CN108003360A - Stem cell is induced into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation - Google Patents
Stem cell is induced into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
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- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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- C08J2451/00—Characterised by the use of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives of such polymers
Abstract
The invention discloses a kind of preparation method for inducing stem cell into the II Collagen Type VI hydrogels of cartilage differentiation, first by II Collagen Type VI side chain lysine ε amino and the amidation process of methacrylic anhydride, the Photocrosslinkable II Collagen Type VIs for possessing Photocrosslinkable and keeping three helical conformation integrality of II Collagen Type VIs have been synthesized；Again under the action of photoinitiator, triggering Photocrosslinkable II Collagen Type VIs polymerization crosslinking to be formed has certain mechanical strength and adjustable one-component II Collagen Type VI hydrogels, and the differentiation microenvironment of bionic is provided for stem cell.
Description
Technical field
The invention belongs to technical field of biomedical materials, is related to the inductivity hydrogel material for cartilage defect repair
Preparation method and application.
Background technology
In view of articular cartilage tissue is without blood vessel and the features such as overall metabolic rate and relatively low competent cell density, when patient meets with
By cartilage related fields disease or wound when its self-repairing capability it is extremely limited.Common repair of cartilage clinical means include
Marrow stimulation, bone cartilage transplantation and prosthesis replacement etc., but these means would generally cause the formation of fibrocartilage tissue, by
There is larger difference with native Zona cartilaginous tissue in fibrocartilage tissue structure composition and mechanical strength, therefore common cartilage is repaiied
Multiple clinical means are difficult to realize permanently effective therapeutic effect.And the cell therapy based on organizational project, by by cartilage cell
Or cartilage precursor cells are inoculated in the biocompatible scaffold of bionic, be conducive to rebuild cartilage microenvironment；It is wherein biological
Biomimetic scaffolds are maintained for chondrocyte phenotype and mesenchymal stem cell (Bone marrow Mesenchymal Stem
Cells, BMSCs) cartilage differentiation is most important.
Important composition component of the II Collagen Type VIs as cartilage cell epimatrix, there is excellent biocompatibility and biology to drop
Xie Xing.But the fiber assembling poor ability of II Collagen Type VIs, it is difficult to gel is formed, must so that cannot be provided steadily in the long term for cell
The microenvironment for Proliferation, Differentiation wanted.The common crosslinking method that gel is prepared into using II Collagen Type VIs as raw material mainly passes through penta
(N- hydroxysuccinimidyls acyl is sub- by dialdehyde, Geniposide, EDC (1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides)/NHS
Amine) etc. with lyophilized II Collagen Type VI sponge covalent cross-linkings to obtain timbering material (C.-S.Ko, J.- with certain mechanical strength
P.Huang,C.-W.Huang and I.-M.Chu,Journal of bioscience and bioengineering,
2009,107,177-182、X.Zhou,Y.Tao,J.Wang,D.Liu,C.Liang,H.Li and Q.Chen,Journal of
Biomedical Materials Research Part A,2016,104,1687-1693、J.Pieper,P.Van der
Kraan,T.Hafmans,J.Kamp,P.Buma,J.Van Susante,W.Van Den Berg,J.Veerkamp and
T.Van Kuppevelt, Biomaterials, 2002,23,3183-3192 or M.G.Haugh, C.M.Murphy,
R.C.McKiernan,C.Altenbuchner and F.J.O'Brien,Tissue Engineering Part A,2011,
17,1201-1208).But the timbering material prepared with upper type, since glutaraldehyde, Geniposide, EDC/NHS are poisonous so that branch
There are potential cytotoxicity for frame material；And sponge structure lacks to the bionical of three-dimensional cartilage microenvironment, it is impossible to is provided for cell
Suitable growth and differentiation environment.In addition, the use of composite gel material is not enough to prominent II Collagen Type VIs to influencing and instructing carefully
The important function of born of the same parents' behavior.
The content of the invention
The purpose of the present invention is intended to be directed to above-mentioned deficiency of the prior art, there is provided one kind induces stem cell into cartilage differentiation
II Collagen Type VI hydrogels preparation method, to obtain the biocompatible hydrogel based on II Collagen Type VI one-components, structure
One three-dimensional bionic microenvironment can more preferable simulation cartilage cell epimatrix, so as to fulfill induction BMSCs into cartilage
Differentiation.
Induction stem cell provided by the invention into the II Collagen Type VI hydrogels of cartilage differentiation preparation method, with from natural soft
The II Collagen Type VIs extracted in bone pass through the epsilon-amino and methyl on II Collagen Type VI side chain lysines first as chondrocyte induction matrix
The amidation process of acrylic anhydride, synthesis possess Photocrosslinkable and keep three helical conformation integrality of II Collagen Type VIs can light hand over
Join II Collagen Type VIs；Again under photoinitiator catalytic action, triggering Photocrosslinkable II Collagen Type VIs polymerization crosslinking to be formed has certain force
Intensity and adjustable one-component II Collagen Type VI hydrogels are learned, is comprised the following steps that：
(1) II Collagen Type VI solution is prepared
At normal pressure, 0-4 DEG C, it is 1- that II Collagen Type VIs, which are dissolved in, and obtain mass concentration in the acid solution that pH value is 1-3
The II Collagen Type VI solution of 4mg/mL；
(2) Photocrosslinkable II Collagen Type VIs are synthesized
At normal pressure, 0-4 DEG C, the pH value of II Collagen Type VI solution is adjusted with phosphate buffer or/and sodium hydroxide solution
For 7 ＜ pH value≤8, then add methacrylic anhydride and supplement hydrogen-oxygen in normal pressure, 0-4 DEG C of stirring reaction 2-12h, reaction process
Changing sodium solution makes the pH value of reaction system meet 7 ＜ pH value≤8, and the addition of methacrylic anhydride is pressed in II Collagen Type VI solution
The molar ratio of amino and methacrylic anhydride is 1:(0.2-5) is measured；
Unreacted methacrylic anhydride in gained reaction solution is removed after reaction, then will remove unreacted methyl
Reaction solution after acrylic anhydride is freeze-dried to obtain Photocrosslinkable II Collagen Type VIs, or after unreacted methacrylic anhydride will be removed
Reaction solution load bag filter, at normal pressure, 0-4 DEG C by the Sheng liquid bag filter immerse mass concentration be 10%-50% poly- second
4-24h is concentrated in two alcohol solutions and obtains the concentrate containing Photocrosslinkable II Collagen Type VIs；
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
At normal pressure, 0-4 DEG C, Photocrosslinkable II Collagen Type VIs are dissolved to obtain Photocrosslinkable with the acid solution that pH value is 1-3
II Collagen Type VI solution, and with sodium hydroxide solution adjust pH value to 7.0-7.5, then to adjust pH value after Photocrosslinkable II types
Photoinitiator is added in collagen solution, is configured to the final concentration of 4-10mg/mL of Photocrosslinkable II Collagen Type VIs, photoinitiator final concentration
For the gel pre-polymerization liquid of 0.2-1mg/mL, then gel pre-polymerization liquid is placed under ultraviolet light under atmospheric pressure at room and irradiates 10-60s,
Obtain II Collagen Type VI hydrogels；
Or under the conditions of normal pressure, 0-4 DEG C, by the concentrate sodium hydroxide solution tune containing Photocrosslinkable II Collagen Type VIs
PH value is saved to 7.0-7.5, then photoinitiator is added into the Photocrosslinkable II Collagen Type VI solution after adjusting pH value, being configured to can
The gel pre-polymerization liquid of the final concentration of 4-10mg/mL of photo-crosslinking II Collagen Type VIs, the final concentration of 0.2-1mg/mL of photoinitiator, Ran Hou
Gel pre-polymerization liquid is placed under ultraviolet light under atmospheric pressure at room and irradiates 10-60s, that is, obtains II Collagen Type VI hydrogels.
