety of amino is removed for shitosan macromolecule；R2For hydrogen atom, alkyl or alkylidene；R3For hydroxyl
Base, hydroxy derivatives, amino or aminoderivative；R4The residue moiety of amino and carboxyl is removed for collagenous polymer；R5For alkylene
Base or substituted alkylene.
Application of the above-mentioned hydrogel on production biologic medical material or tissue engineering material or 3D printing material.
The beneficial effect of the hydrogel of the embodiment of the present invention and preparation method thereof is: by gathering the acylated shell of maleic anhydride
Sugar is used as raw material with the collagen of sulfhydrylation, can realize raw material by liquid to solid quick by Michael addition reaction
Transformation, substantially increases the printability of material, and hydrogel elastic modulus after solidifying can be realized by the concentration of both adjustings
It adjusts, the application range of material can be expanded.The hydrogel of this method preparation has important in biomedical and field of tissue engineering technology
Purposes, curing rate is fast, and good biocompatibility, mechanical strength are adjustable, stability in the medium is good, biodegradation rate
Adjustable, application range is big.
Detailed description of the invention
It, below will be to use required in embodiment in order to illustrate more clearly of the technical solution of embodiment of the present invention
Attached drawing be briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not to be seen as
It is the restriction to range, it for those of ordinary skill in the art, without creative efforts, can be with root
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is Fourier's infared spectrum (FTIR) comparative analysis of the collagen and sulfhydrylation collagen in the embodiment of the present invention 1
Figure；
Fig. 2 is Fourier's infared spectrum of the chitosan and maleic anhydride modification chitosan in the embodiment of the present invention 1
(FTIR) comparative analysis figure；
Fig. 3 is scanning electron microscope (SEM) surface microstructure figure of the sulfhydrylation collagen in the embodiment of the present invention 1；
Fig. 4 is scanning electron microscope (SEM) surface microstructure figure of the MCS product in the embodiment of the present invention 1；
Fig. 5 is scanning electron microscope (SEM) surface microstructure of hydrogel after the solidification in the embodiment of the present invention 1
Figure；
Fig. 6 is surface apertures scanning electron microscope (SEM) figure of hydrogel after the solidification in the embodiment of the present invention 1；
Fig. 7 is the hydrogel mechanical test curve graph in the embodiment of the present invention 1.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Hydrogel of the embodiment of the present invention and preparation method thereof is specifically described below.
A kind of preparation method of hydrogel comprising: maleic anhydride modification chitosan and sulfhydrylation collagen are carried out
Michael addition reaction；The general formula of maleic anhydride modification chitosan are as follows:Sulfhydrylation collagen leads to
Formula are as follows:
Wherein, R1The residue moiety of amino is removed for shitosan macromolecule；R2For hydrogen atom, alkyl or alkylidene；R3For hydroxyl
Base, hydroxy derivatives, amino or aminoderivative；R4The residue moiety of amino and carboxyl is removed for collagenous polymer；R5For alkylene
Base or substituted alkylene.
Chitosan, also known as chitosan are the products of de-acetyl chitin, are a kind of widely used natural more
Sugar.Chitosan is the unique alkaline polysaccharide of nature, has a pair of unshared electricity on the free amine group nitrogen-atoms on strand
Son is right, can be in conjunction with a Hydrogen Proton, so that chitosan be made to become positively charged polyelectrolyte.Chitosan has good biology
Compatibility, biological degradability, bactericidal properties etc. are a kind of safe natural polymers.Free ammonia in chitosan molecule
Base chemical activity with higher, is easy to by the higher modified with functional group of carboxyl isoreactivity, using maleic anhydride to chitosan into
Row modification, can be obtained water soluble chitosan, while alpha-beta unsaturated carbonyl structure is introduced in chitosan molecule chain, can be by
Addition reaction occurs for nucleopilic reagent attack, successfully realizes the crosslinking of polymer, reaches quick-setting effect.
Specifically, the general formula of maleic anhydride modification chitosan are as follows:
Collagen is a kind of important protein, is usually made of three peptide chains, these peptide chains are referred to as α-chain.Collagen is thin
One of extracellular matrix structural proteins can be divided into the Collagen Type VIs such as I, II, III, IV according to the division of its structure function.Collagen
Hydrogel has been successfully realized culture of the cell on three-dimensional substrate, and one kind is also considered as in organizational project and is answered very much
With the timbering material of prospect.Although collagen itself can form hydrogel, the hydrogel mechanical strength that is individually formed due to it
Weaker, elasticity modulus is smaller, and crosslinking method is unstable, and molecule interchain active force is more easily damaged in the medium and dissolves,
It limits its application.There are free amine groups and carboxylic group on tropocollagen molecule chain, remove modification ammonia with the compound with sulfydryl
Base or carboxyl, can be by collagen sulfhydrylation, and the collagen after modification has the chemical property of sulfydryl, under alkaline condition sulfydryl proton from
Go to can produce sulphur anion, the chitosan acylated with maleic anhydride as nucleopilic reagent attack, in the form of C-S key by collagen and
MCS is quickly grafted together, generates netted composite hydrogel.
Specifically, the general formula of sulfhydrylation collagen are as follows:
Wherein, R5For alkylidene or substituted alkylene.Substituted alkylene can be at least one hydrogen atom by alkyl, carboxylic
The alkylidene that at least one of base, amino, alkoxy, aromatic radical, ester group and halogenated alkyl group replaces.It can be alkylidene
In there is a hydrogen atom to be replaced by one of alkyl, carboxyl, amino, alkoxy, aromatic radical, ester group and halogenated alkyl group；
Hydrogen atom is by alkyl, carboxyl, amino, alkoxy, aromatic radical, ester group and halogenated alkyl there are two being also possible in alkylidene
Two kinds of groups replace；Above hydrogen atom is by alkyl, carboxyl, amino, alkoxy, fragrance there are two being also possible in alkylidene
Two or more groups in base, ester group and halogenated alkyl replace；It is also possible in alkylidene multiple hydrogen atoms by alkyl, carboxylic
Multiple same groups in base, amino, alkoxy, aromatic radical, ester group and halogenated alkyl replace or by plurality of different bases
The combination correspondence of group is replaced.
