CN113150313A - Preparation method of neutral-dissolved modified photocured collagen and photocured collagen raw hydrogel - Google Patents
Preparation method of neutral-dissolved modified photocured collagen and photocured collagen raw hydrogel Download PDFInfo
- Publication number
- CN113150313A CN113150313A CN202010012313.4A CN202010012313A CN113150313A CN 113150313 A CN113150313 A CN 113150313A CN 202010012313 A CN202010012313 A CN 202010012313A CN 113150313 A CN113150313 A CN 113150313A
- Authority
- CN
- China
- Prior art keywords
- collagen
- solution
- modified
- acid
- cured
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H1/00—Macromolecular products derived from proteins
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2471/00—Characterised by the use of polyethers obtained by reactions forming an ether link in the main chain; Derivatives of such polymers
- C08J2471/02—Polyalkylene oxides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Dispersion Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Preparation (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a preparation method of neutral-dissolved modified photo-cured collagen and photo-cured collagen water gel, belonging to the technical field of biological materials. The modified collagen is capable of dissolving under neutral conditions. Can be rapidly solidified into collagen hydrogel through thiol-ene reaction, and has excellent gel strength and biocompatibility. The cross-linking agent is a compound containing two or more thiol groups, which can form a gel by a photoinitiator or other free radical generating reaction. In addition, the gel can be used for conveniently modifying functional molecules, such as protein drugs, antibodies, polypeptides and the like.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of neutral-dissolved modified photocured collagen and photocured collagen hydrogel.
Background
The photocurable gel is a gel formed by appropriately modifying a molecule and rapidly polymerizing the molecule by irradiation with light in the presence of an initiator. The modified group of the material generally contains double bonds, and can generate polymerization reaction under the initiation of free radicals, such as photosensitive hyaluronic acid, sodium alginate, gelatin, polyethylene glycol and the like. Currently, photosensitive biomaterials are mainly based on two principles: 1. by auto-polymerization of alkenes such as a methacrylic group, a propylene group, etc., as disclosed in documents 1 to 3[ document 1: lin, H., et al, Application of visual light-based project stereoscopic mapping with designed implementation. biomaterials,2013.34(2) p.331-9; document 2: nichol, J.W., et al, Cell-laden microbial gels biomaterials,2010.31(21): p.5536-44; document 3: jeon, O., et al, Photosprung linked together hydrogels with structural biomaterials,2009.30(14): p.2724-34. ]; 2. thiol-ene reactions are polymerized based on one of the click chemistries, as described in references 4-6[ reference 4: fu, Y, et al, 3D cell entry in cross linked protein-poly (ethylene glycol) diacid polyesters, biomaterials,2012.33(1): p.48-58; document 5: lin, C.C., A.Raza and H.Shih, PEG hydrogels for used by thio-ethylene chemistry and the ir effect on the formation and recovery of insulin-dependent cell adhesives, biomaterials,2011.32(36) p.9685-95; document 6: guvendriren, M.and J.A.Burdick, Stiffening hydrogels to probe short-and long-term cellular responses to dynamic machinery. Nat Commun,2012.3: p.792 ]. However, photosensitive materials have certain disadvantages: 1. the polysaccharide macromolecules have no adhesion sites, and the cell extension can be supported by grafting adhesive polypeptide and the like; 2. protein materials such as gelatin have a low molecular weight and a high minimum concentration for gel formation, so that the growth state of cells in the protein materials is poor. The photocuring system based on thiol-ene reaction has fast speed and small dosage of initiator, but the problems can not be overcome, so the improvement of compatibility is not obvious.
Collagen is a biological material with good biocompatibility, is widely applied to the fields of tissue engineering and regenerative medicine, and a series of products such as collagen repair membranes, collagen bone scaffolds and the like have clinically obtained application permission. In experimental studies, acid-soluble collagen has been widely used, and undenatured collagen is dissolved in an acidic solution and forms a gel at 37 ℃ at a certain salt concentration after neutralization. However, the conventional collagen gel has the following disadvantages: 1. the concentration of rat tail collagen commonly used in experimental research is generally 0.3% w/v, and the rat tail collagen is not easy to be neutralized uniformly after the concentration is increased; 2. the collagen is difficult to exist stably after being neutral, 0.3% w/v rat tail collagen can form gel at a slightly high temperature under a neutral condition, and the gel speed is accelerated when the concentration is increased, so that the collagen is required to be prepared in situ for each use, the operation time is limited, and the operation is complex; 3. the strength is poor, and the gel is formed by depending on the entanglement of collagen fibers and the action of non-covalent bonds and is easy to deform and separate out water; 4. the gel is slow, and the collagen forms hydrogel by means of fiber entanglement and non-covalent bond after assembly, and the gel speed is slow, usually more than 15 min. Therefore, it is necessary to modify the acid-soluble collagen appropriately to improve its handleability and mechanical strength.
The literature reports the synthesis of photocurable collagen modified with NHS-activated methacrylate [ Gaudet, I.D. and D.I.Shreiber, Characterisation of methylated type-I collagen as a dynamic, photoactive hydrogel.biointerographs, 2012.7(1-4): p.25] or methacrylic anhydride [ Brinkman, W.T., et al, Photo-crosslinking-linking of type I collagen in the present of cosmetic cells: mechanical properties, cell viability, and function. collagen, 2003.4(4): p.890-5] to obtain photocurable photosensitive collagen. However, the collagen can only be dissolved in an acidic solution, so that the application range of the collagen is limited, and a large amount of citation and report are not found.