Above-mentioned induction stem cell into the II Collagen Type VI hydrogels of cartilage differentiation preparation method, in the step (2), reaction
After dialysing to remove in gained reaction solution be by way of unreacted methacrylic anhydride：It is 1-3's first to pH value
Ethanol is added in acid solution and is configured to serial dialysis liquid, in serial dialysis liquid, the volume fraction of ethanol of each dialyzate reduces successively
10%, the volume fraction of ethanol is no more than 50% in the dialyzate of volume fraction of ethanol maximum；Then the saturating of reaction solution will be filled
Analysis bag is put into dialyzate is dialysed several times in 0-4 DEG C under agitation, the body of ethanol in the dialyzate that dialysis is replaced every time
Fraction reduces by 10%, and each dialysis time is 3-7 days；It is finally the sour molten of 2-3 in pH value by the bag filter for filling reaction solution
Dialyse 1-3 days in liquid.The molecular weight that shuts off of the bag filter is 8000-14000 dalton.
Above-mentioned induction stem cell is into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, and in the step (3), light draws
Hair agent is irgacure2959 (2- hydroxyls -4'- (2- hydroxy ethoxies) -2- methyl phenyl ketones), irgacure184 (1- hydroxyl rings
Hexyl phenyl ketone), irgacure127 (1,1'- (methylene two -4,1- phenylenes) double [2- hydroxy-2-methyls -1- third
Ketone]), irgacure651 (benzoin dimethylether) or rose Bengal (tetrachlorotetraiodo-fluorescein disodium).
Above-mentioned induction stem cell into the II Collagen Type VI hydrogels of cartilage differentiation preparation method, in the step (3), for
Photocrosslinkable II Collagen Type VIs, acid solution dosage are limited with being at least completely dissolved Photocrosslinkable II Collagen Type VIs.Photoinitiator is added
Afterwards, can by add phosphate buffer be met can light join the gel of II Collagen Type VIs concentration and photoinitiator final concentration requirement
Pre-polymerization liquid.
Above-mentioned induction stem cell into the II Collagen Type VI hydrogels of cartilage differentiation preparation method, it is ultraviolet in the step (3)
Luminous intensity is 4-8W/cm2, under the light-intensity conditions, it is only necessary to which 10-60s irradiation times, can realize Photocrosslinkable II collagens
Polymerization crosslinking, obtains II Collagen Type VI hydrogels.
Above-mentioned induction stem cell is into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, the step (2) and step
(3) in, the molar concentration of sodium hydroxide solution is 0.5-5M.
Above-mentioned induction stem cell is into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, the step (2) and step
(3) molar concentration of phosphate buffer is 0.2-2M in；The phosphate buffer is disodium phosphate soln or di(2-ethylhexyl)phosphate
Hydrogen sodium solution.
Above-mentioned induction stem cell into the II Collagen Type VI hydrogels of cartilage differentiation preparation method, the step (1), (2) and
(3) acid solution is hydrochloric acid solution or acetum in.
When being applied to promote stem cell Osteoblast Differentiation by II Collagen Type VIs hydrogel provided by the invention, first pancreas egg can will be used
Mescenchymal stem cell after white enzymic digestion is uniformly mixed with the pre-polymerization liquid that step (2) obtains, then gained mixed liquor is placed in ultraviolet
Irradiation obtains the II Collagen Type VI hydrogels of parcel cell under light, will wrap up the II Collagen Type VI hydrogels of cell afterwards in culture medium
Culture；It is real by being cultivated in the II Collagen Type VIs hydrogel implantation respective organization of the parcel cell taken out from culture medium when to be used
Existing repair of cartilage；The II Collagen Type VI hydrogels that step (3) are prepared can also be implanted directly into culture in respective organization to realize
Repair of cartilage.
Compared with prior art, the invention has the advantages that：
1st, the method for the invention passes through the epsilon-amino and the acid amides of methacrylic anhydride on II Collagen Type VI side chain lysines
Change reaction, not only so that II collagens possess Photocrosslinkable, and maintain the integrality of original three helical conformation of II Collagen Type VIs, then
The one-component II Collagen Type VI hydrogels with certain mechanical strength are obtained by further polymerization crosslinking, biology is provided for stem cell
Bionical differentiation microenvironment.
2nd, the method for the invention makes II using the high response of the Lysine s-amino groups on II Collagen Type VI side chain lysines
Collagen Type VI has Photocrosslinkable property, and the shortcomings that improving II Collagen Type VI self assembly scarce capacities by covalent cross-linking mode is to help
The regulation and control of mechanical strength in a big way just can be realized in the formation of later stage hydrogel, and by adjusting gel pre-polymerization liquid concentration.
3rd, II Collagen Type VI hydrogels prepared by the method for the invention, since II Collagen Type VIs come from cartilaginous tissue, itself tool
Have good cell compatibility, can promote bone marrow mesenchymal stem cells into cartilage differentiation, improve cartilage specificity matrix
Expression.
4th, II collagen hydrogels preparation method of the present invention, based on conventional equipment i.e. can be achieved, and it is raw materials used it is nontoxic,
Environmental protection, is advantageously implemented industrialized production.
Brief description of the drawings
Fig. 1 is the polyacrylamide gel electrophoresis detection and analysis figure of native type II collagen neutral solution.