Wherein, R5Carbon atom number can be 1~20.That is R5Can for C1, C2, C3, C4, C5, C6, C7, C8, C9,
The alkylidene or substituted alkylene of C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20.
The collagen for carrying out sulfhydrylation is selected from collagen, alkaline gelatin albumen, acidic gelatin albumen, genetic recombination collagen egg
White, basic group is because recombinant gelatins albumen, acidic groups are because of recombinant gelatins albumen, core protein, polysaccharide laminin and fiber
One of binding protein.There is free amino and carboxyl on these strands, so as to by with carboxyl or amino into
Row reaction achievees the purpose that carry out modification to tropocollagen molecule chain.
The sulfhydryl compound to react with collagen is the compound simultaneously with carboxyl and sulfydryl, or has ammonia simultaneously
The compound of base and sulfydryl；Above-mentioned sulfhydryl compound can be by directly generating through hydrolysis, ammonolysis reaction etc., or can lead to
The compound containing disulfide bond is crossed to obtain after carrying out reduction.For example, having the compound of carboxyl and sulfydryl can be with simultaneously are as follows: dimercapto fourth
Diacid, dimercaptosuccinic acid, mercaptopropionic acid, thioglycolic acid, 2- sulfydryl-acidum nicotinicum etc..There is amino and sulfydryl simultaneously
Compound can be with are as follows: acthiol-J, Ethitanin, 4- amino -3- mercaptobenzoic acid etc..
Further, some embodiments of the present invention also provide the preparation method of hydrogel, comprising: by maleic acid
Acid anhydride modifies chitosan solution and sulfhydrylation collagen solution carries out Michael addition reaction.
According to some embodiments, maleic anhydride modification chitosan solution is that maleic anhydride modification chitosan is dissolved in acid
Solution and maleic anhydride that the concentration that is made into is 10~100mg/ml modifies chitosan acid solution；Sulfhydrylation collagen solution is by mercapto
The sulfhydrylation collagen aqueous slkali for 10~100mg/ml that base collagenolysis is made into aqueous slkali.By above-mentioned concentration and
The setting of its solvent, so that maleic anhydride modification chitosan and sulfhydrylation collagen can be good at being dissolved, and can
It is more conducive to the progress of Michael addition reaction between the two.
According to certain embodiments of the present invention, the preparation of hydrogel is carried out by following steps:
(1) maleic anhydride modification chitosan (MCS) is prepared.
The chemical equation of maleic anhydride modification chitosan are as follows:
It according to some embodiments, dissolves chitosan in acid solution, maleic anhydride is dissolved in polar solvent, shell
The anhydride molar ratio of free amine group and maleic anhydride is preferably 1:1~3 in polysaccharide chains, by the maleic anhydride of dissolution in magnetic agitation
It is slowly added in chitosan acid solution on device, is sufficiently mixed uniformly, is reacted under the conditions of 10~90 DEG C at room temperature, preferably 40~
90 DEG C, 2~10h of reaction time.After reaction by dialysis 2~4 days, it is freeze-dried 2~4 days, chitosan after being modified
Product.
According to some embodiments, the acid solution for dissolving chitosan can be organic acid soln, preferably acetic acid solution, more excellent
Selection of land, the mass fraction of acetic acid solution are 0.01~30%.Dissolution maleic anhydride polar solvent include acetone, butanone, water,
DMSO, DMF etc., preferably acetone, and the dosage of acetone is to guarantee for maleic anhydride to be completely dissolved.By will be on chitosan chain
The anhydride molar ratio of free amine group and maleic anhydride is set as 1:1~3, enables the acid anhydrides of maleic anhydride preferably poly- with shell
Free amine group is reacted on sugar chain, to obtain maleic anhydride modification chitosan to chitosan-modified.Meanwhile by the horse of dissolution
Carry out acid anhydrides to stir on magnetic stirring apparatus, and be slowly added in chitosan acid solution, enables to the two can be more fully
It is contacted, and then reaches better reaction effect.Operation is dialysed and is freeze-dried after the completion of reaction to reaction solution, it can
It is preferably removed so that maleic anhydride is modified remaining small molecular weight impurity in chitosan, is repaired with obtaining the higher maleic anhydride of purity
Chitosan is adornd, and then promotes the more preferable progress of subsequent Michael addition reaction.
(2) sulfhydrylation collagen (COL-SH) is prepared.
Sulfhydrylation collagen (COL-SH) is generated by following formulas:
According to some embodiments, the preparation method of sulfhydrylation collagen (COL-SH) be by the solution containing collagen with contain
The solution of sulfhydryl compound is reacted under the action of carboxyl activator.
According to some embodiments, when using the sulfhydryl compound of carboxyl is contained as raw material, sulfhydrylation collagen (COL-
SH it) can be prepared by the following method:
A. the solution containing collagen is prepared: by collagenolysis in the acid solution that mass fraction is 0.01~30%.
B. it prepares the solution of compounds containing thiol groups: sulfhydryl compound is dissolved in distilled water or alkali soluble according to its dissolubility
In liquid or acid solution.
C. carboxyl activator is added in the solution of above-mentioned compounds containing thiol groups, after mixing, adjust its pH value to
4.5~6.5, continues to mix stirring.
D. the solution containing sulfhydryl compound after activation is mixed with the solution containing collagen, sulfhydryl compound with
The molar ratio of amino is 1~10:1 on collagen, stirs, is preferably transferred in round-bottomed flask, and being placed on temperature is 37
DEG C or less carry out isothermal reaction.