Disclosure of Invention
The invention aims to provide a preparation method of modified photo-cured collagen capable of being neutrally dissolved and capable of being polymerized by free radicals and a photo-cured collagen hydrogel, which are capable of neutrally dissolving the modified collagen capable of being initiated and polymerized by the free radicals, and have obviously improved curing speed, mechanical stability and even cell compatibility compared with collagen.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of neutral dissolved modified photo-cured collagen comprises the following steps:
(1) preparation of acid-soluble collagen solution: adding the extracted I type collagen into 0.1-0.5mol/L acid solution to prepare 3-10mg/mL acid-soluble collagen solution by taking the extracted I type collagen as a raw material;
(2) diluting the acid-soluble collagen solution in the step (1) with 0-5 times of water by mass, and adjusting the pH value of the diluted acid-soluble collagen solution to 7-10 by adopting a strong alkaline solution, wherein the temperature is maintained at 0-10 ℃;
(3) preparation of acid anhydride solution: dissolving norbornene dianhydride in a solvent to obtain an anhydride solution;
(4) dropwise adding the anhydride solution obtained in the step (2) into the collagen solution with the pH value of 7-10 and the temperature of 0-10 ℃ in the step (2) for reaction, and dropwise adding a sodium hydroxide solution to maintain the pH value of 7-10; the temperature adjustable range is 0-37 ℃, and the reaction time is 30min-1 day;
(5) and (4) dialyzing the reaction product obtained in the step (3) by using distilled water, and freeze-drying to obtain the neutral dissolved modified photocured collagen.
In the step (1), the type I collagen is type I collagen such as mouse collagen, bovine collagen, pig collagen or fish collagen, and the type I collagen is extracted by an enzymatic method or an acid method; the acid solution is acetic acid solution or hydrochloric acid solution, and the pH of the prepared acid-soluble collagen solution is 1-5.
In the step (2), the strong alkali solution is a sodium hydroxide solution or a potassium hydroxide solution with the concentration of 0.5-6 mol/L; in the step (3), the solvent is ethanol or acetone.
In the step (4), the weight ratio of the acid anhydride in the acid anhydride solution to the collagen in the collagen solution is (0.1-10): 1; the reaction temperature is 0-37 ℃; the volume ratio of the solvent in the acid anhydride solution to the collagen solution is (0.5-2): 20. In the reaction process, the norbornene dianhydride reacts with one or two of free amino and hydroxyl of the modified collagen side chain, and is connected with the collagen through ester bonds and/or amido bonds after the reaction.
The prepared modified light-cured collagen can be dissolved in PBS (phosphate buffer solution) with pH of 7-8, Hanks balanced salt solution or various cell culture media, and can also be dissolved in acidic solution;
the prepared modified photocured collagen can be uniformly blended with other solutions or modified products of other solutions under a neutral condition without forming collagen fibers; the other solution is one or more of high molecular water solutions such as gelatin, sodium alginate, hyaluronic acid, water-soluble cellulose, xanthan gum, gellan gum, carrageenan and polyethylene glycol.
Preparing a collagen hydrogel by using the modified photo-cured collagen, wherein the collagen hydrogel is obtained by a process comprising the following steps (a) to (c):
(a) dissolving the modified photocured collagen in a neutral solution at low temperature to obtain a modified photocured collagen solution with the concentration of 5-10 mg/ml;
(b) adding a cross-linking agent and a photoinitiator into the modified photocuring collagen solution obtained in the step (a); wherein: the concentration of the cross-linking agent in the photo-curing collagen solution is 0.5-10mM, and the mass of the photoinitiator accounts for 0.01-2% of the mass of the modified photo-curing collagen solution.
(c) And (c) illuminating the solution finally obtained in the step (b), wherein the light acts on a photoinitiator to generate free radicals to initiate thiol-ene reaction, so as to obtain the collagen hydrogel.
In the step (b), the cross-linking agent is a small molecule or a macromolecule of a compound containing two or more thiol groups, such as Dithiothreitol (DTT); sulfhydryl-modified double-arm or multi-arm polyethylene glycol with different molecular weights; thiol-modified double-arm or multi-arm polyvinyl alcohols; a synthetic polypeptide, genetically engineered peptide or protein comprising 2 or more free thiol groups. )
In the step (b), the photoinitiator is a water-soluble photoinitiator such as phenyl-2, 4, 6-trimethylbenzoyllithium phosphite (LAP) or 2-hydroxy-1- [4- (2-hydroxyethoxy) phenyl ] -2-methyl-1-propanone (irgacure 2959).
In the step (c), the light intensity is adjustable by using the optimal wavelength matched with the photoinitiator.
