CN104892962B - A kind of preparation method and applications of the controllable self-crosslinking hyaluronic acid gel of sulfydryl/disulfide bond - Google Patents
A kind of preparation method and applications of the controllable self-crosslinking hyaluronic acid gel of sulfydryl/disulfide bond Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and applications of the controllable self-crosslinking hyaluronic acid gel of sulfydryl/disulfide bond, belong to technical field of biological material.The hydrogel is using the sulfydryl hyaluronic acid compound with free sulfhydryl group as raw material, by adjusting sulfydryl density（10%—60%）, gelling temp（4 DEG C, 37 DEG C）With the molecular weight of hyaluronic acid（0.1M, 0.3M, 1M）, using the redox conversion characteristic between sulfydryl and disulfide bond, so as to control formation time, degradation time and the mechanical characteristic of gel, construct the controllable injection type progress in Intelligent Hydrogel with good three-dimensional network cross-linked structure.Meanwhile such hydrogel component composition is single, plastic is front and rear to be introduced without exogenous toxicant, has good biocompatibility and degradation property.Research is found based on more than, and such controllable self-crosslinking high molecular polymer can be used for minimally invasive reparation and the adjustable Three-dimensional cell culture support of structure intelligence of organizational project damage in situ.
Description
Technical field
The invention belongs to technical field of biological material, and in particular to a kind of controllable self-crosslinking hyaluronic acid water-setting of sulfydryl/disulfide bond
Glue and its preparation method and application.
Technical background
Organizational engineering（tissue engineering）, also someone be called " regenerative medicine ", refer to living using biology
Property material, by vitro culture or the method for structure, reconstruction or the technology for repairing organ and tissue.This concept is by state of the U.S.
The science fund committee of family proposed in 1987, was by its formal definition within 1988：Using life science and the principle of engineering science
With technology, on the basis of the institutional framework and functional relationship under the correctly normal and pathology two states of understanding mammal,
Research and develop the biosubstitute for repairing, safeguarding, promote the function after the various tissues of human body or organ damage and form
One new branch of science.
Bio-medical material（Biomedical Materials）, may be also referred to simply as biomaterial（Biomaterials）,
It is for pathological tissues, organ or the new high-tech material for promoting its function to be diagnosed, treated, repaired or replaced to organism
Material.And one of key technology of organizational engineering is prepared with good biocompatibility and can dropped by human body with biomaterial
Solve the cytoskeleton absorbed.Gel state is the intermediateness of solid and liquid, and hydrogel is to refer to that swelling occurs simultaneously in water
Hydrophilic crosslinked Space network of polymer of the large quantity of moisture without dissolving can be kept.Hydrogel is a kind of preferably biological material
Material, by being simply modified, similar with natural extracellular matrix, gratifying physics and chemical property can be obtained, simultaneously
Good permeability is shown for oxygen, nutriment, cell metabolite and water-soluble metal ion.Prepare the parent of hydrogel
Aqueous high molecular is by sources divided into natural polymer and synthesis macromolecule.Natural hydrogel include collagen, gelatin, fibrin,
Polysaccharide etc., artificial synthesized hydrogel include synthesis class polypeptide, PEG and its derivative, PMMA and its derivative, PLGA and its derivative
Thing etc..
Hyaluronic acid（Hyaluronic acid, HA）It is one kind in glycosaminoglycan, possesses good water solubility, biology
Compatibility and degradation property, it played an important role in the secretion of extracellular matrix and proteoglycans forming process is promoted；
However, fragile physical and mechanical properties and degradation speed too fast in vivo limits its application prospect.In order to be had
The natural polymer hydrogel of preferable physical and mechanical properties and biodegradation rate, the mode of chemical crosslinking are widely used in
In preparation process.The higher functional group of the chemisms such as carboxyl, hydroxyl, amino is often applied in chemical crosslink reaction, is commonly used
Chemical cross-linking agent typically contain difunctional, such as diamines, two hydrazines, dialdehyde, glycol etc., but these crosslinking agents generally have it is thin
Cellular toxicity, if residual will influence the biocompatibility of hydrogel material.Need to study a kind of new chemical crosslinking macromolecule
Hydrogel, to avoid the cytotoxicity added additional chemical material in cross-linking reaction and brought.Meanwhile modern medicine requires biology
Material can have plastic and controllability in use, realize minimally invasive therapeutic effect.
The content of the invention
In view of the above-mentioned problems, the invention provides a kind of controllable self-crosslinking hyaluronic acid gel of sulfydryl/disulfide bond.It is described
Hydrogel physical property is controllable, has good cell compatibility and biodegradability, available for organizational project damage in situ
Reparation or the three-dimensional cell support for building controlled absorbed.
The present invention is achieved through the following technical solutions：
A kind of controllable self-crosslinking hyaluronic acid gel of sulfydryl/disulfide bond, with sulfydryl-hyaluronic acid with free sulfhydryl group
Compound is raw material, by dissolution of raw material, stable disulfide bond is then formed by oxidation reaction between sulfydryl, so as to build three-dimensional
The progress in Intelligent Hydrogel of chemical crosslinking, by adjusting sulfydryl-hyaluronic acid high molecular polymer surface sulfydryl density, gelling temp
With the molecular weight of hyaluronic acid, using the redox conversion characteristic between sulfydryl and disulfide bond, so as to control the formation of gel
Time, degradation time and mechanical characteristic, controllable intelligent type hydrogel of the structure with good three-dimensional network cross-linked structure.As
Optional mode, the reaction condition of the three dimensional chemical crosslinking is pH value 7.4~8.0.Under reducing agent effect, in the hydrogel
Disulfide bond can be broken, the rapid avalanche of gel structure, be degraded to the good hyaluronic acid monomolecular substance of biocompatibility.