E. the reaction solution obtained after reaction is subjected to dialysis 2~5 days, then obtained dialysis product is subjected to freeze-drying 2
After~5 days, sulfhydrylation collagen is obtained.It can remove remaining small molecular weight impurity by the means of dialysis, improve resulting collagen
The purity of thiolated derivative.
It should be noted that can also first carry out step b and c in the above process, then step a is carried out, will not influence most
The generation of final product.
According to some embodiments, it is molten that the acid solution in sulfhydrylation collagen (COL-SH) preparation process can be organic acid
Liquid, preferably acetic acid solution.I.e. preferably by collagenolysis in the acetic acid solution that mass fraction is 0.01~30%.
According to some embodiments, aqueous slkali can be strong base solution, for example, sodium hydroxide solution, potassium hydroxide solution,
Calcium hydroxide solution etc., or weak caustic solution, such as ammonia spirit, sodium carbonate liquor, sodium bicarbonate solution etc., preferably hydrogen
Sodium hydroxide solution.Distilled water can make dissolved solution impurity content few, and purity is higher, and subsequent reactions effect is better.It is molten
The acid solution of solution sulfhydryl compound can be organic acid soln, preferably acetic acid solution.
According to some embodiments, carboxyl activator includes EDC and NHS, EDC, that is, 1- (3- dimethylamino-propyl) -3- second
Base carbodiimide hydrochloride, EDC are soluble in the carbodiimide of water, and the activating reagent of carboxyl is used as in amide synthesis, is also used
In the crosslinking of activated phosphate group, protein and nucleic acid and producing for immune conjugate, normal and n-hydroxysuccinimide
(NHS) or N- hydroxy thiosuccinimide is used in conjunction, to improve coupling efficiency.NHS, that is, n-hydroxysuccinimide, activated carboxyl
In favor of the formation of amido bond.For synthesizing amino acid protective agent, semi-synthetic kanamycins and medicine intermediate.The present invention is implemented
Carboxyl activator in example can be prepared according to the ratio that the mass ratio of EDC and NHS is 1~10:1, and this ratio is conducive to
The two reaches better coupling efficiency, so that the activated carboxylic effect to sulfhydryl compound is more preferable.
Preferably, EDC and NHS can be added to the solution of above-mentioned compounds containing thiol groups under magnetic stirring apparatus effect
In, to reach better mixed effect.
According to some embodiments, after mixing carboxyl activator, the mercapto added with carboxyl activator is adjusted with alkali or acid solution
The pH value of based compound solution, so that its pH value is 4.5~6.5.Preferably, it is adjusted with 1M sodium hydroxide or 1M hydrochloric acid solution
Save pH value.EDC and NHS can reach the activation effect best to carboxyl under conditions of pH value is 4.5~6.5.
According to some embodiments, adjusting the time being mixed after pH value is 10~150min, so that carboxyl activator
Can the carboxyl to sulfhydryl compound adequately activated, preferably to modify collagen.
Wherein, preferably 10~30 DEG C of reaction temperature, further preferred 15~20 DEG C, the reaction time can be 1~8h, preferably
2~6h, more preferably 4~5h.
In the other better embodiments of the present invention, when using the sulfhydryl compound that contains amino as raw material, collagen mercapto
Base derivative can also be prepared by the following method:
A. the solution containing collagen is prepared: by collagenolysis in the acid solution that mass fraction is 0.01~30%.
According to some embodiments, acid solution can be organic acid soln, preferably acetic acid solution.I.e. preferably by the collagen
It is dissolved in the acetic acid solution that mass fraction is 0.01~30%.
B. it prepares the solution of compounds containing thiol groups: sulfhydryl compound is dissolved in distilled water or alkali soluble according to its dissolubility
In liquid or acid solution.
According to some embodiments, aqueous slkali can be strong base solution, for example, sodium hydroxide solution, potassium hydroxide solution,
Calcium hydroxide solution etc., or weak caustic solution, such as ammonia spirit, sodium carbonate liquor, sodium bicarbonate solution etc., preferably hydrogen
Sodium hydroxide solution.Distilled water can make dissolved solution purity higher, and subsequent reactions effect is better.Dissolve sulfhydryl compound
Acid solution can be organic acid soln, preferably acetic acid solution.
C. carboxyl activator is added in the above-mentioned solution containing collagen, after mixing, adjusts its pH value to 4.5-
6.5, continues to mix stirring.
According to some embodiments, carboxyl activator includes EDC and NHS.Carboxyl activator in embodiment of the present invention
It can be prepared according to the ratio that the mass ratio of EDC and NHS is 10:1~1:1, this ratio is conducive to the two and reaches better
Coupling efficiency, so that the activated carboxylic effect to collagen is more preferable.
According to some embodiments, EDC and NHS can be taken to be added under magnetic stirring apparatus effect above-mentioned containing the molten of collagen
In liquid, to reach better mixed effect.
According to some embodiments, after mixing carboxyl activator, the glue added with carboxyl activator is adjusted with alkali or acid solution
The pH value of original solution, so that its pH value is 4.5-6.5.Preferably, pH value is adjusted with 1M sodium hydroxide or 1M hydrochloric acid solution.
EDC and NHS can reach the activation effect best to carboxyl under conditions of pH value is 4.5-6.5.
According to some embodiments, adjusting the time being mixed after pH value is 10~150min, so that carboxyl activator
Can the carboxyl to collagen adequately activated, preferably to modify collagen.
D. the solution containing sulfhydryl compound after activation is mixed with the solution containing collagen, sulfhydryl compound with
The molar ratio of carboxyl is 1~10:1 on collagen, is stirred, mixing time preferably 10~150min, and preferably will mixing
Liquid is transferred in round-bottomed flask, and being placed on temperature is 37 DEG C or less progress isothermal reactions.
According to some embodiments, preferably 10~30 DEG C of reaction temperature, further preferred 15~20 DEG C, the reaction time can be with
For 1~8h, preferably 2~6h, more preferably 4~5h, wherein reaction temperature is not limited to 37 DEG C or less.