Fig. 2 for hydrogen nuclear magnetic resonance spectrogram (1H-NMR), wherein, (a) be native type II collagen and embodiment 1 prepare can
The hydrogen nuclear magnetic resonance spectrogram of photo-crosslinking II Collagen Type VIs, (b) figure are the proton magnetic of Photocrosslinkable II Collagen Type VIs prepared by embodiment 5
Resonance light spectrogram, (c) figure are the hydrogen nuclear magnetic resonance spectrogram of Photocrosslinkable II Collagen Type VIs prepared by embodiment 8.
Fig. 3 is plastic design sketch and storage modulus collection of illustrative plates, wherein, (a) is that the plastic of native type II collagen neutral solution is imitated
Fruit is schemed, and (b) is the plastic design sketch of II Collagen Type VI hydrogels prepared by embodiment 1, and (c) is embodiment 1, embodiment 5 and implements
The storage modulus collection of illustrative plates of II Collagen Type VI hydrogels prepared by example 8.
Fig. 4 is that bone marrow mesenchymal stem cells are wrapped in the FDA/PI dyes cultivated in II Collagen Type VI hydrogels in application examples 1
Color result figure, wherein, (a) is the microscope imaging picture after cell culture 1 day, (b) be microscope after cell culture 4 days into
As picture, (c) is the microscope imaging picture after cell culture 7 days, and (d) is the microscope imaging figure after cell culture 14 days
Piece, (e) are the microscope imaging picture after cell culture 21 days.
Fig. 5 is that bone marrow mesenchymal stem cells were wrapped in II Collagen Type VI hydrogels In vivo culture after 14 days in application examples 2
Microscope imaging picture, wherein (a) for histotomy using safranin O (SO) dye microscope imaging picture, (b) cuts for tissue
Piece is using toluidine blue (TB) dyeing microphotograph.
Embodiment
Clear, complete description is carried out to technical scheme by the following examples and with reference to attached drawing, it is clear that institute
The embodiment provided is only the part of the embodiment of the present invention, instead of all the embodiments.Based on the implementation in the present invention
Example, those of ordinary skill in the art's obtained all other embodiment on the premise of creative work is not made, all belongs to
In the scope that the present invention is protected.
In following embodiments, II Collagen Type VIs are purchased from Chondrex companies of the U.S., and II Collagen Type VIs are one kind of collagen, right
It carries out nuclear magnetic resonance spectroscopy, shown in gained hydrogen nuclear magnetic resonance figure such as Fig. 2 (a).II Collagen Type VIs are first dissolved in concentration 20mM's
Hydrochloric acid solution is configured to the II Collagen Type VI solution of concentration 2mg/mL, then disodium hydrogen phosphate buffer solution and concentration 5M with concentration 0.2M
Sodium hydroxide solution adjust the pH value of II Collagen Type VI solution and obtain natural II collagens neutral solution to 7, its in solution state such as
Shown in Fig. 3 (a).Polyacrylamide gel electrophoresis detection, its testing result are carried out to obtained native type II collagen neutral solution
It is control with M (Marker) and Col-I (type i collagen), analysis result is as shown in Figure 1, the tropocollagen molecule amount distribution of II Collagen Type VIs
It is characterized with tri- chains of α, β and γ, the molecular weight of β and γ chains is respectively 250kDa and 300kDa；Compared to the I type glue of heterogeneous type
Former (there are two α bands), the α chains of II Collagen Type VIs belong to homotrimer, therefore reflection only exists a α into electrophoretic band
Chain.
In following embodiments, methacrylic anhydride, photoinitiator irgacure2959 is purchased from sigma companies of the U.S.,
Irgacure651 is purchased from German BASF BASF.
Bone marrow mesenchymal stem cells (BMSCs) may be referred to the conventional method extraction that this area has disclosed and obtain【Ginseng
See L.Zhang, T.Yuan, L.Guo and X.Zhang, Journal of Biomedical Materials Research
Part A,2012,100,2717-2725】, the BMSCs used in following embodiments is from extracting method for self-carry：To newborn family
Rabbit injection overdose of sodium pentobarbital is lethal, and is soaked 10-20 minutes and sterilized with 0.5% (volume fraction) bromogeramine, Ran Hou
Shin bone and femur are removed together with epiphysis end cartilage, reject and cartilaginous tissue and sudden and violent is peeled off after muscle and fibr tissue under aseptic condition
Undisguised pulp cavity, extracts the α-MEM culture mediums containing 20% hyclone with syringe and rinses ossis (culture volume 10mL/
Ware), and myeloid tissue is blown and beaten repeatedly, bone marrow cell is well-dispersed in culture medium, it is left to be supplemented culture volume 1%
The penicillin and streptomysin (HyClone) of right 100U/mL, is put in 37 DEG C and contains 5% (volume fraction) CO afterwards2Incubator in
Adhere-wall culture 24h, changes liquid and removes no adherent haemocyte, rejoins the α-MEM culture mediums containing 20% hyclone and continues
Culture, changes liquid once in every 2 days, and primary BMSCs, which was cultivated to the 4th day, to pass on.α-MEM culture mediums are purchased from HyClone companies of the U.S.；Tire
Cow's serum is the Gibco hyclones of Life Technologies companies of the U.S..
Embodiment 1
The step of the present embodiment, is as follows：
(1) II Collagen Type VI solution is prepared
In normal pressure under the conditions of 4 DEG C, II Collagen Type VIs are configured to the II Collagen Type VIs of concentration 2mg/mL with 20mM hydrochloric acid solutions
Solution, stirring 12 make II collagenolysises when small.
(2) Photocrosslinkable II Collagen Type VIs are synthesized
In normal pressure under the conditions of 4 DEG C, II Collagen Type VIs are adjusted with 0.2M disodium hydrogen phosphates buffer solution and 5M sodium hydroxide solutions
The pH value of solution is then 1 according to the molar ratio of amino in collagen and methacrylic anhydride to 7.5:5, after being adjusted to pH value
Methacrylic anhydride is added in II Collagen Type VI solution, when persistently stirring reaction 4 is small, 5M sodium hydroxide solutions are supplemented in reaction process
The pH value of reaction system is set to be maintained at 7.5.
Unreacted methacrylic anhydride in the reaction solution as obtained by dialysing and remove, concrete mode are after reaction：It is first
Ethanol is first added into 20mM hydrochloric acid solutions and is configured to the dialysis that volume fraction of ethanol is 10%, 20%, 30%, 40%, 50%
Liquid；Then the bag filter for filling gained reaction solution is put into dialyzate according to the order that volume fraction of ethanol in dialyzate reduces
Into dialysing several times under 4 DEG C of stirring conditions, each dialysis time is 3-7 days；The bag filter of gained reaction solution will finally be filled
Dialyse 2 days in 20mM hydrochloric acid solutions.