Alternately, sulfydryl-hyaluronic acid compound with free sulfhydryl group is using hyaluronic acid as raw material, is passed through
Half Guang ammonia is modified what is obtained.
Alternately, the molecular weight of the hyaluronic acid is：0.1MDa~1.0MDa.
Alternately, half Guang ammonia grafting rate in the sulfydryl-hyaluronic acid compound is；10%~60%；
Alternately, the sulfydryl-hyaluronic acid compound concentration is 1 ~ 5%；Further can be 1.0% or
2.0% or 3.0%.
By the molecular weight for adjusting hyaluronic acid（0.1 MDa、0.3 MDa、1 MDa）, half Guang ammonia grafting rate
（11.28%、12.41%、14.69%、29.13%、33.54%、37.47%、51.47%、55.44%、60.56%）, sulfydryl-hyalomitome
The concentration of acid compound（1.0 wt %, 2.0 wt% or 3.0%）, gel environment temperature (4 DEG C, 37 DEG C), can accurately control
The time that hydrogel is formed（From 6 minutes to more than 2 hours）.
Alternately, the disulfide bond in the hydrogel can be in dithiothreitol (DTT)（DTT）Or glutathione（GSH）Deng
Under the conditions of reproducibility small molecule is existing, disconnect the disulfide bond between hyaluronic acid, so that reductive cleavage occurs for hydrogel.
Further, the concentration in system of the reproducibility small molecule is 0.1 ~ 10mM.By adjust hyaluronic acid molecular weight,
The grafting rate of half Guang ammonia, the concentration of reproducibility small molecule, the degree of hydrogel reductive cleavage can be controlled（From a few minutes to several small
When）.
Alternately, the hydrogel can be specific shape by mould molding.
Alternately, the hydrogel is injectable, can by controlling the gel time or environment temperature of material
It is in before the injection fluid state to make product, forms gel state after injection.The hydrogel have injectable, can it is in situ into
Glue, gel process do not produce or remained the advantages that containing cytotoxic substance.The hydrogel can be planted by way of injection
Enter in vivo, and hydrogel is formed in site of tissue damage, realize the in-situ immobilization of tissue damage.
The invention provides a kind of hydrogel plastic based on sulfydryl-hyaluronic acid compound with free sulfhydryl group with splitting
Enzymatic hydrolysis system, the hydrogel plastic include the above-mentioned controllable self-crosslinking hyaluronic acid gel of sulfydryl/disulfide bond with cracking system
With reproducibility small molecule.Its gelation time can be adjusted flexibly with cracking degree according to using needs, and the system has well thin
Born of the same parents' compatibility, gel process do not produce the cytotoxic material of tool, the introducing of no exogenous material, are advantageous to the increasing of cell
Grow；With good biodegradability, reducing substances caused by cell itself can also accelerate disulfide bond degraded, gel degradation
Process does not produce the cytotoxic material of tool, is advantageous to the secretion of cellular matrix and the reparation of site of tissue damage.
Present invention also offers a kind of preparation method of the hyaluronic acid gel of above-mentioned disulfide bond crosslinking, with hyalomitome
Acid is raw material, is modified to obtain the sulfydryl with free sulfhydryl group-hyaluronic acid high molecular polymer by half Guang ammonia（It is spongy solid
Body）, dissolved, disulfide bond, water of the structure with three dimensional chemical cross-linked structure then formed by oxidation reaction between sulfydryl
Gel.
As optional, sulfydryl-hyaluronic acid compound after dissolving can be placed in corresponding mould and carry out gel reaction
To obtain the hydrogel of given shape；Parenteral solution can also be made in sulfydryl-hyaluronic acid compound after dissolving, made after injection
It is in target area gel in-situ.
Alternately, in above-mentioned preparation method, using hyaluronic acid as raw material, being modified by half Guang ammonia is had
The sulfydryl of free sulfhydryl group-hyaluronic acid high molecular polymer, is dissolved in the PBS of pH=7.4, then adjusts pH
Value, by forming the reaction of disulfide bond between sulfydryl, forms the hydrogel of three dimensional chemical crosslinking to 7.4~8.0.
As optional, using the pH value for the NaOH solution for adding 1M.
Alternately, in above-mentioned preparation method, following steps are specifically included：
1）Half Guang ammonia of hyaluronic acid is modified：
The carboxyl on amino and hyaluronic acid on half Guang ammonia is in N- succinimides（NHS）, 1- ethyls-(3- dimethyl
Aminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC HCl) catalysis under carry out acid amides reaction, prepare raw material sulfydryl-hyalomitome
Acid compound；
2）Chemical crosslinking:
By step 1）In preparation the dissolving of half Guang ammonia modified hyaluronic acid, then pass through and disulfide bond formed between sulfydryl
Reaction, form the hydrogel of three dimensional chemical crosslinking.