E. the reaction solution obtained after reaction is subjected to dialysis 2~5 days, then obtained dialysis product is subjected to freeze-drying 2
After~5 days, collagen thiolated derivative is obtained.It can remove remaining small molecular weight impurity by the means of dialysis, improve gained
Collagen thiolated derivative purity.
It should be noted that can also first carry out step b in the above process, then step a is carried out, will not influence final production
The generation of object.
(3) maleic anhydride modification chitosan solution and sulfhydrylation collagen solution are subjected to Michael addition reaction.
According to some embodiments, carry out Michael addition reaction can be by maleic anhydride modification chitosan solution with
The direct mixed-forming of sulfhydrylation collagen solution utilizes 3D printer sample introduction, and squeezes out contact moudling.It is mixed by 3D printer
It closes maleic anhydride modification chitosan solution and with sulfhydrylation collagen solution the two can be sufficiently mixed according to a certain percentage,
To be more conducive to the progress of reaction and the adjusting of the performance to the hydrogel of generation.
Specifically, firstly, by maleic anhydride that step (1) obtains modification chitosan be dissolved in mass fraction be 0.1~
In 30% acid solution, it is made into the solution of 10~50mg/ml of concentration；Wherein, acid solution can be with organic acid soln, and preferably acetic acid is molten
Liquid.
Secondly, the sulfhydrylation collagenolysis that step (2) obtains is made into the solution of 10~50mg/ml in aqueous slkali, and
Sulfhydrylation collagen in solution is handled through reducing agent.
Wherein, aqueous slkali can be strong base solution, such as sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution
Deng, or weak caustic solution, such as ammonia spirit, sodium carbonate liquor, sodium bicarbonate solution etc., preferably sodium hydroxide solution.
Reducing agent is metal Zn, dithiothreitol (DTT) or hydroquinone.Carry out time preferably 5~50min of reduction treatment.
Then, the N-maleoyl chitosan solution of configuration and sulfhydrylation collagen solution are utilized into 3D printer sample introduction,
Control speed and mixed proportion and the sample position out of liquid discharge sample by computer program, curing molding after two kinds of liquid contacts,
Realize the preparation of novel 3D printing hydrogel.
Above-mentioned preparation process is grafted alpha-beta unsaturated acids structure in chitosan molecule chain respectively, is grafted in tropocollagen molecule chain
Mercapto groups are modified by the active structure to two kinds of biomaterials, using Michael addition reaction, with chemical crosslinking
Mechanism realizes the rapid curing of hydrogel, to realize the 3D printability of material, and by changing the dense of after modifying material
It spends to adjust the mechanical strength and elasticity modulus of hydrogel.The 3D biometric print hydrogel curing rate of preparation of the embodiment of the present invention
Fastly, good biocompatibility, mechanical strength are adjustable, stability in the medium is good, and biodegradation rate is adjustable, application range
Greatly.Its compared with current ultraviolet light cured hydrogel, PH response water gel, temperature-sensitive hydrogel, Ion response hydrogel etc.,
Both improve curing rate, also improve the mechanical strength and elasticity of hydrogel so that the hydrogel of 3D printing have it is good
Supportive and fidelity prevent colloid from collapsing in a short time and the deformation of high degree.In addition, above-mentioned preparation process also provides
A kind of new collagen thiolated modified derivatives, the branched structure and chemical property of the analog derivative can be according to sulfhydryl compound tune
Section；Further, the product of a kind of new sulfydryl and the grafting of alpha-beta unsaturated structure is also provided, the product is in biomedical and group
Engineering field is knitted with important use.
Some embodiments of the present invention additionally provide above-mentioned hydrogel in production biologic medical material or organizational project material
Application on material or 3D printing material.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The preparation method of hydrogel provided in this embodiment includes:
(1) chitosan 1.5g is accurately weighed, is dissolved in the acetic acid that mass fraction is 0.01%.Under magnetic stirring apparatus effect
Substantially after being uniformly dissolved, it is placed in ultrasonator ultrasonic 30min.Maleic anhydride 1.8g is accurately weighed, 5ml acetone, which is added, makes it
It is completely dissolved.By in the addition chitosan acetic acid solution of dissolved maleic anhydride uniform speed slow, stirred under magnetic stirring apparatus
20min or so mixes well it.Uniformly mixed liquid is transferred in 150ml round-bottomed flask, heat-collecting magnetic stirring is placed in
In device, it is arranged 40 DEG C of temperature, heated at constant temperature reacts 2h.Reaction is dialysed three days after stopping, and changes a dialyzate every 5h.Freezing is dry
Obtain MCS product within dry three days.
Chemical equation:
(2) dimercaptosuccinic acid 1.5g is accurately weighed, is added in sodium hydroxide solution and is dissolved.Weigh 1.2g EDC
It is added in above-mentioned solution simultaneously with 300mg NHS, after being sufficiently mixed, adjusts mixed solution PH to 5 with sodium hydroxide solution, set magnetic
10min is stirred on power blender.1.5g collagen is accurately weighed, is dissolved in the acetic acid solution that mass fraction is 0.01%, by collagen
Solution slowly uniformly pours into above-mentioned mixed liquor, and stirring keeps mixing complete.Above-mentioned mixed liquor is transferred in 250ml round-bottomed flask,
It is placed in heat-collecting magnetic stirring device, is arranged 30 DEG C of temperature, heated at constant temperature reacts 4h.Reaction is dialysed three days after stopping, every 5h
Change a dialyzate.Freeze-drying obtains collagen thiol product in three days.
Chemical equation:
(3) MCS 60mg is accurately weighed, is dissolved with the acetic acid solution of mass fraction 1%, successively carries out ultrasonic vibration, nitrogen
Bubble removing is made into the solution that concentration is 10mg/ml.COL-SH60mg is accurately weighed again, is dissolved with sodium hydroxide solution, then
Ultrasonic vibration, nitrogen bubble removing are carried out, the solution that concentration is 10mg/ml is made into, handles 10min with Zn, concussion is uniformly mixed.It will
The MCS solution and COL-SH solution prepared on 3D printer by sampling system sample introduction, computer program control out sample speed and
Printing model is uniformly mixed with to obtain 3D biometric print hydrogel.