The reaction solution after methacrylic anhydride is removed to be freeze-dried to obtain Photocrosslinkable II Collagen Type VIs.
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
In normal pressure under the conditions of 4 DEG C, by Photocrosslinkable II collagenolysises and it is configured to concentration with 20mM hydrochloric acid solutions and is
The Photocrosslinkable II Collagen Type VI solution of 10mg/mL, and pH value is adjusted to 7.0 with 5M sodium hydroxide solutions, to after adjusting pH value
Photocrosslinkable II Collagen Type VI solution in add photoinitiator irgacure2959, be formulated as Photocrosslinkable II Collagen Type VI final concentrations
For 7mg/mL, the gel pre-polymerization liquid of the final concentration of 0.5mg/mL of photoinitiator, if having added the gel pre-polymerization after photoinitiator
Liquid concentration is higher, does not reach Photocrosslinkable II Collagen Type VIs and photoinitiator final concentration requirement, can add respective volume 0.2M phosphoric acid
Disodium hydrogen buffer solution.Then it is 8W/cm exposed to intensity by gel pre-polymerization liquid2Ultraviolet light under irradiate 30 seconds, you can realize pre-
Poly- liquid cures, and obtains II Collagen Type VI hydrogels.
Embodiment 2
The step of the present embodiment, is as follows：
(1) II Collagen Type VI solution is prepared
In normal pressure under the conditions of 0 DEG C, II Collagen Type VIs are configured to the II Collagen Type VIs of concentration 3mg/mL with 20mM acetums
Solution, stirring 12 make II collagenolysises when small.
(2) Photocrosslinkable II Collagen Type VIs are synthesized
In normal pressure under the conditions of 0 DEG C, II Collagen Type VIs are adjusted with 0.5M phosphate sodium dihydrogen buffer solutions and 3M sodium hydroxide solutions
The pH value of solution is then 1 according to the molar ratio of amino in collagen and methacrylic anhydride to 8:2.5, after being adjusted to pH value
Methacrylic anhydride is added in II Collagen Type VI solution, when persistently stirring reaction 8 is small, 3M sodium hydroxide solutions are supplemented in reaction process
The pH value of reaction system is set to be maintained at 8.
Unreacted methacrylic anhydride in the reaction solution as obtained by dialysing and remove, concrete mode are after reaction：It is first
Ethanol is first added into 20mM acetums and is configured to the dialysis that volume fraction of ethanol is 10%, 20%, 30%, 40%, 50%
Liquid；Then the bag filter for filling gained reaction solution is put into dialyzate according to the order that volume fraction of ethanol in dialyzate reduces
Into dialysing several times under 0 DEG C of stirring condition, each dialysis time is 3-7 days；The bag filter of gained reaction solution will finally be filled
Dialyse 1 day in 20mM acetums.
The reaction solution after methacrylic anhydride is removed to be freeze-dried to obtain Photocrosslinkable II Collagen Type VIs.
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
In normal pressure under the conditions of 0 DEG C, with 20mM acetic acid is by Photocrosslinkable II collagenolysises and to be configured to concentration be 10mg/
The Photocrosslinkable II Collagen Type VI solution of mL, and pH value is adjusted to 7.0 with 5M sodium hydroxide solutions, to adjust after pH value can light
It is crosslinked in II Collagen Type VI solution and adds photoinitiator irgacure651, is formulated as the final concentration of 4mg/ of Photocrosslinkable II Collagen Type VIs
The gel pre-polymerization liquid of mL, the final concentration of 0.2mg/mL of photoinitiator, if having added the gel pre-polymerization liquid concentration after photoinitiator
It is higher, do not reach Photocrosslinkable II Collagen Type VIs and photoinitiator final concentration requirement, respective volume 0.5M sodium dihydrogen phosphates can be added
Buffer solution.Then it is 4W/cm exposed to intensity by gel pre-polymerization liquid2Ultraviolet light under irradiate 60 seconds, you can realize that pre-polymerization liquid is consolidated
Change, obtain II Collagen Type VI hydrogels.
Embodiment 3
The step of the present embodiment, is as follows：
(1) II Collagen Type VI solution is prepared
In normal pressure under the conditions of 4 DEG C, II Collagen Type VIs are configured to the II Collagen Type VIs of concentration 4mg/mL with 100mM hydrochloric acid solutions
Solution, stirring 12 make II collagenolysises when small.
(2) Photocrosslinkable II Collagen Type VIs are synthesized
In normal pressure under the conditions of 4 DEG C, it is molten to adjust II Collagen Type VIs with 1M disodium hydrogen phosphates buffer solution and 1M sodium hydroxide solutions
The pH value of liquid is then 1 according to the molar ratio of amino in collagen and methacrylic anhydride to 7.5:0.5, after being adjusted to pH value
Methacrylic anhydride is added in II Collagen Type VI solution, it is molten to supplement 1M sodium hydroxides when persistently stirring reaction 12 is small, in reaction process
Liquid makes the pH value of reaction system be maintained at 7.5.
Unreacted methacrylic anhydride in the reaction solution as obtained by dialysing and remove, concrete mode are after reaction：It is first
Ethanol is first added into 100mM hydrochloric acid solutions and is configured to the dialysis that volume fraction of ethanol is 10%, 20%, 30%, 40%, 50%
Liquid；Then the bag filter for filling gained reaction solution is put into dialyzate according to the order that volume fraction of ethanol in dialyzate reduces
Into dialysing several times under 4 DEG C of stirring conditions, each dialysis time is 3-7 days；The bag filter of gained reaction solution will finally be filled
Dialyse 3 days in 100mM hydrochloric acid solutions.
In normal pressure under the conditions of 4 DEG C, by fill remove methacrylic anhydride after reaction solution bag filter immerse mass concentration
The concentrate containing Photocrosslinkable II Collagen Type VIs is obtained to concentrate 24h in 10% PEG aqueous solutions.
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
In normal pressure under the conditions of 4 DEG C, the 1M sodium hydroxide solutions of the concentrate containing Photocrosslinkable II collagens are adjusted into pH
Value adds photoinitiator irgacure2959 into the Photocrosslinkable II Collagen Type VI solution after adjusting pH value, is formulated as to 7.5
The gel pre-polymerization liquid of the final concentration of 10mg/mL of Photocrosslinkable II Collagen Type VIs, the final concentration of 1mg/mL of photoinitiator, if added
Gel pre-polymerization liquid concentration after photoinitiator is higher, does not reach Photocrosslinkable II Collagen Type VIs and photoinitiator final concentration requirement, can
Add respective volume 1M disodium hydrogen phosphate buffer solutions.Then it is 8W/cm exposed to intensity by gel pre-polymerization liquid2Ultraviolet light under shine
Penetrate 10 seconds, you can realize that pre-polymerization liquid cures, obtain II Collagen Type VI hydrogels.