Alternately, in above-mentioned preparation method, following steps are specifically included：
1）Half Guang ammonia of hyaluronic acid is modified：
Sodium Hyaluronate is in N- succinimides（NHS）, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloric acid
Salt
(EDCHCl) under catalysis with half Guang ammonia（CSH·HCl）Hydrochloric acid reactant salt, then adds dithiothreitol (DTT)
（DTT）, after reaction terminates, dialyse in deionized water, be freeze-dried, obtain half Guang ammonia modified hyaluronic acid；
2）Chemical crosslinking:
By step 1）In half Guang ammonia modified hyaluronic acid of preparation be dissolved in the PBS of pH=7.4, Ran Houtong
Cross mercapto
The reaction of disulfide bond is formed between base, forms the hydrogel of three dimensional chemical crosslinking.
Dithiothreitol (DTT) is added in the preparation process of half Guang ammonia modified hyaluronic acid（DTT）It can avoid in the middle part of preparation process
Point half Guang ammonia modified hyaluronic acid occurs that disulfide bond crosslinking causes to obtain pars shape product after freeze-drying can not be follow-up
Dissolved in step.Dialysis procedure can remove unnecessary dithiothreitol (DTT) again（DTT）, avoid its residual from influenceing follow-up step in the product
Rapid effect.
Alternately, in above-mentioned preparation method, the step 1）Specially：
Sodium Hyaluronate is dissolved in deionized water, is firstly added N- succinimides（NHS）, fully dissolving；Then
1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC HCl) powder is added, with 1M NaOH and 1M
HCl solution adjusts pH=4.75 of reaction solution, reacts 2 hours；Then add half Guang ammonia（CSH•HCl）HCI solution, reaction
24 hours；PH=8.5 of reaction solution finally are adjusted with 1M NaOH solution, add dithiothreitol (DTT)（DTT）Solution, reaction 12
Individual hour.
After reaction terminates, pH=3.0-3.5 of reaction solution is adjusted with 1M HCl solution, in pH=3.0-3.5 deionized water
Middle dialysis 72h, freeze-drying, obtains half Guang ammonia modified hyaluronic acid.
Alternately, in above-mentioned preparation method, carboxyl in Sodium Hyaluronate：N- succinimides：1- ethyls-
(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate：Half Guang ammonia salt hydrochlorate：The ratio between amount of material of dithiothreitol (DTT) is 1：
2：2~4:1~4:3~12.The ratio between amount of material by adjusting half Guang ammonia, hyaluronic acid, NHS and EDCI, can control half Guang ammonia
Substitution value.Alternately, the molecular weight of above-mentioned hyaluronic acid is：0.1MDa~1.0MDa.
Alternately, above-mentioned steps 2）Middle sulfydryl-hyaluronic acid compound solution concentration is 1.0%-3.0%.
Present invention also offers a kind of application of the hyaluronic acid gel of above-mentioned disulfide bond crosslinking, it is characterised in that
It is used as Three-dimensional cell culture support or is made into injection aquagel.Further, the Three-dimensional cell culture support
Organizational project can be used as especially cartilage tissue engineered；The injection aquagel can be used for beauty, prevent wound adhesion, original position
Injury repair, organizational project etc..
Present invention also offers the hyaluronic acid water-setting of a kind of Three-dimensional cell culture system, including above-mentioned disulfide bond crosslinking
Glue and the cell being uniformly distributed therein.Cell can be in hydrogel forms whole three-dimensional rack uniformly in the cultivating system
Distribution, overcomes cell more difficult the defects of moving to internal stent in existing three-dimensional rack, and the hydrogel also has good
Mechanical performance, and after cell is bred to certain scale in the bracket, reducing substances caused by cell itself or exogenous
The reproducibility small molecule of addition can make the disulfide bonds in hydrogel, and hydrogel cracks, hyalomitome caused by cracking
The 3-D solid structure for the similar three-dimensional tissue being made up of completely cell with extracellular matrix can be formed after acid degradation, and production of degrading
Thing can quickly be absorbed by body, cultivating system is had good biocompatibility, the dimensional culture available for various kinds of cell.It is logical
Cultivating system can be realized by crossing the regulation and control molecular weight of hyaluronic acid, the grafting rate of half Guang ammonia, the parameter such as concentration of reproducibility small molecule
The controlled degradation of middle hydrogel scaffold.
Present invention also offers a kind of construction method of Three-dimensional cell culture system, comprise the following steps：
（1）Sulfydryl-hyaluronic acid compound with free sulfhydryl group is dissolved in culture medium, cell is then added and hangs
Liquid, it is well mixed, forms mixed liquor；
（2）By forming the reaction of disulfide bond between sulfydryl, the hyaluronic acid in gained mixed liquor is set to form three dimensional chemical
The hydrogel of crosslinking and by cell encapsulation in wherein.
Alternately, in the construction method of above-mentioned Three-dimensional cell culture system, step（2）Specially：By step
（1）Obtained mixed liquor is added in mould, then in 37 DEG C, 0.5%CO2Plastic process is carried out in cell culture incubator, finally
The hydrogel for wrapping up cell is removed from mould, added in culture medium, in 37 DEG C, 0.5%CO2Trained in cell culture incubator
Support；
Or directly by step（1）Obtained mixed liquor is expelled in organisms, makes hyaluronic acid in organisms
Generation three dimensional chemical is cross-linked to form hydrogel.
Alternately, it is 1 ~ 3%, preferably 3% that the sulfydryl-hyaluronic acid compound, which is dissolved in the concentration after culture medium,.