Embodiment 2
The preparation method of hydrogel provided in this embodiment includes:
(1) chitosan 1.5g is accurately weighed, is dissolved in the acetic acid that mass fraction is 30%.The base under magnetic stirring apparatus effect
Originally after being uniformly dissolved, it is placed in ultrasonator ultrasonic 35min.Maleic anhydride 4.5g is accurately weighed, 15ml acetone, which is added, makes it
It is completely dissolved.By in the addition chitosan acetic acid solution of dissolved maleic anhydride uniform speed slow, stirred under magnetic stirring apparatus
30min mixes well it.Uniformly mixed liquid is transferred in 150ml round-bottomed flask, is placed in heat-collecting magnetic stirring device,
90 DEG C of temperature of setting, heated at constant temperature react 10h.Reaction is dialysed two days after stopping, and changes a dialyzate every 4h.Freeze-drying two
It obtains MCS product.
Chemical equation:
(2) dimercaptosuccinic acid 1.5g is accurately weighed, is added in sodium hydroxide solution and is dissolved.Weigh 1.2g EDC
It is added in above-mentioned solution simultaneously with 300mg NHS, after being sufficiently mixed, it is left to adjust mixed solution PH to 6.5 with sodium hydroxide solution
The right side sets and stirs 10min on magnetic stirring apparatus.1.5g collagen is accurately weighed, is dissolved in the acetic acid solution that mass fraction is 10%, it will
Collagen solution slowly uniformly pours into above-mentioned mixed liquor, and stirring keeps mixing complete.Above-mentioned mixed liquor is transferred to 250ml round-bottomed flask
In, it is placed in heat-collecting magnetic stirring device, is arranged 20 DEG C of temperature, heated at constant temperature reacts 4h.Reaction is dialysed three days after stopping, every
5h changes a dialyzate.Freeze-drying obtains collagen thiol product in three days.
Chemical equation:
(3) accurately weigh MCS 60mg, with mass fraction be 10% acetic acid solution dissolve, successively carry out ultrasonic vibration,
Nitrogen bubble removing is made into the solution that concentration is 10mg/ml.COL-SH 60mg is accurately weighed again, is carried out with sodium hydroxide solution molten
Solution, then ultrasonic vibration, nitrogen bubble removing are carried out, it is made into the solution that concentration is 10mg/ml, handles 15min with Zn, concussion mixing is equal
It is even.By the MCS solution prepared and COL-SH solution by sampling system sample introduction on 3D printer, computer program controls out sample speed
Degree and printing model, are uniformly mixed with to obtain 3D biometric print hydrogel.
Embodiment 3
The preparation method of hydrogel provided in this embodiment includes:
(1) chitosan 1.5g is accurately weighed, is dissolved in the acetic acid of mass fraction 30%.It is basic under magnetic stirring apparatus effect
After being uniformly dissolved, it is placed in ultrasonator ultrasonic 150min.Maleic anhydride 5.4g is accurately weighed, 20ml acetone, which is added, keeps its complete
Fully dissolved.By in the addition chitosan acetic acid solution of dissolved maleic anhydride uniform speed slow, stirred under magnetic stirring apparatus
150min mixes well it.Uniformly mixed liquid is transferred in 150ml round-bottomed flask, heat-collecting magnetic stirring device is placed in
In, it is arranged 90 DEG C of temperature, heated at constant temperature reacts 10h.Reaction is dialysed three days after stopping, and changes a dialyzate every 5h.Freezing is dry
Obtain MCS product within dry three days.
Chemical equation:
(2) 1.5g dimercaptosuccinic acid is accurately weighed, a certain amount of distilled water is added and is completely dissolved.Weigh 1.2g EDC and
300mg NHS is added in above-mentioned solution simultaneously, after being sufficiently mixed, adjusts mixed solution PH to 5 or so with sodium hydroxide solution, sets
10min is stirred on magnetic stirring apparatus.1.5g collagen is accurately weighed, the acetic acid solution of 0.1% mass fraction is dissolved in, by collagen solution
Above-mentioned mixed liquor is slowly uniformly poured into, stirring keeps mixing complete.Above-mentioned mixed liquor is transferred in 250ml round-bottomed flask, is placed in
In heat-collecting magnetic stirring device, it is arranged 36 DEG C of temperature, heated at constant temperature reacts 4h.Reaction is dialysed three days after stopping, and changes one every 5h
Secondary dialyzate.Freeze-drying obtains COL-SH product in three days.
Chemical equation:
(3) MCS 240mg is accurately weighed, is dissolved with the acetic acid solution that mass fraction is 1%, ultrasonic vibration, nitrogen degasification
Bubble is made into the solution that concentration is 100mg/ml.COL-SH 240mg is accurately weighed, is dissolved with sodium hydroxide solution, ultrasonic vibration,
Nitrogen bubble removing is made into the solution that concentration is 100mg/ml, handles 100min with reducing agent dithiothreitol, concussion mixing is equal
It is even.By MCS the and COL-SH solution prepared by sampling system sample introduction on 3D printer, computer program control out sample speed and
Printing model is uniformly mixed and 3D biometric print hydrogel is prepared.
Embodiment 4
The preparation method of hydrogel provided in this embodiment includes:
(1) chitosan 1.5g is accurately weighed, is dissolved in the acetic acid of mass fraction 15%.It is basic under magnetic stirring apparatus effect
After being uniformly dissolved, it is placed in ultrasonator ultrasonic 150min.Maleic anhydride 1.2g is accurately weighed, 6ml acetone, which is added, keeps its complete
Fully dissolved.By in the addition chitosan acetic acid solution of dissolved maleic anhydride uniform speed slow, stirred under magnetic stirring apparatus
40min mixes well it.Uniformly mixed liquid is transferred in 150ml round-bottomed flask, is placed in heat-collecting magnetic stirring device,
Temperature 60 C is set, and heated at constant temperature reacts 6h.Reaction is dialysed three days after stopping, and changes a dialyzate every 4h.Freeze-drying three
It obtains MCS product.