Embodiment 4
The step of the present embodiment, is as follows：
(1) II Collagen Type VI solution is prepared
In normal pressure under the conditions of 0 DEG C, II Collagen Type VIs are configured to the II Collagen Type VIs of concentration 2mg/mL with 10mM hydrochloric acid solutions
Solution, stirring 12 make II collagenolysises when small.
(2) Photocrosslinkable II Collagen Type VIs are synthesized
In normal pressure under the conditions of 0 DEG C, II Collagen Type VIs are adjusted with 2M phosphate sodium dihydrogen buffer solutions and 0.5M sodium hydroxide solutions
The pH value of solution is then 1 according to the molar ratio of amino in collagen and methacrylic anhydride to 7.5:3, after being adjusted to pH value
Methacrylic anhydride is added in II Collagen Type VI solution, it is molten to supplement 0.5M sodium hydroxides when persistently stirring reaction 2 is small, in reaction process
Liquid makes the pH value of reaction system be maintained at 7.5.
Unreacted methacrylic anhydride in the reaction solution as obtained by dialysing and remove, concrete mode are after reaction：It is first
Ethanol is first added into 10mM hydrochloric acid solutions and is configured to the dialysis that volume fraction of ethanol is 10%, 20%, 30%, 40%, 50%
Liquid；Then the bag filter for filling gained reaction solution is put into dialyzate according to the order that volume fraction of ethanol in dialyzate reduces
Into dialysing several times under 0 DEG C of stirring condition, each dialysis time is 3-7 days；The bag filter of gained reaction solution will finally be filled
Dialyse 1 day in 10mM hydrochloric acid solutions.
In normal pressure under the conditions of 0 DEG C, by fill remove methacrylic anhydride after reaction solution bag filter immerse mass concentration
To be concentrated to give the concentrate containing Photocrosslinkable II Collagen Type VIs in 50% PEG aqueous solutions.
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
In normal pressure under the conditions of 0 DEG C, the 5M sodium hydroxide solutions of the concentrate containing Photocrosslinkable II collagens are adjusted into pH
Value adds photoinitiator irgacure2959 into the Photocrosslinkable II Collagen Type VI solution after adjusting pH value, is formulated as to 7.5
The gel pre-polymerization liquid of the final concentration of 7mg/mL of Photocrosslinkable II Collagen Type VIs, the final concentration of 1mg/mL of photoinitiator, if added
Gel pre-polymerization liquid concentration after photoinitiator is higher, does not reach Photocrosslinkable II Collagen Type VIs and photoinitiator final concentration requirement, can
Add respective volume 2M phosphate sodium dihydrogen buffer solutions.Then it is 6W/cm exposed to intensity by gel pre-polymerization liquid2Ultraviolet light under shine
Penetrate 30 seconds, you can realize that pre-polymerization liquid cures, obtain II Collagen Type VI hydrogels.
Embodiment 5
The step of the present embodiment, is as follows：
(1) II Collagen Type VI solution is prepared
In normal pressure under the conditions of 4 DEG C, II Collagen Type VIs are configured to the II Collagen Type VIs of concentration 2mg/mL with 20mM hydrochloric acid solutions
Solution, stirring 12 make II collagenolysises when small.
(2) Photocrosslinkable II Collagen Type VIs are synthesized
In normal pressure under the conditions of 4 DEG C, II Collagen Type VIs are adjusted with 0.2M disodium hydrogen phosphates buffer solution and 5M sodium hydroxide solutions
The pH value of solution is then 1 according to the molar ratio of amino in collagen and methacrylic anhydride to 7.5:5, after being adjusted to pH value
Methacrylic anhydride is added in II Collagen Type VI solution, when persistently stirring reaction 4 is small, 5M sodium hydroxide solutions are supplemented in reaction process
The pH value of reaction system is set to be maintained at 7.5.
Unreacted methacrylic anhydride in the reaction solution as obtained by dialysing and remove, concrete mode are after reaction：It is first
Ethanol is first added into 20mM hydrochloric acid solutions and is configured to the dialysis that volume fraction of ethanol is 10%, 20%, 30%, 40%, 50%
Liquid；Then the bag filter for filling gained reaction solution is put into dialyzate according to the order that volume fraction of ethanol in dialyzate reduces
Into dialysing several times under 4 DEG C of stirring conditions, each dialysis time is 3-7 days；The bag filter of gained reaction solution will finally be filled
Dialyse 2 days in 20mM hydrochloric acid solutions.
The reaction solution after methacrylic anhydride is removed to be freeze-dried to obtain Photocrosslinkable II Collagen Type VIs.
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
In normal pressure under the conditions of 4 DEG C, by Photocrosslinkable II collagenolysises and it is configured to concentration with 20mM hydrochloric acid solutions and is
The Photocrosslinkable II Collagen Type VI solution of 10mg/mL, and pH value is adjusted to 7.0 with 5M sodium hydroxide solutions, to after adjusting pH value
Photocrosslinkable II Collagen Type VI solution in add photoinitiator irgacure2959, be formulated as Photocrosslinkable II Collagen Type VI final concentrations
For 4mg/mL, the gel pre-polymerization liquid of the final concentration of 0.5mg/mL of photoinitiator, if having added the gel pre-polymerization after photoinitiator
Liquid concentration is higher, does not reach Photocrosslinkable II Collagen Type VIs and photoinitiator final concentration requirement, can add respective volume 0.2M phosphoric acid
Disodium hydrogen buffer solution.Then it is 8W/cm exposed to intensity by gel pre-polymerization liquid2Ultraviolet light under irradiate 30 seconds, you can realize pre-
Poly- liquid cures, and obtains II Collagen Type VI hydrogels.
In order to detect whether methacrylic anhydride is successfully grafted on II collagens, to the II type optical cements without any processing
Photocrosslinkable II Collagen Type VIs carry out nuclear magnetic resonance spectroscopy, obtained hydrogen nuclear magnetic resonance figure such as Fig. 1 (b) institutes obtained by former and step (2)
Show, it can be seen from the figure that being deposited in the corresponding hydrogen nuclear magnetic resonance figure of Photocrosslinkable collagen that step (2) obtains between 5~6ppm
At a double bond peak, show that methacrylic anhydride is successfully grafted on collagen.
Embodiment 6
The step of the present embodiment, is as follows：
(1) II Collagen Type VI solution is prepared
In normal pressure under the conditions of 4 DEG C, the II Collagen Type VIs that II Collagen Type VIs are configured to concentration 1mg/mL with 1mM hydrochloric acid solutions are molten
Liquid, stirring 12 make II collagenolysises when small.