Base-hyaluronic acid compound molecular weight is 0.1MDa~1.0MDa, preferably 0.3MDa.
Alternately, cell-seeding-density is 5 × 106cells/mL.The cell can be that nascent rabbit cartilage is thin
Born of the same parents（chondrocytes）, nascent rabbit bone mescenchymal stem cell（MSCs）, l cell（L929 cells）Deng.
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.
Beneficial effects of the present invention：
The raw material hyaluronic acid of hydrogel application of the present invention is natural polymer, has good water solubility, water suction
Property, gained hydrogel physical property is controllable, has easy gel formats, and gel process is not produced containing cytotoxic thing
Matter, there is good cell compatibility and biodegradability, available for organizational project original position injury repair or for building three
Tie up cytoskeleton.
Brief description of the drawings：
Fig. 1 is the syntheti c route figure of hydrogel of the present invention；
Fig. 2 is the mechanical property testing result of each hydrogel material prepared in embodiment 1-9；
Fig. 3 is the degradation characteristic under the conditions of the reducing agent DTT of various concentrations（Weight change）, three points from top to bottom in figure
The DTT concentration of figure is followed successively by 0.1mM, 1mM, 10Mm；
Fig. 4 is rabbit cartilage cell of being come into being described in embodiment 12（chondrocytes）Trained altogether with hydrogel of the present invention
Support result（It is respectively from top to bottom：3 days, 7 days, 14 days, 21 days；It is respectively from left to right：It is copolymerized under the conditions of burnt different multiples and sees
Examine, tem observation）；
Fig. 5 is rabbit bone mescenchymal stem cell of being come into being described in embodiment 13（MSCs）Cultivation results（Distinguish from top to bottom
For：3 days, 7 days, 14 days, 21 days；It is respectively from left to right：It is copolymerized observation, tem observation under the conditions of burnt different multiples）；
Fig. 6 is the cultivation results of MSC cells described in embodiment 13（It is respectively from top to bottom：3 days, 7 days, 14 days, 21
My god；It is respectively from left to right：It is copolymerized observation, tem observation under the conditions of burnt different multiples）；
Fig. 7 is that the hydrogels of chondrocyte cells is wrapped up in embodiment 16 is (in figure left in small white mouse In vivo culture 14 days
Side), 28 days (in figure right side) laser co-focusing photo afterwards；
Fig. 8 is that the hydrogels of chondrocyte cells is wrapped up in embodiment 16 is (in figure left in small white mouse In vivo culture 14 days
Side), 28 days (in figure right side) laser co-focusing photo afterwards.
Embodiment：
Embodiment by the following examples is described in further detail to the above of the present invention again.But
The scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.The spiritual and former of the present invention is not being departed from
Any modification made within then, and the equivalent substitution made according to ordinary skill knowledge and customary means or change
Enter, all should include within the scope of the present invention.
Embodiment 1
（1）400mg Sodium Hyaluronates are dissolved in 100mL deionized waters, are firstly added 230mg N- succinimides
（NHS）, fully dissolving；Then 385mg 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC is added
HCl) powder, pH=4.75 of reaction solution is adjusted with 1M NaOH and 1M HCl solution, react 2 hours；Then add 10mL
11.36mg/mL half Guang ammonia（CSH·HCl）Hydrochloride, react 24 hours；Finally with 1M NaOH solution adjustment reaction solution
PH=8.5, add 10mL46.5mg/ml dithiothreitol (DTT)（DTT）Solution, react 12 hours.
（2）After reaction terminates, pH=3.0-3.5 of reaction solution is adjusted with 1M HCl solution, pH=3.0-3.5 go from
Dialyse 72h in sub- water, freeze-drying, obtains half Guang ammonia modified hyaluronic acid.
Wherein, Sodium Hyaluronate molecular weight is 0.1MDa, carboxyl in Sodium Hyaluronate：N- succinimides：1- ethyls-
(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate：Half Guang ammonia salt hydrochlorate：The ratio between amount of material of dithiothreitol (DTT) is 1:
2:2:1:3。
（3）30mg sulfydryls-hyaluronic acid compound is dissolved in the PBS of 1mL pH=7.4, first in frozen water
1M NaOH solution adjustment pH=7.4~8.0 are added under the conditions of bath（The solution remains to keep under the conditions of 4 DEG C after 2 hours
Good mobility）, solution is then poured into a diameter of 8.5mm, highly in 3.0mm cylindrical die, to be finally placed on
In 37 DEG C of incubator, react to form disulfide bond and prepare hydrogel by sulfydryl.Gel time under the conditions of 37 DEG C is 34 minutes.
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.1MDa, and half Guang ammonia grafting rate is 14.69%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 3.3kPa.
Embodiment 2
（1）400mg Sodium Hyaluronates are dissolved in 100mL deionized waters, are firstly added 230mg N- succinimides
（NHS）, fully dissolving；Then 575mg 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC is added
HCl) powder, pH=4.75 of reaction solution is adjusted with 1M NaOH and 1M HCl solution, react 2 hours；Then add 10mL
22.72mg/mL half Guang ammonia（CSH·HCl）Hydrochloride, react 24 hours；Finally with 1M NaOH solution adjustment reaction solution
PH=8.5, add 10mL93.0mg/ml dithiothreitol (DTT)（DTT）Solution, react 12 hours.