In the process of forming gel in the step (c), the crosslinking agent molecules with sulfydryl can react with the norbornene groups of the modified collagen side chains to form a network skeleton structure, namely, the collagen molecules are crosslinked into the network structure to form gel; the collagen hydrogel can fix functional molecules which are not used as a gel skeleton structure, wherein the functional molecules are protein, peptide, small molecule medicine, nucleic acid, carbohydrate or polysaccharide molecules and the like; the functional molecule has 1 or more than 1 reactive groups, and the reactive groups are free sulfydryl or/and alkene groups (such as methacrylate, acrylate, maleimide, norbornenyl and the like).
The reactive groups are divided into two types, namely a reactive group carried by a functional molecule and a modified reactive group.
The reaction group carried by the functional molecule is mainly free sulfhydryl or alkene group, including but not limited to various proteins, synthetic polypeptides, antibodies, cells, etc.; the modified reactive groups are divided into thiol reactive groups and alkene reactive groups, and are modified on functional molecules in various ways.
The dry modified photocured collagen and the modified photocured collagen hydrogel prepared by the method are applied to 3D printing, in-situ injection, gel dressing, hemostatic and the like in the modes of three-dimensional culture, extrusion, photocuring, ink jetting and the like of cells.
The invention has the following advantages and beneficial effects:
1. the invention synthesizes the light-curable collagen which can be dissolved under the neutral condition for the first time, and further prepares the raw hydrogel, and the prepared gel has good mechanical property and excellent biocompatibility.
2. The modified collagen is synthesized in water phase, and the synthesis method is simple and has low cost. Compared with polysaccharide light-cured materials, the cell adhesion is promoted without modifying the adhesion polypeptide. Compared to other thiol-ene photocurable materials, no crosslinker with degradation sites is required. Compared with the light-cured protein material, the cell has faster proliferation speed and expansion capability.
3. The gel speed of the common collagen is more than 15min, the curing speed of the modified collagen gel prepared by the invention is as fast as a second grade, and the modified collagen gel can be stably stored before free radicals are introduced. Greatly improves the collagen operability in a neutral state.
4. The modified collagen prepared by the invention can be conveniently mixed with other materials without causing collagen fibrosis, and the modification space of the material is greatly improved.
5. The modified collagen of the invention forms hydrogel after being solidified, and can conveniently modify active substances, such as medicines, growth factors, polypeptides and the like.
6. Realizes collagen printing containing cells in various modes.
Drawings
FIG. 1 shows nuclear magnetic spectrum of modified collagen.
FIG. 2 is modified collagen and collagen; wherein: (a) SDS electrophoretogram; (b) a Zeta potential; (c) solubility.
FIG. 3 shows the appearance of the gel and the growth state of fibroblasts at 7 days; wherein: (a) before photocuring; (b) gelling after photocuring; (c) solidifying in an injector and then extruding; (d) fibroblast phalloidin staining in the gel.
FIG. 4 is a graph showing the effect of different types and concentrations of cross-linking agents on fibroblast survival and expansion; wherein: (a) 1 and 7 days of SH-PEG-SH dead-live staining; (b) 1day and 7 days of dead-live staining of the polypeptide; (c)1 and 7 days of dead and live staining for DTT; (d) survival rate after 1 day.
FIG. 5 is a rheological curve of photo-cured modified collagen gel with different initiator dosages.
FIG. 6 is a rheological curve of photo-cured modified collagen gel at different concentrations.
FIG. 7 is an extruded 3D printing of photo-activated modified collagen of example 4; wherein: (a) a schematic diagram; (b) printing the appearance of the structure; (c) dying and alive in 1 day; (d) DAPI staining for 7 days; (e)7 days phalloidin staining; (f) merge.
FIG. 8 is an extruded 3D printing of photo-activated modified collagen according to example 5; wherein: (a) a schematic diagram; (b) printing the appearance of the structure; (c) dying and alive in 1 day; (d) DAPI staining for 7 days; (e)7 days phalloidin staining; (f) merge.
FIG. 9 is the spreading of fibroblasts in alginate/modified collagen, alginate/collagen gel at 7 days; wherein: (a) sodium alginate/modified collagen gel; (b) sodium alginate/collagen gel.
Detailed Description
For a further understanding of the present invention, the following description is given in conjunction with the examples which are set forth to illustrate, but are not to be construed to limit the present invention, features and advantages.
Example 1:
extracting collagen:
bovine achilles tendon was crushed and degreased, immersed in a 0.16mol/L acetic acid solution, and pepsin (3mg/mL) was added. After stirring at 4 ℃ for 72 hours, the precipitate was salted out with 4mol/L sodium chloride solution, and the resulting precipitate was dialyzed against 0.16mol/L acetic acid with a cut-off of 8000Da in a dialysis bag. The concentration was adjusted using acetic acid solution (0.16mol/L) to give 6mg/mL bovine achilles tendon collagen, all in sterile procedures.
Collagen modification:
1. 150mL of the bovine Achilles tendon was taken and added with 150mL of cold pure water to obtain 300mL of a 3mg/mL solution of bovine Achilles tendon.
2. 0.9g of nadic anhydride was dissolved in 15mL of ethanol.
3. The pH of the collagen solution was adjusted to 9 using 3M sodium hydroxide and maintained at 4 ℃ in an ice bath.
4. And dropwise adding the anhydride solution into the collagen solution, simultaneously dropwise adding 3M sodium hydroxide to maintain the pH of 9, heating the reaction system to 25 ℃, and continuously dropwise adding sodium hydroxide to maintain the pH of 9 until the pH is unchanged after the anhydride is dropwise added.