Chemical equation:
(2) 1.5g dimercaptosuccinic acid is accurately weighed, a certain amount of l distilled water is added and is completely dissolved.Weigh 1.2g EDC and
300mg NHS is added in above-mentioned solution simultaneously, after being sufficiently mixed, adjusts mixed solution PH to 6 with sodium hydroxide solution, sets magnetic force
8min is stirred on blender.1.5g collagen is accurately weighed, the acetic acid solution of 2% mass fraction is dissolved in, collagen solution is slowly equal
Even to pour into above-mentioned mixed liquor, stirring keeps mixing complete.Above-mentioned mixed liquor is transferred in 250ml round-bottomed flask, heat collecting type is placed in
In magnetic stirring apparatus, it is arranged 15 DEG C of temperature, heated at constant temperature reacts 4h.Reaction is dialysed three days after stopping, and changes primary dialysis every 4h
Liquid.Freeze-drying obtains COL-SH product in three days.
Chemical equation:
(3) MCS 240mg is accurately weighed, is dissolved with the acetic acid solution that mass fraction is 1%, ultrasonic vibration, nitrogen degasification
Bubble is made into the solution that concentration is 100mg/ml.COL-SH 240mg is accurately weighed, is dissolved with sodium hydroxide solution, ultrasonic vibration,
Nitrogen bubble removing is made into the solution that concentration is 100mg/ml, handles 100min with reducing agent hydroquinone, concussion is uniformly mixed.
By MCS the and COL-SH solution prepared by sampling system sample introduction on 3D printer, computer program controls out sample speed and beats
Watermark patterns are uniformly mixed and 3D biometric print hydrogel are prepared.
Embodiment 5
The preparation method of hydrogel provided in this embodiment includes:
(1) chitosan 1.5g is accurately weighed, is dissolved in the acetic acid of mass fraction 15%.It is dissolved under magnetic stirring apparatus effect
After uniformly, it is placed in ultrasonator ultrasonic 30min.Maleic anhydride 3.6g is accurately weighed, 20ml acetone, which is added, keeps it completely molten
Solution.By in the addition chitosan acetic acid solution of dissolved maleic anhydride uniform speed slow, stirring 80min under magnetic stirring apparatus makes
It is mixed well.Uniformly mixed liquid is transferred in 150ml round-bottomed flask, is placed in heat-collecting magnetic stirring device, setting temperature
60 DEG C of degree, heated at constant temperature react 5h.Reaction is dialysed three days after stopping, and changes a dialyzate every 3h.Freeze-drying 4 days or so
To MCS product.
Chemical equation:
(2) 1.5g collagen is accurately weighed, is dissolved in the acetic acid solution that mass fraction is 1%, weighs 1.2g EDC and 300mg
NHS is added in above-mentioned solution simultaneously, after being sufficiently mixed, adjusts mixed solution PH to 6 or so with sodium hydroxide solution, sets magnetic force and stir
It mixes and stirs 90min on device.5.3g Ethitanin is accurately weighed, 150ml distilled water is added and is completely dissolved, by cysteine second
Ester solution slowly uniformly pours into above-mentioned mixed liquor, and stirring keeps mixing complete.Above-mentioned mixed liquor is transferred to 250ml round-bottomed flask
In, it is placed in heat-collecting magnetic stirring device, is arranged 20 DEG C of temperature, heated at constant temperature reacts 8h.Reaction is dialysed three days after stopping, every
4h changes a dialyzate.Freeze-drying obtains COL-SH product in three days or so.
Chemical equation:
(3) MCS 120mg is accurately weighed, is dissolved with the acetic acid solution that mass fraction is 1%, ultrasonic vibration, nitrogen degasification
Bubble is made into the solution that concentration is 50mg/ml.COL-SH 120mg is accurately weighed, is dissolved with sodium hydroxide solution, ultrasonic vibration,
Nitrogen bubble removing is made into the solution that concentration is 50mg/ml, and with reducing agent processing 50min or so, concussion is uniformly mixed.It will prepare
MCS and COL-SH solution on 3D printer by sampling system sample introduction, computer program controls out sample speed and printing model,
It is uniformly mixed and 3D biometric print hydrogel is prepared.
Embodiment 6
The preparation method of hydrogel provided in this embodiment includes:
(1) chitosan 1.5g is accurately weighed, is dissolved in the acetic acid of mass fraction 15%.It is dissolved under magnetic stirring apparatus effect
After uniformly, it is placed in ultrasonator ultrasonic 30min.Maleic anhydride 3.6g is accurately weighed, 20ml butanone, which is added, keeps it completely molten
Solution.By in the addition chitosan acetic acid solution of dissolved maleic anhydride uniform speed slow, stirring 90min under magnetic stirring apparatus makes
It is mixed well.Uniformly mixed liquid is transferred in 150ml round-bottomed flask, is placed in heat-collecting magnetic stirring device, setting temperature
10 DEG C of degree, heated at constant temperature react 6h.Reaction is dialysed three days after stopping, and changes a dialyzate every 5h.Freeze-drying 4 days or so
To MCS product.
Chemical equation:
(2) 1.5g collagen is accurately weighed, is dissolved in the acetic acid solution that mass fraction is 1%, weighs 1.2g EDC and 300mg
NHS is added in above-mentioned solution simultaneously, after being sufficiently mixed, adjusts mixed solution PH to 6 or so with sodium hydroxide solution, sets magnetic force and stir
It mixes and stirs 90min on device.5.3g Ethitanin is accurately weighed, a certain amount of distilled water is added and is completely dissolved, by cysteine
Ethyl ester solution slowly uniformly pours into above-mentioned mixed liquor, and stirring keeps mixing complete.Above-mentioned mixed liquor is transferred to 250ml round-bottomed flask
In, it is placed in heat-collecting magnetic stirring device, is arranged 3 DEG C of temperature, heated at constant temperature reacts 8h.Reaction is dialysed three days after stopping, every
3h changes a dialyzate.Freeze-drying obtains COL-SH product in three days or so.