（2）After reaction terminates, pH=3.0-3.5 of reaction solution is adjusted with 1M HCl solution, pH=3.0-3.5 go from
Dialyse 72h in sub- water, freeze-drying, obtains half Guang ammonia modified hyaluronic acid.
Wherein, Sodium Hyaluronate molecular weight is 0.1MDa, carboxyl in Sodium Hyaluronate：N- succinimides：1- ethyls-
(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate：Half Guang ammonia salt hydrochlorate：The ratio between amount of material of dithiothreitol (DTT) is 1:
2:3:2:6。
（3）30mg sulfydryls-hyaluronic acid compound is dissolved in the PBS of 1mL pH=7.4, first in frozen water
1M NaOH solution adjustment pH=7.4~8.0 are added under the conditions of bath,（The solution remains to keep under the conditions of 4 DEG C after 2 hours
Good mobility）Then solution is poured into a diameter of 8.5mm, highly in 3.0mm cylindrical die, to be finally placed on 37
DEG C incubator in, react to form disulfide bond and prepare hydrogel by sulfydryl.Gel time under the conditions of 37 DEG C is 28 minutes
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.1MDa, and half Guang ammonia grafting rate is 37.47%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 12.4kPa.
Embodiment 3
（1）400mg Sodium Hyaluronates are dissolved in 100mL deionized waters, are firstly added 230mg N- succinimides
（NHS）, fully dissolving；Then 770mg 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC is added
HCl) powder, pH=4.75 of reaction solution is adjusted with 1M NaOH and 1M HCl solution, react 2 hours；Then add 10mL
45.44mg/mL half Guang ammonia（CSH·HCl）Hydrochloride, react 24 hours；Finally with 1M NaOH solution adjustment reaction solution
PH=8.5, add 10mL186.0mg/ml dithiothreitol (DTT)（DTT）Solution, react 12 hours.
（2）After reaction terminates, pH=3.0-3.5 of reaction solution is adjusted with 1M HCl solution, pH=3.0-3.5 go from
Dialyse 72h in sub- water, freeze-drying, obtains half Guang ammonia modified hyaluronic acid.
Wherein, Sodium Hyaluronate molecular weight is 0.1MDa, carboxyl in Sodium Hyaluronate：N- succinimides：1- ethyls-
(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate：Half Guang ammonia salt hydrochlorate：The ratio between amount of material of dithiothreitol (DTT) is 1:
2:4:4:12。
（3）30mg sulfydryls-hyaluronic acid compound is dissolved in the PBS of 1mL pH=7.4, first in frozen water
1M NaOH solution adjustment pH=7.4~8.0 are added under the conditions of bath,（The solution remains to keep under the conditions of 4 DEG C after 2 hours
Good mobility）Then solution is poured into a diameter of 8.5mm, highly in 3.0mm cylindrical die, to be finally placed on 37
DEG C incubator in, react to form disulfide bond and prepare hydrogel by sulfydryl.Gel time under the conditions of 37 DEG C is 18 minutes
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.1MDa, and half Guang ammonia grafting rate is 60.56%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 22.0kPa.
Embodiment 4
With reference to the methods described of embodiment 1, it the difference is that only：Sodium Hyaluronate molecular weight is 0.3MDa.
Gained sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 12.41%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 7.7kPa.
Obtained product is still keeping good mobility under the conditions of 4 DEG C after 2 hours before forming gel, 37 DEG C
Under the conditions of gel time be 25 minutes.
Embodiment 5
With reference to the methods described of embodiment 2, it the difference is that only：Sodium Hyaluronate molecular weight is 0.3MDa.
Gained sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 33.54%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 16.2kPa.
Obtained product is still keeping good mobility, 37 DEG C of bars under the conditions of 4 DEG C after 2 hours before forming gel
Gel time under part is 18 minutes.
Embodiment 6
With reference to the methods described of embodiment 2, it the difference is that only：Sodium Hyaluronate molecular weight is 0.3MDa
Gained sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 55.44%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 35.2kPa.
Obtained product is still keeping good mobility under the conditions of 4 DEG C before gel is formed after 2 hours, and 37
Gel time under the conditions of DEG C is 11 minutes.
Embodiment 7
With reference to the methods described of embodiment 1, it the difference is that only：Sodium Hyaluronate molecular weight is 1.0 MDa.
Gained sulfydryl-hyaluronic acid compound molecular weight is 1.0MDa, and half Guang ammonia grafting rate is 11.28%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 9.2kPa.
Obtained product is still keeping good mobility under the conditions of 4 DEG C after 2 hours before forming gel, 37 DEG C
Under the conditions of gel time be 25 minutes.
Embodiment 8
With reference to the methods described of embodiment 2, it the difference is that only：Sodium Hyaluronate molecular weight is 1.0 MDa.
Gained sulfydryl-hyaluronic acid compound molecular weight is 1.0MDa, and half Guang ammonia grafting rate is 29.13%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 17.7kPa.
Obtained product was still keeping good mobility under the conditions of 4 DEG C in 116 minutes before forming gel, 37 DEG C
Under the conditions of gel time be 15 minutes.