(2) Photocrosslinkable II Collagen Type VIs are synthesized
In normal pressure under the conditions of 4 DEG C, II Collagen Type VIs are adjusted with 0.2M phosphate sodium dihydrogen buffer solutions and 5M sodium hydroxide solutions
The pH value of solution is then 1 according to the molar ratio of amino in collagen and methacrylic anhydride to 8:2, the II after being adjusted to pH value
Methacrylic anhydride is added in Collagen Type VI solution, when persistently stirring reaction 6 is small, supplement 5M sodium hydroxide solutions make in reaction process
The pH value of reaction system is maintained at 8.
Unreacted methacrylic anhydride in the reaction solution as obtained by dialysing and remove, concrete mode are after reaction：It is first
Ethanol is first added into 1mM hydrochloric acid solutions and is configured to the dialysis that volume fraction of ethanol is 10%, 20%, 30%, 40%, 50%
Liquid；Then the bag filter for filling gained reaction solution is put into dialyzate according to the order that volume fraction of ethanol in dialyzate reduces
Into dialysing several times under 4 DEG C of stirring conditions, each dialysis time is 3-7 days；The bag filter of gained reaction solution will finally be filled
Dialyse 2 days in 1mM hydrochloric acid solutions.
The reaction solution after methacrylic anhydride is removed to be freeze-dried to obtain Photocrosslinkable II Collagen Type VIs.
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
In normal pressure under the conditions of 4 DEG C, with 1mM hydrochloric acid is by Photocrosslinkable II collagenolysises and to be configured to concentration be 10mg/mL
Photocrosslinkable II Collagen Type VI solution, and adjust pH value to 7.5 with 5M sodium hydroxide solutions, to adjust after pH value can light hand over
Join and photoinitiator irgacure2959 is added in II Collagen Type VI solution, be formulated as the final concentration of 6mg/ of Photocrosslinkable II Collagen Type VIs
The gel pre-polymerization liquid of mL, the final concentration of 0.8mg/mL of photoinitiator, if having added the gel pre-polymerization liquid concentration after photoinitiator
It is higher, do not reach Photocrosslinkable II Collagen Type VIs and photoinitiator final concentration requirement, respective volume 0.2M sodium dihydrogen phosphates can be added
Buffer solution.Then it is 8W/cm exposed to intensity by gel pre-polymerization liquid2Ultraviolet light under irradiate 30 seconds, you can realize that pre-polymerization liquid is consolidated
Change, obtain II Collagen Type VI hydrogels.
Embodiment 7
The step of the present embodiment, is as follows：
(1) II Collagen Type VI solution is prepared
In normal pressure under the conditions of 0 DEG C, II Collagen Type VIs are configured to the II Collagen Type VIs of concentration 2mg/mL with 20mM hydrochloric acid solutions
Solution, stirring 12 make II collagenolysises when small.
(2) Photocrosslinkable II Collagen Type VIs are synthesized
In normal pressure under the conditions of 0 DEG C, II Collagen Type VIs are adjusted with 0.2M disodium hydrogen phosphates buffer solution and 5M sodium hydroxide solutions
The pH value of solution is then 1 according to the molar ratio of amino in collagen and methacrylic anhydride to 8:0.2, after being adjusted to pH value
Methacrylic anhydride is added in II Collagen Type VI solution, when persistently stirring reaction 4 is small, 5M sodium hydroxide solutions are supplemented in reaction process
The pH value of reaction system is set to be maintained at 8.
Unreacted methacrylic anhydride in the reaction solution as obtained by dialysing and remove, concrete mode are after reaction：It is first
Ethanol is first added into 20mM hydrochloric acid solutions and is configured to the dialysis that volume fraction of ethanol is 10%, 20%, 30%, 40%, 50%
Liquid；Then the bag filter for filling gained reaction solution is put into dialyzate according to the order that volume fraction of ethanol in dialyzate reduces
Into dialysing several times under 0 DEG C of stirring condition, each dialysis time is 3-7 days；The bag filter of gained reaction solution will finally be filled
Dialyse 2 days in 20mM hydrochloric acid solutions.
The reaction solution after methacrylic anhydride is removed to be freeze-dried to obtain Photocrosslinkable II Collagen Type VIs.
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
In normal pressure under the conditions of 4 DEG C, with 20mM hydrochloric acid is by Photocrosslinkable II collagenolysises and to be configured to concentration be 10mg/
The Photocrosslinkable II Collagen Type VI solution of mL, and pH value is adjusted to 7.5 with 5M sodium hydroxide solutions, to adjust after pH value can light
It is crosslinked in II Collagen Type VI solution and adds photoinitiator irgacure2959, is formulated as the final concentration of 4mg/ of Photocrosslinkable II Collagen Type VIs
The gel pre-polymerization liquid of mL, the final concentration of 0.5mg/mL of photoinitiator, if having added the gel pre-polymerization liquid concentration after photoinitiator
It is higher, do not reach Photocrosslinkable II Collagen Type VIs and photoinitiator final concentration requirement, respective volume 0.2M disodium hydrogen phosphates can be added
Buffer solution.Then it is 4W/cm exposed to intensity by gel pre-polymerization liquid2Ultraviolet light under irradiate 60 seconds, you can realize that pre-polymerization liquid is consolidated
Change, obtain II Collagen Type VI hydrogels.
Embodiment 8
The step of the present embodiment, is as follows：
(1) II Collagen Type VI solution is prepared
In normal pressure under the conditions of 4 DEG C, II Collagen Type VIs are configured to the II Collagen Type VIs of concentration 2mg/mL with 20mM hydrochloric acid solutions
Solution, stirring 12 make II collagenolysises when small.
(2) Photocrosslinkable II Collagen Type VIs are synthesized
In normal pressure under the conditions of 4 DEG C, II Collagen Type VIs are adjusted with 0.2M disodium hydrogen phosphates buffer solution and 5M sodium hydroxide solutions
The pH value of solution is then 1 according to the molar ratio of amino in collagen and methacrylic anhydride to 7.5:5 to pH value adjust after II
Methacrylic anhydride is added in Collagen Type VI solution, when persistently stirring reaction 4 is small, supplement 5M sodium hydroxide solutions make in reaction process
The pH value of reaction system is maintained at 7.5.