Chemical equation:
(4) MCS 120mg is accurately weighed, is dissolved with the acetic acid solution that mass fraction is 10%, ultrasonic vibration, nitrogen degasification
Bubble is made into the solution that concentration is 50mg/ml.COL-SH 120mg is accurately weighed, is dissolved with sodium hydroxide solution, ultrasonic vibration,
Nitrogen bubble removing is made into the solution that concentration is 50mg/ml, and with reducing agent processing 50min or so, concussion is uniformly mixed.It will prepare
MCS and COL-SH solution on 3D printer by sampling system sample introduction, computer program controls out sample speed and printing model,
It is uniformly mixed and 3D biometric print hydrogel is prepared.
To in embodiment 1 collagen and sulfhydrylation collagen and chitosan and maleic anhydride modification chitosan carry out Fu in
Leaf infrared analysis map respectively obtains Fig. 1 and Fig. 2, and collagen can be seen with sulfhydrylation collagen infared spectrum comparative analysis in Fig. 1
Out, in wave number 900cm-1Near, sulfhydrylation collagen has the weak absorbing peak for being clearly distinguishable from collagen, illustrates on sulfhydrylation collagen
There are C-S key, show that sulfhydryl compound is successfully introduced on collagen chain；Chitosan and maleic anhydride grafted chitosan in Fig. 2
Infared spectrum comparative analysis can see place, in 3000cm-1Near, MCS has any different in the characteristic absorption peak of C-S key, is herein carbon
The stretching vibration peak of c h bond in carbon double bond.In 1650cm-1There is the absorption peak more sharp than C-S key in place, MCS, are herein carbon carbon
The stretching vibration peak of double bond and the absorption overlap of peaks of amide, the stronger absorption at realization near the same wave number.In 1000cm-1It is attached
Closely, MCS occurs being clearly distinguishable from the absorption peak of C-S key, is herein the out-of-plane bending vibration peak of C-H bond on carbon-carbon double bond.?
3400cm-1Place, it is herein the stretching vibration peak of O-H on carboxyl that the wide absorption peak for being clearly distinguishable from C-S key, which occurs, in MCS.By with
Upper comparative analysis shows that maleic anhydride is successfully grafted on chitosan.Meanwhile by scanning electron microscope to the glue in embodiment 1
The surface texture and surface apertures of MCS product in former thiolated derivative and embodiment 11 and the hydrogel after solidifying are swept
Retouch Electronic Speculum (SEM) analysis.As a result successively such as Fig. 3, Fig. 4, Fig. 5, shown in Fig. 6.Hydrogel surface occurs after sem analysis shows solidification
Hole after many crosslinkings, illustrates that cross-linking reaction carries out very complete, further illustrates that the sulfhydrylation collagen that is prepared can be with
Successfully prepare rapid-solidified hydrogel.
Further, it by being tested with instron mechanics tester hydrogel obtained in embodiment 11, uses
10N sensor is placed in sensor compress substrate, sets test parameter according to the size of sample, test curve is mutated
Afterwards, stop compression, the automatically derived elastic mould value of system.
Test parameter are as follows: long 10mm, wide 8mm, high 5mm, compression speed 0.8mm/min.Test results are shown in figure 7,
Compression displacement is 3.38mm, and compression stress is broken when being 0.049MPa, so that measuring elasticity modulus is 0.107MPa, because
This, it can be seen that the hydrogel has preferable mechanical strength and elastic property.
In conclusion passing through by using the collagen of the acylated chitosan of maleic anhydride and sulfhydrylation as raw material
Michael addition reaction can realize that raw material by liquid to solid fast transition, substantially increase the printability of material, lead to
The concentration both overregulated can be realized solidify after hydrogel elastic modulus adjusting, the application range of material, this method can be expanded
The hydrogel of preparation has important use in biomedical and field of tissue engineering technology, and curing rate is fast, good biocompatibility, machine
Tool intensity is adjustable, stability in the medium is good, and biodegradation rate is adjustable, and application range is big.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of preparation method of hydrogel characterized by comprising by maleic anhydride modification chitosan and sulfhydrylation collagen into
Row Michael addition reaction；The general formula of the maleic anhydride modification chitosan are as follows:The sulfydryl
Change the general formula of collagen are as follows:
Wherein, R1The residue moiety of amino is removed for shitosan macromolecule；R2For hydrogen atom, alkyl or alkylidene；R3For hydroxyl,
Hydroxy derivatives, amino or aminoderivative；R4The residue moiety of amino and carboxyl is removed for collagenous polymer；R5For alkylidene
Or substituted alkylene.
2. the preparation method of hydrogel according to claim 1, which is characterized in that before progress Michael addition reaction
The sulfhydrylation collagen is handled by reducing agent, it is preferable that the reducing agent is for metal Zn, dithiothreitol (DTT) or to benzene
Diphenol.
3. the preparation method of hydrogel according to claim 1, which is characterized in that maleic anhydride is modified chitosan solution
Michael addition reaction is carried out with sulfhydrylation collagen solution, it is preferable that the concentration of maleic anhydride modification chitosan solution is
10~100mg/ml, pH value is less than 7；Preferably, 10~100mg/ml of the concentration of the sulfhydrylation collagen solution, pH value are greater than
7。
4. the preparation method of hydrogel according to claim 3, which is characterized in that carry out the Michael addition reaction
It is by maleic anhydride modification chitosan solution and the direct mixed-forming of the sulfhydrylation collagen solution or to utilize 3D printer
Sample introduction, and make the maleic anhydride modification chitosan solution and sulfhydrylation collagen solution haptoreaction in extrusion process
After form.