Embodiment 9
With reference to the methods described of embodiment 2, it the difference is that only：Sodium Hyaluronate molecular weight is 1.0 MDa
Gained sulfydryl-hyaluronic acid compound molecular weight is 1.0MDa, and half Guang ammonia grafting rate is 51.47%, sulfydryl-transparent
Matter acid compound solution concentration 3.0%, 1% deformation quantity, under the conditions of 10Hz frequency detectings, storage modulus 42.8kPa.
Obtained product was still keeping good mobility under the conditions of 4 DEG C before gel is formed in 105 minutes, and 37
Gel time under the conditions of DEG C is 6 minutes.
Embodiment 10
（1）400mg Sodium Hyaluronates are dissolved in 100mL deionized waters, are firstly added 230mg N- succinimides
（NHS）, fully dissolving；Then 385mg 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC is added
HCl) powder, pH=4.75 of reaction solution is adjusted with 1M NaOH and 1M HCl solution, react 2 hours；Then add 10mL
11.36mg/mL half Guang ammonia（CSH·HCl）Hydrochloride, react 24 hours.
（2）After reaction terminates, pH=3.0-3.5 of reaction solution is adjusted with 1M HCl solution, pH=3.0-3.5 go from
Dialyse 72h in sub- water, freeze-drying, obtains half Guang ammonia modified hyaluronic acid.
Wherein, Sodium Hyaluronate molecular weight is 0.1MDa, carboxyl in Sodium Hyaluronate：N- succinimides：1- ethyls-
(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate：Half Guang ammonia salt hydrochlorate：The ratio between amount of material of dithiothreitol (DTT) is 1:
2:2:1:3。
（3）30mg sulfydryls-hyaluronic acid compound is dissolved in the PBS of 1mL pH=7.4, first in frozen water
1M NaOH solution adjustment pH=7.4~8.0 are added under the conditions of bath, solution is then poured into a diameter of 8.5mm, is highly 3.0mm
Cylindrical die in, be finally placed in 37 DEG C of incubator, react to form disulfide bond and prepare hydrogel by sulfydryl.
Gained hydrogel combination property and product in embodiment 1 are basically identical.Simply in step（2）In generating unit sometimes
Divide continuous shape desciccate, it is difficult to be dissolved in subsequent step.
Embodiment 11
Obtained hydrogel in Example 1 ~ 9 respectively, adds reproducibility small molecule DTT（It is to DTT concentration respectively
The degraded situation of hydrogel is detected under the conditions of 0.1mM, 1mM, 10Mm）, the extension that gel can be over time progressively drops
Solution, until being changed into solution state.
As a result it is as shown in Figure 3：Hydrogel of the present invention can be accelerated to degrade in a reducing environment, and degradation rate is with reduction
Agent concentration increases and accelerated.
In addition, above-mentioned reproducibility small molecule is changed into glutathione by DTT（GSH）Essentially identical degraded can also be reached
Effect.
Embodiment 12
（1）By nascent rabbit cartilage cell（chondrocytes）It is evenly distributed in culture dish, in 37 DEG C, 0.5%CO2 Carefully
Cultivated in born of the same parents' incubator.After cell covers with culture dish, cell is collected, is prepared into 100 μ L concentration as 5.5 × 107 cells/
ML cell suspension.
（2）33mg sulfydryls-hyaluronic acid compound is dissolved in 1mL α-MEM culture mediums, is firstly added 1M NaOH
Solution adjusts pH=7.4~8.0, then adds 100 μ L chondrocyte cell suspensions of above-mentioned preparation, is well mixed（Water-setting
Glue final concentration of 3%, cell-seeding-density are 5 × 106cells/mL）, solution is poured into mould, then 37 DEG C, 0.5%
CO2 Plastic process is carried out in cell culture incubator, finally removes the hydrogel for wrapping up cell from mould, is added to α-MEM trainings
Support in base, in 37 DEG C, 0.5%CO2 Cultivated in cell culture incubator.
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 55.44%.
After the hydrogel culture 1 day, 3 days, 7 days, 14 days, 21 days for wrapping up chondrocyte cells, optical microphotograph is used respectively
The growing state of mirror, laser co-focusing and the scanning micro- sem observation cell of orbital electron.As a result it is as shown in Figure 4：Co-cultivation process
In, chondrocyte cells remain rounded form；After co-culturing 7 days, the appearance of paired cell can be substantially observed；
After co-culturing 14 days, cell mass and matrix secretion phenomenon can be substantially observed；After co-culturing 21 days, base can be substantially observed
A large amount of secretions of matter and the cell mass of matrix parcel.Observation indicate that chondrocyte cells can be in co-culture system
Normally bred, and keep secreting the normal biological functions such as matrix.
Embodiment 13
（1）By nascent rabbit bone mescenchymal stem cell（MSCs）It is evenly distributed in culture dish, in 37 DEG C, 0.5%CO2 Cell
Cultivated in incubator.After cell covers with culture dish, cell is collected, is prepared into 100 μ L concentration as 5.5 × 107 cells/mL
Cell suspension.
（2）33mg sulfydryls-hyaluronic acid compound is dissolved in 1mL α-MEM culture mediums, is firstly added 1M NaOH
Solution adjusts pH=7.4~8.0, then adds 100 μ L MSCs cell suspensions of above-mentioned preparation, is well mixed（Hydrogel is dense eventually
Spend for 3%, cell-seeding-density is 5 × 106cells/mL）, solution is poured into mould, then in 37 DEG C, 0.5%CO2 Cell
Plastic process is carried out in incubator, finally the hydrogel for wrapping up cell is removed from mould, is added in α-MEM culture mediums,
In 37 DEG C, 0.5%CO2 Cultivated in cell culture incubator.