Unreacted methacrylic anhydride in the reaction solution as obtained by dialysing and remove, concrete mode are after reaction：It is first
Ethanol is first added into 20mM hydrochloric acid solutions and is configured to the dialysis that volume fraction of ethanol is 10%, 20%, 30%, 40%, 50%
Liquid；Then the bag filter for filling gained reaction solution is put into dialyzate according to the order that volume fraction of ethanol in dialyzate reduces
Into dialysing several times under 4 DEG C of stirring conditions, each dialysis time is 3-7 days；The bag filter of gained reaction solution will finally be filled
Dialyse 2 days in 20mM hydrochloric acid solutions.
The reaction solution after methacrylic anhydride is removed to be freeze-dried to obtain Photocrosslinkable II Collagen Type VIs.
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
In normal pressure under the conditions of 4 DEG C, by Photocrosslinkable II collagenolysises and it is configured to concentration with 20mM hydrochloric acid solutions and is
The Photocrosslinkable II Collagen Type VI solution of 14mg/mL, and pH value is adjusted to 7.0 with 5M sodium hydroxide solutions, to after adjusting pH value
Photocrosslinkable II Collagen Type VI solution in add photoinitiator irgacure2959, be formulated as Photocrosslinkable II Collagen Type VI final concentrations
For 10mg/mL, the gel pre-polymerization liquid of the final concentration of 0.5mg/mL of photoinitiator, if having added the gel pre-polymerization after photoinitiator
Liquid concentration is higher, does not reach Photocrosslinkable II Collagen Type VIs and photoinitiator final concentration requirement, can add respective volume 0.2M phosphoric acid
Disodium hydrogen buffer solution.Then it is 8W/cm exposed to intensity by gel pre-polymerization liquid2Ultraviolet light under irradiate 30 seconds, you can realize pre-
Poly- liquid cures, and obtains II Collagen Type VI hydrogels.
In order to detect whether methacrylic anhydride is successfully grafted on II collagens, to the II type optical cements without any processing
Photocrosslinkable II Collagen Type VIs carry out nuclear magnetic resonance spectroscopy obtained by step (2) in former and embodiment 1, embodiment 5, embodiment 8, obtain
Hydrogen nuclear magnetic resonance figure as shown in Fig. 2, as can be seen that δ 5.3 and 5.6ppm (C=CH2), and δ in figure from Fig. 2 (a)
1.85ppm (CH3) correspond to the characteristic peak through the modified II Collagen Type VIs of methacrylic anhydride, show methacrylic anhydride by
Successfully it is grafted on collagen.Equally, the corresponding proton magnetic of Photocrosslinkable collagen that step (2) obtains in embodiment 5, embodiment 8
There are a double bond peak between 5~6ppm in resonance figure【As shown in Fig. 2 (b) and (c)】, show methacrylic anhydride by into
Work(is grafted on collagen.
In order to be characterized to the gelling performance of II Collagen Type VIs hydrogel provided by the invention, embodiment 1 is acquired first
The photo of the II Collagen Type VI hydrogels of preparation, as shown in Fig. 3 (b), it can be seen from the figure that being obtained by preparation method of the present invention
II Collagen Type VIs hydrogel be in gelatinous.At the same time to embodiment 1, embodiment 5, embodiment 8 prepare II Collagen Type VIs hydrogel into
Row DMA (Dynamic mechanical analysis) is analyzed, can from figure shown in obtained storage modulus figure such as Fig. 3 (c)
To find out, the storage modulus of II Collagen Type VI hydrogels prepared by embodiment 1 is about 2~3kPa, II Collagen Type VIs prepared by embodiment 5
The storage modulus of hydrogel is about 1kPa, and the storage modulus of II Collagen Type VI hydrogels prepared by embodiment 8 is about 6~7kPa, this
The II Collagen Type VIs hydrogel for showing to obtain by preparation method provided by the invention has certain mechanical strength, and passes through adjusting
Photocrosslinkable II Collagen Type VI final concentrations in gel pre-polymerization liquid, it is possible to achieve to the tune of the mechanical strength of gained II collagen hydrogels
Section.
1 cytoactive of application examples is tested
Using the lyophilized obtained Photocrosslinkable II Collagen Type VIs sample of step (2) in above-described embodiment 1 in normal pressure in 4 DEG C of conditions
Under, the solution of 10mg/mL is dissolved as with sterile 20mM hydrochloric acid solutions in super-clean bench, after sample is completely dissolved, use is sterile
5M sodium hydroxide solutions adjust pH value to 7.0, add sterile photoinitiator irgacure2959, be formulated as II Collagen Type VIs end
Concentration is 7mg/mL, the II Collagen Type VI pre-polymerization liquid of the modification of the final concentration of 0.5mg/mL of photoinitiator, if addition photoinitiator
Pre-polymerization liquid concentration afterwards is higher, can add 0.2 sterile disodium hydrogen phosphate salt buffer of respective volume.
It will cultivate to the bone marrow mesenchymal stem cells in P2 generations 1ml/ wares (each culture dish addition 1ml trypsase) pancreas
It is uniformly mixed after protease digestion 30s with gel pre-polymerization liquid, parcel cell density is every milliliter of pre-polymerization liquid product parcel 1 × 107
A cell.Then it is some groups of samples according to every 30 μ L, mono- components by the gel pre-polymerization liquid of parcel cell, each sample is exposed to
Ultraviolet ray intensity is 8W/cm2Lower irradiation 30 seconds, obtains the II Collagen Type VI hydrogels of three-dimensional parcel cell, sample is respectively in culture 1
My god, after 4 days, 7 days, 14 days and 21 days, it is imaged with the dyeing of FDA/PI solution using laser confocal microscope, as a result such as Fig. 4 institutes
Show, figure 4, it is seen that cell hydrogel show it is good sprawl and situation of rising in value, illustrate photo-crosslinking II Collagen Type VIs
Hydrogel is conducive to cell Proliferation, shows that II Collagen Type VIs hydrogel has excellent cell compatibility.
2 cell of application examples secretes matrix experiments
Using the lyophilized obtained photo-crosslinking Collagen specimens of step (2) in above-described embodiment 1 in normal pressure under the conditions of 4 DEG C,
The solution of 10mg/mL is dissolved as with sterile 20mM hydrochloric acid solutions in super-clean bench, after sample is completely dissolved, with sterile 5M
Sodium hydroxide solution adjusts pH value to 7.0, adds sterile photoinitiator irgacure2959, is formulated as II Collagen Type VI final concentrations
For 7mg/mL, the II Collagen Type VI pre-polymerization liquid of the modification of the final concentration of 0.5mg/mL of photoinitiator, if after addition photoinitiator
Pre-polymerization liquid concentration is higher, can add 0.2 sterile disodium hydrogen phosphate salt buffer of respective volume.