5. the preparation method of hydrogel according to claim 1, which is characterized in that the maleic anhydride modification chitosan
It is reacted after preparing the following steps are included: the acid solution containing chitosan and the ketone solution containing maleic anhydride are mixed, then
It dialysed, be freeze-dried；Preferably, on the chitosan chain molar ratio of free amine group and the maleic anhydride be 1:1~
10。
6. the preparation method of hydrogel according to claim 5, which is characterized in that the acid solution containing chitosan and
The reaction temperature that the ketone solution containing maleic anhydride is reacted be 10~90 DEG C, preferably 40~90 DEG C, the reaction time
For 2~10h.
7. the preparation method of hydrogel according to claim 1, which is characterized in that the preparation of the sulfhydrylation collagen includes
Following steps: by containing the collagen solution with containing the sulfhydryl compound solution under the action of carboxyl activator into
Row reaction；Preferably, the solution containing the collagen is that the collagenolysis is molten for 0.01~30% acid in mass fraction
In liquid, the solution containing the sulfhydryl compound is that the sulfhydryl compound according to its dissolubility, is dissolved in distilled water or alkali
In solution or acid solution, the carboxyl activator includes EDC and NHS, and the mass ratio of the EDC and the NHS are 1~10:1；
It is further preferred that the molar ratio of amino or carboxyl is 1~10:1 on the sulfhydryl compound and the collagen；Preferably, instead
Answering temperature is 37 DEG C hereinafter, more preferably 10~30 DEG C, further preferably 15~20 DEG C, the reaction time is 4~12h, and is incited somebody to action
Mixed liquor after reaction is dialysed, is freeze-dried；Preferably, the sulfhydryl compound is to contain carboxyl and sulfydryl simultaneously
Or the compound containing amino and sulfydryl simultaneously, it is highly preferred that the sulfhydryl compound includes acetylcysteine, half Guang ammonia
Acid, dimercaptosuccinic acid, dimercaptosuccinic acid, 2- sulfydryl-acidum nicotinicum, acthiol-J, Ethitanin and 4- ammonia
Any one of base -3- mercaptobenzoic acid.
8. the preparation method of hydrogel according to claim 7, which is characterized in that the preparation of the sulfhydrylation collagen is first
The carboxyl in the solution containing the sulfhydryl compound or the collagen is activated using the carboxyl activator, is then mixed
It closes the solution containing the sulfhydryl compound and the solution containing the collagen is reacted.
9. a kind of hydrogel, which is characterized in that be prepared by the preparation method of hydrogel according to any one of claims 1 to 8
It arrives, the general formula of the hydrogel are as follows:
Any one of；
Wherein, R1The residue moiety of amino is removed for shitosan macromolecule；R2For hydrogen atom, alkyl or alkylidene；R3For hydroxyl,
Hydroxy derivatives, amino or aminoderivative；R4The residue moiety of amino and carboxyl is removed for collagenous polymer；R5For alkylidene
Or substituted alkylene.
10. hydrogel according to claim 9 is in production biologic medical material or tissue engineering material or 3D printing material
On application.
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| CN110680959A (en) * | 2019-10-31 | 2020-01-14 | 江苏地韵医疗科技有限公司 | Hydrogel for repairing multiple cross-linked meniscus and preparation method thereof |
| CN111187429A (en) * | 2019-06-18 | 2020-05-22 | 吾奇生物医疗科技(镇江)有限公司 | Double-crosslinking collagen hydrogel material, and preparation method and application thereof |
| CN111333878A (en) * | 2019-05-23 | 2020-06-26 | 吾奇生物医疗科技(镇江)有限公司 | Double-crosslinked chitosan hydrogel and preparation method and application thereof |
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| CN112126080A (en) * | 2019-06-24 | 2020-12-25 | 中国科学院苏州纳米技术与纳米仿生研究所 | Photocuring hydrogel based on sulfydryl-alkene click reaction, and preparation method and application thereof |
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| CN115975071A (en) * | 2023-01-12 | 2023-04-18 | 大连大学 | Maleylation chitosan macromolecule cross-linking agent and preparation method thereof |
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| CN109749098A (en) * | 2019-01-30 | 2019-05-14 | 福州大学 | Double cross-linked network high intensity gelatin hydrogels of a kind of physical/chemical and preparation method thereof |
| CN109749098B (en) * | 2019-01-30 | 2021-05-18 | 福州大学 | Physical/chemical double-crosslinking-network high-strength gelatin hydrogel and preparation method thereof |
| CN111333878A (en) * | 2019-05-23 | 2020-06-26 | 吾奇生物医疗科技(镇江)有限公司 | Double-crosslinked chitosan hydrogel and preparation method and application thereof |
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| CN112126080A (en) * | 2019-06-24 | 2020-12-25 | 中国科学院苏州纳米技术与纳米仿生研究所 | Photocuring hydrogel based on sulfydryl-alkene click reaction, and preparation method and application thereof |
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| CN110680959A (en) * | 2019-10-31 | 2020-01-14 | 江苏地韵医疗科技有限公司 | Hydrogel for repairing multiple cross-linked meniscus and preparation method thereof |
| CN110680959B (en) * | 2019-10-31 | 2021-12-03 | 江苏地韵医疗科技有限公司 | Hydrogel for repairing multiple cross-linked meniscus and preparation method thereof |
| CN111617747A (en) * | 2020-05-11 | 2020-09-04 | 贵州大学 | Chitosan/nano-metal composite hydrogel and preparation method and application thereof |
| CN111617747B (en) * | 2020-05-11 | 2022-05-31 | 贵州大学 | Chitosan/nano-metal composite hydrogel and preparation method and application thereof |
| CN115975071A (en) * | 2023-01-12 | 2023-04-18 | 大连大学 | Maleylation chitosan macromolecule cross-linking agent and preparation method thereof |
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