5. After reacting for 2 hours, transferring the solution into a dialysis bag with the molecular weight cut-off of 8000, dialyzing for 4 days by using distilled water, changing the water twice every day, and freeze-drying the dialyzed solution to obtain the soluble modified collagen.
6. The nuclear magnetic resonance hydrogen spectrum (figure 1) and SDS gel electrophoresis (figure 2(a)) of the modified collagen obtained by the reaction prove that the norbornene group is successfully modified, and the collagen still retains the three-strand helical structure.
7. The isoelectric points and solubilities of the unmodified collagen solution and the modified collagen solution were measured at different pH as shown in FIGS. 2(b) - (c).
8. The collagen was re-dissolved in the DPBS solution at a concentration of 6 mg/mL.
9. Adding SH-PEG-SH (molecular weight 1000) into the modified collagen solution to a final concentration of 4mM, adding photoinitiator LAP to a final concentration of 0.05mg/mL, and adding fibroblast to a final concentration of 5 x 105and/mL. Mixing well, and using 365nm, 10mw/cm2Irradiating with light for 30s to obtain cell-containing collagen hydrogel, culturing for 7d, and staining with phalloidin and DAPIThe results of the cells in the colored gel are shown in FIG. 3 (d).
Example 2:
extracting collagen:
bovine achilles tendon was crushed and degreased, immersed in a 0.16mol/L acetic acid solution, and pepsin (3mg/mL) was added. After stirring at 4 ℃ for 72 hours, the precipitate was salted out with 4mol/L sodium chloride solution, and the resulting precipitate was dialyzed against 0.16mol/L acetic acid with a cut-off of 8000Da in a dialysis bag. The concentration was adjusted using acetic acid solution (0.16mol/L) to give 6mg/mL bovine achilles tendon collagen, all in sterile procedures.
Collagen modification:
1. 150mL of the bovine Achilles tendon was taken and added with 150mL of cold pure water to obtain 300mL of a 3mg/mL solution of bovine Achilles tendon.
2. 0.9g of nadic anhydride was dissolved in 15mL of ethanol.
3. The pH of the collagen solution was adjusted to 9 using 3M sodium hydroxide and maintained at 4 ℃ in an ice bath.
4. And dropwise adding the anhydride solution into the collagen solution, simultaneously dropwise adding 3M sodium hydroxide to maintain the pH of 9, heating the reaction system to 25 ℃, and continuously dropwise adding sodium hydroxide to maintain the pH of 9 and keep the pH unchanged after the anhydride is dropwise added.
5. After reacting for 2 hours, transferring the solution into a dialysis bag with the molecular weight cut-off of 8000, dialyzing for 4 days by using distilled water, changing the water twice every day, and freeze-drying the dialyzed solution to obtain the soluble modified collagen.
6. The collagen was re-dissolved in the DPBS solution at a concentration of 6 mg/mL.
7. Adding cross-linking agents with different types and concentrations into the modified collagen solution: adding SH-PEG-SH (molecular weight 1000) to a final concentration of 4mM and 8mM, adding Dithiothreitol (DTT) to a final concentration of 4mM and 8mM, adding a synthetic polypeptide (amino acid sequence GCREG PQGIWGQ ERCG) having a thiol group at the terminal end thereof and having a MMP enzyme degradation site to a final concentration of 4mM and 8mM, adding a photoinitiator LAP to a final concentration of 0.05mg/mL, adding fibroblasts to a final concentration of 5.105and/mL. Mixing well, and using 365nm, 10mw/cm2Irradiating for 30s to obtain collagen hydrogel containing cells with different cross-linking agents and concentrations, and culturingThe results of the dying and alive staining after 1day of cultivation and the survival rate and the dying and alive staining after 7 days of cultivation are shown in FIG. 4.
8. Adding SH-PEG-SH (molecular weight is 1000) with different concentrations into the modified collagen solution, wherein the final concentrations are 0, 2,4,6 and 8mM respectively, adding a photoinitiator LAP, the final concentration is 0.05mg/mL, measuring the change of storage modulus and loss modulus in the photocuring process by using a rheometer, the light wavelength is 365nm, and the light intensity is 20mW/cm2The rheological curve is shown in FIG. 5.
9. Adding DPBS into the modified collagen solution to dilute the solution to 2mg/mL, 3mg/mL or 4mg/mL, adding SH-PEG-SH (molecular weight 1000) to a final concentration of 4mM, adding a photoinitiator LAP to a final concentration of 0.05mg/mL, and measuring changes of storage modulus and loss modulus in a photocuring process by using a rheometer, wherein the light wavelength is 365nm, and the light intensity is 20mW/cm2The rheological curve is shown in FIG. 6.
Example 3:
extracting collagen: the rat tendon was taken, crushed and degreased, and immersed in 0.02M acetic acid solution. Dissolving at 4 deg.C for 3 weeks, centrifuging, collecting supernatant, salting out with sodium chloride, dialyzing the obtained precipitate with 0.02M acetic acid, and dialyzing with dialysis bag having molecular weight cutoff of 8000 Da. The concentration was adjusted using acetic acid solution to obtain 3mg/mL bovine achilles tendon collagen, all in sterile procedures.