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 55.44%.
After the hydrogel culture 1 day, 3 days, 7 days, 14 days, 21 days for wrapping up MSCs cells, respectively with light microscope, laser
Copolymerization is burnt and scans the growing state of the micro- sem observation cell of orbital electron.As a result it is as shown in Figure 5：During co-cultivation, L929
Cell remains rounded form；After co-culturing 7 days, the appearance of paired cell can be substantially observed；, can after co-culturing 14 days
Substantially to observe cell mass and matrix secretion phenomenon；After co-culturing 21 days, it can substantially observe and largely be wrapped up by matrix
Cell mass.Observation indicate that L929 cells can normally be bred in co-culture system, and keep secreting matrix
Etc. normal biological function.
Embodiment 14
（1）By l cell（L929 cells）It is evenly distributed in culture dish, in 37 DEG C, 0.5%CO2 Cell
Cultivated in incubator.After cell covers with culture dish, cell is collected, is prepared into 100 μ L concentration as 5.5 × 107 cells/mL
Cell suspension.
（2）33mg sulfydryls-hyaluronic acid compound is dissolved in 1mL α-MEM culture mediums, is firstly added 1M NaOH
Solution adjusts pH=7.4~8.0, then adds 100 μ L L929 cell suspensions of above-mentioned preparation, is well mixed（Hydrogel is dense eventually
Spend for 3%, cell-seeding-density is 5 × 106cells/mL）, solution is poured into mould, then in 37 DEG C, 0.5%CO2 Cell
Plastic process is carried out in incubator, finally the hydrogel for wrapping up cell is removed from mould, is added in α-MEM culture mediums,
In 37 DEG C, 0.5%CO2 Cultivated in cell culture incubator.
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 55.44%.
After the hydrogel culture 1 day, 3 days, 7 days, 14 days, 21 days for wrapping up L929 cells, respectively with light microscope, laser
Copolymerization is burnt and scans the growing state of the micro- sem observation cell of orbital electron.As a result it is as shown in Figure 6：During co-cultivation, MSCs
Cell remains rounded form, shows certain multiplication capacity.Observation indicate that MSCs cells can co-culture body
Normally bred in system.
Embodiment 15
1 is dissolved in 30mg sulfydryls-hyaluronic acid compound in 1mL α-MEM culture mediums, is firstly added 1M PBS bufferings
Solution adjusts pH=7.4,（Hydrogel final concentration of 3%）, 0.1mL solution is then expelled to BALB/C small white mouse skins with syringe
Under.
2 wherein, and sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 33.54%.
3 hydrogels take out after small white mouse In vivo culture 7 days, 28 days, observe the degraded situation of hydrogel, and use laser
The growing state of the burnt observation cell of copolymerization.
Experimental result finds that the gel can preferably keep its gel state in vivo, occurs without conspicuousness and shrinks and molten
It is swollen.
Embodiment 16
1 by nascent rabbit cartilage cell（chondrocytes）It is evenly distributed in culture dish, in 37 DEG C, 0.5%CO2Cell
Cultivated in incubator.After cell covers with culture dish, cell is collected, is prepared into 100 μ L concentration as 5.5 × 107 Cells/mL's
Cell suspension.
2 are dissolved in 33mg sulfydryls-hyaluronic acid compound in 1mL α-MEM culture mediums, are firstly added 1M PBS bufferings
Solution adjusts pH=7.4, then adds 100 μ L chondrocyte cell suspensions of above-mentioned preparation, is well mixed（Hydrogel is whole
Concentration is 3%, and cell-seeding-density is 5 × 106cells/mL）, 0.1mL solution is expelled to BALB/C small white mouses with syringe
Subcutaneously.
3 wherein, and sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 33.54%.
The hydrogel of 4 parcel chondrocyte cells takes out, is total to laser after small white mouse In vivo culture 14 days, 28 days
Focus on the growing state of observation cell（As shown in Figure 7）.
Experimental result is found, can should preferably keep its gel state in vivo in Cellular gels, occurs without conspicuousness receipts
Contracting and swelling.Cell can be good at breeding inside gel.
Embodiment 17
1 by nascent rabbit cartilage cell（chondrocytes）It is evenly distributed in culture dish, in 37 DEG C, 0.5%CO2 cells
Cultivated in incubator.After cell covers with culture dish, cell is collected, is prepared into 100 μ L concentration as 27.5 × 107 cells/mL
Cell suspension.
2 are dissolved in 33mg sulfydryls-hyaluronic acid compound in 1mL α-MEM culture mediums, are firstly added 1M PBS bufferings
Solution adjusts pH=7.4, then adds 100 μ L chondrocyte cell suspensions of above-mentioned preparation, is well mixed（Hydrogel is whole
Concentration is 3%, and cell-seeding-density is 2.5 × 107cells/mL）, 0.1mL solution is expelled to BALB/C little Bai with syringe
Mouse is subcutaneous.
3 wherein, and sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia grafting rate is 33.54%.
The hydrogel of 4 parcel chondrocyte cells takes out, is total to laser after small white mouse In vivo culture 14 days, 28 days
Focus on the growing state of observation cell.（As shown in Figure 8）
Experimental result is found, can should preferably keep its gel state in vivo in Cellular gels, occurs without conspicuousness receipts
Contracting and swelling.Cell can be good at breeding inside gel.