The bone marrow mesenchymal stem cells to P2 generations will be cultivated with pre- with two kinds of gels after 1ml/ ware Trypsin Induceds 30s
Poly- liquid is uniformly mixed, and parcel cell density is every milliliter of pre-polymerization liquid product parcel 1 × 107A cell.Then cell will be wrapped up
Gel pre-polymerization liquid is some groups of samples according to every 100 μ L, mono- components, and each sample is 8W/cm exposed to ultraviolet ray intensity2Lower photograph
Penetrate 30 seconds, obtain the II Collagen Type VI hydrogels of three-dimensional parcel cell, cultivate in the α-MEM culture mediums containing 10% (v/v) serum.
After in vitro culture 1 day, take out sample implantation nude mice by subcutaneous and carry out In vivo culture.After 14 days, sample preparation is taken out into 10 μm of thickness
Histotomy, with the sarranine-O and Toluidine blue staining of preparation, as shown in figure 5, from figure 5 it can be seen that polysaccharide matrix has
Obvious deposition, the II Collagen Type VIs hydrogel for showing to prepare can promote BMSCs to break up into cartilage direction.
Claims (8)
1. a kind of preparation method for inducing stem cell into the II Collagen Type VI hydrogels of cartilage differentiation, it is characterised in that step is as follows：
(1) II Collagen Type VI solution is prepared
At normal pressure, 0-4 DEG C, it is 1-4mg/mL that II Collagen Type VIs, which are dissolved in, and obtain mass concentration in the acid solution that pH value is 1-3
II Collagen Type VI solution；
(2) Photocrosslinkable II Collagen Type VIs are synthesized
At normal pressure, 0-4 DEG C, the pH value that II Collagen Type VI solution is adjusted with phosphate buffer or/and sodium hydroxide solution is 7 ＜
PH value≤8, then add methacrylic anhydride and supplement sodium hydroxide in normal pressure, 0-4 DEG C of stirring reaction 2-12h, reaction process
Solution makes the pH value of reaction system meet 7 ＜ pH value≤8, and the addition of methacrylic anhydride presses the amino in II Collagen Type VI solution
Molar ratio with methacrylic anhydride is 1:(0.2-5) is measured；
Unreacted methacrylic anhydride in gained reaction solution is removed after reaction, then will remove unreacted metering system
Reaction solution after acid anhydrides is freeze-dried to obtain Photocrosslinkable II Collagen Type VIs, or anti-after unreacted methacrylic anhydride by removing
Answer liquid to load bag filter, the Sheng liquid bag filter is immersed into the polyethylene glycol that mass concentration is 10%-50% at normal pressure, 0-4 DEG C
4-24h is concentrated in aqueous solution and obtains the concentrate containing Photocrosslinkable II Collagen Type VIs；
(3) photo-crosslinking II Collagen Type VI hydrogels are prepared
At normal pressure, 0-4 DEG C, Photocrosslinkable II Collagen Type VIs are dissolved with the acid solution that pH value is 1-3 to obtain Photocrosslinkable II types
Collagen solution, and with sodium hydroxide solution adjust pH value to 7.0-7.5, then to adjust pH value after Photocrosslinkable II Collagen Type VIs
Photoinitiator is added in solution, it is final concentration of to be configured to the final concentration of 4-10mg/mL of Photocrosslinkable II Collagen Type VIs, photoinitiator
Gel pre-polymerization liquid, is then placed under ultraviolet light and irradiates 10-60s, i.e., by the gel pre-polymerization liquid of 0.2-1mg/mL under atmospheric pressure at room
Obtain II Collagen Type VI hydrogels；
Or under the conditions of normal pressure, 0-4 DEG C, the sodium hydroxide solution of the concentrate containing Photocrosslinkable II Collagen Type VIs is adjusted into pH
Value adds photoinitiator to 7.0-7.5, then into the Photocrosslinkable II Collagen Type VI solution after adjusting pH value, and being configured to can light friendship
Join the final concentration of 4-10mg/mL of II Collagen Type VIs, the gel pre-polymerization liquid of the final concentration of 0.2-1mg/mL of photoinitiator, then in normal pressure
Gel pre-polymerization liquid is placed under ultraviolet light at room temperature and irradiates 10-60s, that is, obtains II Collagen Type VI hydrogels.
2. stem cell is induced according to claim 1 into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, its feature
It is in the step (2), passes through the side of unreacted methacrylic anhydride in reaction solution obtained by removing of dialysing after reaction
Formula is：Ethanol is added into the acid solution that pH value is 1-3 be configured to serial dialysis liquid first, in serial dialysis liquid, each dialyzate
Volume fraction of ethanol reduce by 10% successively, the volume fraction of ethanol is no more than in the dialyzate of volume fraction of ethanol maximum
50%；Then the bag filter for filling reaction solution is put into dialyzate and is dialysed several times in 0-4 DEG C under agitation, every time thoroughly
Analysing the volume fraction of ethanol in the dialyzate replaced reduces by 10%, and each dialysis time is 3-7 days；To finally reaction solution be filled
Bag filter is dialysed 1-3 days in the acid solution that pH value is 2-3.
3. for induction stem cell according to claim 1 or claim 2 into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, it is special
Sign be in the step (3), photoinitiator irgacure2959, irgacure184, irgacure127,
Irgacure500, irgacure651 or rose Bengal.
4. for induction stem cell according to claim 1 or claim 2 into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, it is special
Sign is in the step (3) after addition photoinitiator, by add phosphate buffer be met can light join II Collagen Type VIs
Final concentration and the gel pre-polymerization liquid of photoinitiator final concentration requirement.
5. stem cell is induced according to claim 4 into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, its feature
It is in the step (3) that ultraviolet ray intensity is 4-8W/cm2。
6. for induction stem cell according to claim 1 or claim 2 into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, it is special
Sign is in the step (2) and step (3) that the molar concentration of sodium hydroxide solution is 0.5-5M.
7. for induction stem cell according to claim 1 or claim 2 into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, it is special
Sign is that the molar concentration of phosphate buffer in the step (2) and step (3) is 0.2-2M；The phosphate buffer is
Disodium phosphate soln or sodium dihydrogen phosphate.
8. for induction stem cell according to claim 1 or claim 2 into the preparation method of the II Collagen Type VI hydrogels of cartilage differentiation, it is special
Sign is in the step (1), (2) and (3) that acid solution is hydrochloric acid solution or acetum.
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| CN110790950A (en) * | 2019-10-21 | 2020-02-14 | 南京理工大学 | Photo-crosslinking recombinant collagen hydrogel, preparation method and application thereof in 3D bioprinting |
| CN112126027A (en) * | 2020-09-24 | 2020-12-25 | 中山大学 | Hydrogel material and preparation method and application thereof |
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