Collagen modification:
1. 300mL of the above rat tail collagen solution was taken.
2. 0.9g of nadic anhydride was dissolved in 15mL of ethanol.
3. The pH of the collagen solution was adjusted to 9 using 3M sodium hydroxide and maintained at 4 ℃ in an ice bath.
4. And dropwise adding the anhydride solution into the collagen solution, simultaneously dropwise adding 3M sodium hydroxide to maintain the pH value of 9, continuously dropwise adding the sodium hydroxide until the pH value is unchanged after the anhydride is dropwise added, and adjusting the temperature of the reaction system to 25 ℃.
5. After reacting for 2 hours, transferring the solution into a dialysis bag with the molecular weight cut-off of 8000, dialyzing for 4 days by using distilled water, changing the water twice every day, and freeze-drying the dialyzed solution to obtain the soluble modified collagen.
6. The collagen was re-dissolved in the DPBS solution at a concentration of 6 mg/mL.
7. And (3) reacting the basic fibroblast growth factor with sh-peg-nhs to obtain the basic fibroblast growth factor with a sulfhydryl group.
8. Adding SH-PEG-SH (molecular weight 1000) into the modified collagen solution to a final concentration of 4mM, adding photoinitiator LAP to a final concentration of 0.05mg/mL, adding the modified growth factor to a final concentration of 1 μ g/mL, and adding fibroblast to a final concentration of 5 x 105and/mL. Mixing well, and using 365nm, 10mw/cm2And (5) illuminating for 30s to obtain the collagen hydrogel fixed with the growth factors.
Example 4:
extracting collagen: bovine achilles tendon was crushed and defatted, immersed in 0.16M acetic acid solution, and 0.3% pepsin was added. After stirring at 4 ℃ for 72 hours, sodium chloride was salted out, and the resulting precipitate was dialyzed against 0.16M acetic acid with a cut-off of 8000Da in the dialysis bag. The concentration was adjusted using acetic acid solution to obtain 6mg/mL bovine achilles tendon collagen, all in sterile procedures.
Collagen modification:
1. 150mL of the bovine Achilles tendon was taken and added with 150mL of cold pure water to obtain 300mL of a 3mg/mL solution of bovine Achilles tendon.
2. 0.9g of nadic anhydride was dissolved in 15mL of ethanol.
3. The pH of the collagen solution was adjusted to 9 using 3M sodium hydroxide and maintained at 4 ℃ in an ice bath.
4. And dropwise adding the anhydride solution into the collagen solution, simultaneously dropwise adding 3M sodium hydroxide to maintain the pH value of 9, continuously dropwise adding the sodium hydroxide until the pH value is unchanged after the anhydride is dropwise added, and adjusting the temperature of the reaction system to 25 ℃.
5. After reacting for 2 hours, transferring the solution into a dialysis bag with the molecular weight cut-off of 8000, dialyzing for 4 days by using distilled water, changing the water twice every day, and freeze-drying the dialyzed solution to obtain the soluble modified collagen.
6. The collagen was re-dissolved in the DPBS solution at a concentration of 10 mg/mL.
7. Adding SH-PEG-SH into the modified collagen solution to a final concentration of 4mM, adding a photoinitiator LAP to the modified collagen solution to a final concentrationAdding fibroblast at 0.05mg/mL to a final concentration of 5 x 105Gelatin was added to a final concentration of 30mg/mL, and glycerol was added to a final concentration of 30 mg/mL. After being uniformly mixed, the mixture is added into a 3D printer, the temperature of a spray head is 18 ℃, and the temperature of a bottom plate is 10 ℃. After printing was complete, 365nm, 10mw/cm was used2And (5) illuminating for 30s to obtain the printed collagen hydrogel. The structural appearance was printed, 1day dead-live staining, and 7 days phalloidin/DAPI staining are shown in fig. 7.
Example 5:
extracting collagen: bovine achilles tendon was crushed and defatted, immersed in 0.16M acetic acid solution, and 0.3% pepsin was added. After stirring at 4 ℃ for 72 hours, sodium chloride was salted out, and the resulting precipitate was dialyzed against 0.16M acetic acid with a cut-off of 8000Da in the dialysis bag. The concentration was adjusted using acetic acid solution to obtain 6mg/mL bovine achilles tendon collagen, all in sterile procedures.
Collagen modification:
1. 150mL of the bovine Achilles tendon was taken and added with 150mL of cold pure water to obtain 300mL of a 3mg/mL solution of bovine Achilles tendon.
2. 0.9g of nadic anhydride was dissolved in 15mL of ethanol.
3. The pH of the collagen solution was adjusted to 9 using 3M sodium hydroxide and maintained at 4 ℃ in an ice bath.
4. And dropwise adding the anhydride solution into the collagen solution, simultaneously dropwise adding 3M sodium hydroxide to maintain the pH of 9, and continuously dropwise adding the sodium hydroxide until the pH is unchanged after the anhydride is dropwise added.