The preferred embodiments of the present invention are the foregoing is only, are merely illustrative for the purpose of the present invention, and it is nonrestrictive；
Those of ordinary skill in the art understand that can carry out many to it in the spirit and scope that the claims in the present invention are limited changes
Become, modification, or even equivalent change, but fall within protection scope of the present invention.
Claims (9)
1. a kind of preparation method of the controllable self-crosslinking hyaluronic acid gel of sulfydryl/disulfide bond, it is characterised in that including following step
Suddenly：
1）Half Guang ammonia of hyaluronic acid is modified：
Sodium Hyaluronate is dissolved in deionized water, is firstly added N- succinimides（NHS）, fully dissolving；Then add
1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC HCl) powder, the HCl with 1M NaOH and 1M are molten
Liquid adjusts pH=4.75 of reaction solution, reacts 2 hours；Then add half Guang ammonia salt hydrochlorate（CSH•HCl）Solution, react 24
Hour；PH=8.5 of reaction solution finally are adjusted with 1M NaOH solution, add dithiothreitol (DTT)（DTT）Solution, reaction are 12 small
When, it is 10%~60% to control half Guang ammonia grafting rate in gained sulfydryl-hyaluronic acid compound, the dithiothreitol (DTT)（DTT）
The addition phenomenon and can that can either avoid obtaining pars shape product after freeze-drying and can not dissolving in subsequent step reach
The hydrogel that the surface sulfydryl density on effectively control hyaluronic acid surface ensures finally to obtain has controllable self-crosslinking characteristic；Reaction
After end, pH=3.0-3.5 of reaction solution is adjusted with 1M HCl solution, dialyse 72h in pH=3.0-3.5 deionized water, cold
It is lyophilized dry, obtain a kind of sulfydryl-hyaluronic acid compound with free sulfhydryl group, i.e. half Guang ammonia modified hyaluronic acid；
2）By step 1）In half Guang ammonia modified hyaluronic acid of preparation be dissolved in the PBS of pH=7.4, then adjust
PH value, by forming the reaction of disulfide bond between sulfydryl, forms the hydrogel of three dimensional chemical crosslinking to 7.4~8.0.
2. preparation method according to claim 1, it is characterised in that carboxyl in Sodium Hyaluronate：N- succinimides：1-
Ethyl-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate：Half Guang ammonia salt hydrochlorate：The ratio between amount of material of dithiothreitol (DTT)
For 1：2：2~4:1~4:3~12.
3. preparation method according to claim 1, it is characterised in that the step 2）In half Guang ammonia modified hyaluronic acid it is molten
The mass percent concentration of liquid is 1.0%-3.0%.
4. preparation method according to claim 1, it is characterised in that in the step 2）The middle half Guang ammonia by after dissolving changes
Property hyaluronic acid be placed in corresponding mould and carry out gel reaction to obtain the hydrogel of given shape；Or by half after dissolving
Parenteral solution is made in Guang ammonia modified hyaluronic acid, and it is made after injection in target area gel in-situ.
5. a kind of controllable self-crosslinking hyaluronic acid gel of sulfydryl/disulfide bond prepared using claim 1 methods described, it is special
Sign is, the gel time of the hydrogel can accuracy controlling, and it is transparent that the Guang ammonia of part half can be avoided in preparation process to be modified
Matter acid occurs to obtain the phenomenon that pars shape product can not dissolve in subsequent step after disulfide bond crosslinking causes freeze-drying.
6. the application of the hydrogel described in a kind of claim 5, it is characterised in that be used as Three-dimensional cell culture support or incite somebody to action
Injection aquagel is made in it.
7. a kind of Three-dimensional cell culture strutting system, it is characterised in that controllable including sulfydryl/disulfide bond described in claim 5
Self-crosslinking hyaluronic acid gel and the cell being uniformly distributed therein.
8. the construction method of the Three-dimensional cell culture system described in a kind of claim 7, it is characterised in that comprise the following steps：
（1）By step 1）Sulfydryl-hyaluronic acid compound with free sulfhydryl group of middle preparation is dissolved in culture medium, Ran Houjia
Enter cell suspension, be well mixed, form mixed liquor；
（2）The reaction of disulfide bond is formed by being aoxidized between sulfydryl, the hyaluronic acid in gained mixed liquor is formed three dimensional chemical
The hydrogel of crosslinking and by cell encapsulation in wherein.
9. the construction method of Three-dimensional cell culture system according to claim 8, it is characterised in that step（2）Specially：
By step（1）Obtained mixed liquor is added in mould, then in 37 DEG C, 0.5%CO2Plastic mistake is carried out in cell culture incubator
Journey, finally the hydrogel for wrapping up cell is removed from mould, added in culture medium, in 37 DEG C, 0.5%CO2Cell culture incubator
In cultivated；Or directly by step（1）Obtained mixed liquor is expelled in organisms, makes hyaluronic acid in organism
Three dimensional chemical occurs in vivo and is cross-linked to form hydrogel.
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| CN108084461B (en) * | 2017-12-28 | 2020-11-10 | 四川大学 | Controllable self-crosslinking thiolated hyaluronic acid-collagen composite hydrogel and preparation method and application thereof |
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