5. After reacting for 2 hours, transferring the solution into a dialysis bag with the molecular weight cut-off of 8000, dialyzing for 4 days by using distilled water, changing the water twice every day, and freeze-drying the dialyzed solution to obtain the soluble modified collagen.
6. The collagen was re-dissolved in the DPBS solution at a concentration of 10 mg/mL.
7. Adding SH-PEG-SH into the modified collagen solution to a final concentration of 4mM, adding a photoinitiator LAP to a final concentration of 0.05mg/mL, adding fibroblasts to a final concentration of 5 × 105/mL, and adding a certain amount of PBS to adjust the final concentration of the modified collagen to 0.6 mg/mL.
8. Adding the solution into a 3D printer nozzleExtruding at 2mW/cm2And (3) irradiating the gel with 365nm ultraviolet light to obtain the 3D printing gel with a certain shape. The structural appearance was printed, 1day dead-live staining, and 7 days phalloidin/DAPI staining are shown in fig. 8.
Example 6:
extracting collagen: bovine achilles tendon was crushed and defatted, immersed in 0.16M acetic acid solution, and 0.3% pepsin was added. After stirring at 4 ℃ for 72 hours, sodium chloride was salted out, and the resulting precipitate was dialyzed against 0.16M acetic acid with a cut-off of 8000Da in the dialysis bag. The concentration was adjusted using acetic acid solution to obtain 6mg/mL bovine achilles tendon collagen, all in sterile procedures.
Collagen modification:
1. 150mL of the bovine Achilles tendon was taken and added with 150mL of cold pure water to obtain 300mL of a 3mg/mL solution of bovine Achilles tendon.
2. 0.9g of nadic anhydride was dissolved in 15mL of ethanol.
3. The pH of the collagen solution was adjusted to 9 using 3M sodium hydroxide and maintained at 4 ℃ in an ice bath.
4. And dropwise adding the anhydride solution into the collagen solution, simultaneously dropwise adding 3M sodium hydroxide to maintain the pH of 9, and continuously dropwise adding the sodium hydroxide until the pH is unchanged after the anhydride is dropwise added.
5. After reacting for 2 hours, transferring the solution into a dialysis bag with the molecular weight cut-off of 8000, dialyzing for 4 days by using distilled water, changing the water twice every day, and freeze-drying the dialyzed solution to obtain the soluble modified collagen.
6. The collagen was re-dissolved in the DPBS solution at a concentration of 6 mg/mL.
7. Adding metalloprotease into the modified collagen solution to degrade and synthesize polypeptide CGPQGIWGQC to a final concentration of 5mM, adding photoinitiator LAP to a final concentration of 0.05mg/mL, and adding fibroblast to a final concentration of 5 × 105and/mL. Mixing well, and using 365nm, 10mw/cm2And (3) irradiating for 30s to obtain the collagen hydrogel with the metalloprotease degradation sites.
Example 7:
extracting collagen: bovine achilles tendon was crushed and defatted, immersed in 0.16M acetic acid solution, and 0.3% pepsin was added. After stirring at 4 ℃ for 72 hours, sodium chloride was salted out, and the resulting precipitate was dialyzed against 0.16M acetic acid with a cut-off of 8000Da in the dialysis bag. The concentration was adjusted using acetic acid solution to obtain 6mg/mL bovine achilles tendon collagen, all in sterile procedures.
Collagen modification:
1. 150mL of the bovine Achilles tendon was taken and added with 150mL of cold pure water to obtain 300mL of a 3mg/mL solution of bovine Achilles tendon.
2. 0.9g of nadic anhydride was dissolved in 15mL of ethanol.
3. The pH of the collagen solution was adjusted to 9 using 3M sodium hydroxide and maintained at 4 ℃ in an ice bath.
4. And dropwise adding the anhydride solution into the collagen solution, simultaneously dropwise adding 3M sodium hydroxide to maintain the pH of 9, and continuously dropwise adding the sodium hydroxide until the pH is unchanged after the anhydride is dropwise added.
5. After reacting for 2 hours, transferring the solution into a dialysis bag with the molecular weight cut-off of 8000, dialyzing for 4 days by using distilled water, changing the water twice every day, and freeze-drying the dialyzed solution to obtain the soluble modified collagen.
6. The collagen was re-dissolved in the DPBS solution at a concentration of 10 mg/mL.
7. Adding the modified collagen solution into a crosslinking agent SH-PEG-SH with a final concentration of 4mM, adding a photoinitiator LAP with a final concentration of 0.05mg/mL, and adding fibroblasts with a final concentration of 5 x 105Sodium alginate was added to a final concentration of 15mg/mL and modified collagen was added to a final concentration of 5 mg/mL. After mixing well, the mixture was added dropwise to a 200mM calcium chloride solution, and after 5 minutes, 365nm, 10mw/cm was used2And (3) irradiating for 30s, and cleaning calcium chloride with normal saline to obtain the double-skeleton sodium alginate-photosensitive collagen hydrogel. The same concentration of neutralized unmodified collagen and sodium alginate solution was mixed, and the same concentration of cells was added and cross-linked in the same manner as a control. The gel can be ion-crosslinked, and has cell expansion and adhesion superior to those of sodium alginate/common collagen mixture hydrogel. After 7 days the phalloidin/DAPI stained cells in both gels, and the results are shown in fig. 9.
Claims (10)
1. A preparation method of neutral dissolved modified photo-cured collagen is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of acid-soluble collagen solution: adding the extracted I type collagen into 0.1-0.5mol/L acid solution to prepare 3-10mg/mL acid-soluble collagen solution by taking the extracted I type collagen as a raw material;
(2) diluting the acid-soluble collagen solution in the step (1) with 0-5 times of water by mass, adjusting the pH value of the diluted acid-soluble collagen solution to 7-10 by adopting a strong alkali solution, and storing at the temperature of 0-10 ℃ for later use;
(3) preparation of acid anhydride solution: dissolving norbornene dianhydride in a solvent to obtain an anhydride solution;
(4) dropwise adding the anhydride solution obtained in the step (2) into the collagen solution with the pH value of 7-10 and the temperature of 0-10 ℃ in the step (2) for reaction, and dropwise adding a sodium hydroxide solution to maintain the pH value of 7-10; the reaction temperature is 0-37 ℃, and the reaction time is 30min-1 day;
(5) and (4) dialyzing the reaction product obtained in the step (3) by using distilled water, and freeze-drying to obtain the neutral dissolved modified photocured collagen.
2. The method for preparing a neutrally solubilized modified photo-cured collagen according to claim 1, wherein: in the step (1), the type I collagen is rat collagen, bovine collagen, pig collagen or fish collagen, and the extraction mode of the type I collagen is enzyme method or acid method extraction; the acid solution is acetic acid solution or hydrochloric acid solution, and the pH of the prepared acid-soluble collagen solution is 1-5.
3. The method for preparing a neutrally solubilized modified photo-cured collagen according to claim 1, wherein: in the step (2), the strong alkali solution is a sodium hydroxide solution or a potassium hydroxide solution with the concentration of 0.5-6 mol/L; in the step (3), the solvent is ethanol or acetone.
4. The method for preparing a neutrally solubilized modified photo-cured collagen according to claim 1, wherein: in the step (4), the weight ratio of the acid anhydride in the acid anhydride solution to the collagen in the collagen solution is (0.1-10): 1; the reaction temperature is 0-37 ℃; the volume ratio of the solvent in the acid anhydride solution to the collagen solution is (0.5-2): 20. In the reaction process, the norbornene dianhydride reacts with one or two of free amino and hydroxyl of the modified collagen side chain, and is connected with the collagen through ester bonds or/and amido bonds after the reaction.
5. The method for preparing a neutrally solubilized modified photo-cured collagen according to claim 1, wherein: the prepared modified light-cured collagen can be dissolved in PBS solution with pH of 7-8, Hanks balanced salt solution or various cell culture media;
the prepared modified photocured collagen can be uniformly blended with other solutions or modified products of other solutions under a neutral condition without forming collagen fibers; the other solution is one or more of high molecular water solutions such as gelatin, sodium alginate, hyaluronic acid, water-soluble cellulose, xanthan gum, gellan gum, carrageenan and polyethylene glycol.
6. A collagen hydrogel produced by using the modified photo-cured collagen produced by the method of claim 1, wherein: the process for obtaining the collagen hydrogel comprises the following steps (a) to (c):
(a) dissolving the modified light-cured collagen in a D-PBS (Du's phosphate buffer) solution to obtain a modified light-cured collagen solution with the concentration of 2-20 mg/mL;
(b) adding a cross-linking agent and a photoinitiator into the modified photocuring collagen solution obtained in the step (a); wherein: the concentration of the cross-linking agent in the photo-curing collagen solution is 0.5-10mM, and the mass of the photoinitiator accounts for 0.01-2% of the mass of the modified photo-curing collagen solution.
(c) And (c) illuminating the solution finally obtained in the step (b), wherein the light acts on a photoinitiator to generate free radicals to initiate thiol-ene reaction, so as to obtain the collagen hydrogel.
7. The collagen hydrogel according to claim 6, wherein: in the step (b), the crosslinking agent is a compound containing two or more mercapto groups.
8. The collagen hydrogel according to claim 6, wherein: in the step (b), the photoinitiator is a water-soluble photoinitiator such as phenyl-2, 4, 6-trimethyl benzoyl lithium phosphite (LAP) or 2-hydroxy-1- [4- (2-hydroxyethoxy) phenyl ] -2-methyl-1-acetone (Irgacure 2959).
9. The collagen hydrogel according to claim 6, wherein: in the step (c), the illumination wavelength is the optimal wavelength matched with the photoinitiator, and the light intensity is adjustable.
10. The collagen hydrogel according to claim 9, wherein: the collagen hydrogel can fix functional molecules, wherein the functional molecules are proteins, peptides, micromolecular drugs, nucleic acids, carbohydrates or polysaccharide molecules and the like; the functional molecule has 1 or more than 1 reactive group, and the reactive group is free sulfydryl or/and alkene group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010012313.4A CN113150313A (en) | 2020-01-07 | 2020-01-07 | Preparation method of neutral-dissolved modified photocured collagen and photocured collagen raw hydrogel |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010012313.4A CN113150313A (en) | 2020-01-07 | 2020-01-07 | Preparation method of neutral-dissolved modified photocured collagen and photocured collagen raw hydrogel |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113150313A true CN113150313A (en) | 2021-07-23 |
Family
ID=76881328
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010012313.4A Pending CN113150313A (en) | 2020-01-07 | 2020-01-07 | Preparation method of neutral-dissolved modified photocured collagen and photocured collagen raw hydrogel |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113150313A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114874455A (en) * | 2022-02-28 | 2022-08-09 | 中国科学院沈阳自动化研究所 | Construction method of neutral-dissolved modified collagen and gel with self-assembly capability and photo-crosslinking capability |
| CN117180494A (en) * | 2023-11-07 | 2023-12-08 | 四川大学 | Injectable polysaccharide hydrogel capable of reducing fibrotic scar generation and preparation method thereof |
| CN117562828A (en) * | 2024-01-15 | 2024-02-20 | 吉林大学 | Skin care lotion and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108367100A (en) * | 2015-12-02 | 2018-08-03 | 奥塔哥创新有限公司 | It is prepared by the photoactivation of hydrogel |
| CN109553783A (en) * | 2017-09-26 | 2019-04-02 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of photocuring hydrogel and the preparation method and application thereof |
-
2020
- 2020-01-07 CN CN202010012313.4A patent/CN113150313A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108367100A (en) * | 2015-12-02 | 2018-08-03 | 奥塔哥创新有限公司 | It is prepared by the photoactivation of hydrogel |
| US20180355127A1 (en) * | 2015-12-02 | 2018-12-13 | University Of Otago | Light-activated preparation of hydrogels |
| CN109553783A (en) * | 2017-09-26 | 2019-04-02 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of photocuring hydrogel and the preparation method and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114874455A (en) * | 2022-02-28 | 2022-08-09 | 中国科学院沈阳自动化研究所 | Construction method of neutral-dissolved modified collagen and gel with self-assembly capability and photo-crosslinking capability |
| CN114874455B (en) * | 2022-02-28 | 2023-10-27 | 中国科学院沈阳自动化研究所 | Construction method of neutral-dissolution modified collagen and gel with self-assembly capability and photocrosslinking capability |
| CN117180494A (en) * | 2023-11-07 | 2023-12-08 | 四川大学 | Injectable polysaccharide hydrogel capable of reducing fibrotic scar generation and preparation method thereof |
| CN117180494B (en) * | 2023-11-07 | 2024-01-23 | 四川大学 | Injectable polysaccharide hydrogel capable of reducing fibrotic scar generation and preparation method thereof |
| CN117562828A (en) * | 2024-01-15 | 2024-02-20 | 吉林大学 | Skin care lotion and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cai et al. | Polypeptide-based self-healing hydrogels: Design and biomedical applications | |
| Claaßen et al. | Quantification of substitution of gelatin methacryloyl: best practice and current pitfalls | |
| Broguiere et al. | Novel enzymatically cross-linked hyaluronan hydrogels support the formation of 3D neuronal networks | |
| Gonen-Wadmany et al. | Protein–polymer conjugates for forming photopolymerizable biomimetic hydrogels for tissue engineering | |
| CN113150313A (en) | Preparation method of neutral-dissolved modified photocured collagen and photocured collagen raw hydrogel | |
| Lü et al. | An injectable and self-healing hydrogel with covalent cross-linking in vivo for cranial bone repair | |
| US10196602B2 (en) | Biomaterial based on a hydrophilic polymeric carrier | |
| Xu et al. | Injectable, biomolecule-responsive polypeptide hydrogels for cell encapsulation and facile cell recovery through triggered degradation | |
| EP2150282B1 (en) | Compositions and methods for scaffold formation | |
| Kiick | Peptide-and protein-mediated assembly of heparinized hydrogels | |
| US20170232143A1 (en) | Covalently cross linked hydrogels and methods of making and using same | |
| CN114874455B (en) | Construction method of neutral-dissolution modified collagen and gel with self-assembly capability and photocrosslinking capability | |
| CN112062981B (en) | Preparation method of culture medium mediated crosslinked hyaluronic acid-based double-crosslinked hydrogel | |
| Thakur et al. | Crosslinking biopolymers for advanced drug delivery and tissue engineering applications | |
| Echalier et al. | Sol–gel synthesis of collagen-inspired peptide hydrogel | |
| US11130797B2 (en) | Covalent modification of biological macromolecules | |
| Hao et al. | Rapid bioorthogonal chemistry enables in situ modulation of the stem cell behavior in 3d without external triggers | |
| CN101220090B (en) | Multi-modification derivant of glutin and crosslinked material thereof | |
| KR101898229B1 (en) | Method for producing photo-crosslinked silk fibroin, and photo-crosslinked silk fibroin produced by the same | |
| CN114349990B (en) | Hydrogel with adjustable dynamic characteristics and preparation method and application thereof | |
| CN108341976B (en) | Derivative of methacrylated matrix material based on click chemistry and synthesis method | |
| CN102858845A (en) | Hydrogel precursor formulation and production process thereof | |
| CN110753537A (en) | Non-covalently assembled conductive hydrogels | |
| Moon et al. | Photocrosslinkable natural polymers in tissue engineering | |
| Lim et al. | Biosynthetic hydrogels for cell encapsulation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |