CN110585483A - Novel biological ink capable of being crosslinked by multiple methods and preparation method thereof - Google Patents

Abstract

The present invention relates to a novel biological ink which can be cross-linked by multiple methods and a preparation method thereof. The ink includes: methacrylic anhydride modified acellular matrix, pepsin, an initiator or other material that can be chemically cross-linked with the methacrylic anhydride modified acellular matrix, and a cell suspension. The method comprises: adding acellular matrix powder to a solution of pepsin, dropwise adding methacrylic anhydride, amidation reaction, dialysis, freezing, freeze-drying to obtain acellular matrix modified with methacrylic anhydride, adding an initiator or a Other materials and cell suspensions chemically cross-linked with methacrylic anhydride-modified acellular matrices. The ink has the properties of thermal crosslinking, photocrosslinking and chemical crosslinking, and the biocompatibility is basically unchanged.

Description

A novel bioink that can be cross-linked by multiple methods and its preparation method

technical field

The invention belongs to the field of biological ink and its preparation, in particular to a novel biological ink that can be cross-linked by multiple methods and a preparation method thereof.

Background technique

With the development of the aging population and the gradual increase in the incidence of various trauma and diseases, tissue or organ defects have gradually become a common phenomenon, such as bone and cartilage defects, heart valve insufficiency, mastectomy, and extensive skin necrosis. .

Existing methods of treating tissue or organ loss include filling the defect with degradable natural or synthetic materials, adding cellular material composites, or filling with permanent prostheses. The filled material often has a large mechanical difference with the defect site, the shape cannot be completely matched, the chemical composition is very different, and the lack of cell recognition sites is easy to cause allergic and rejection reactions. Therefore, technologies that can match tissue defects in shape, mechanics, chemical composition, and biological signals are the main development trends in the future.

3D printing technology, also known as additive manufacturing technology, is a rapid prototyping technology. It is based on digital model files, using bondable materials such as powders, metals, plastics or hydrogels to construct the desired shape of matter by printing layer by layer. At present, 3D printing technology has been widely studied in the field of tissue engineering. Natural or synthetic biomaterials can be made into bioinks, which can also be mixed with cells or active drugs. By imitating the extracellular matrix of living tissues, this technology has great applications in the field of tissue engineering. At present, 3D printing technology can prepare tissues and organs for drug testing, and tissue engineering scaffolds.

The acellular matrix generally refers to the components and structures of the extracellular matrix without the existence of cells prepared by the allogeneic tissue after cell inactivation treatment, and its components mainly include collagen, structural protein, glucosamine and elastin. Acellular matrix has the same components of natural extracellular matrix, low immunogenicity, good histocompatibility, and is conducive to cell adhesion, proliferation and differentiation, and is an ideal tissue engineering scaffold material.

At present, the 3D printing of acellular matrix is mainly mixed with other materials or used as a coating of synthetic polymers. Due to poor mechanical properties and lack of in-situ cross-linking methods, pure acellular matrix bioinks, and acellular matrix There are relatively few studies on the main raw material.

Therefore, by grafting methacrylic acid functional groups on the decellularized extracellular matrix, on the basis of the original thermal crosslinking, the properties of photocrosslinking and chemical crosslinking are given to it, and the biocompatibility is basically unchanged. The application and industrialization of extracellular matrix in 3D printed tissue engineering scaffolds is of great significance.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a novel bio-ink that can be cross-linked by multiple methods and a preparation method thereof, so as to overcome the deficiencies in the prior art, such as the lack of a gel-forming method for decellularized extracellular matrix.

The invention provides a bio-ink that can be cross-linked by multiple methods. The bio-ink comprises: acellular matrix modified with methacrylic anhydride, pepsin, an initiator or chemical cross-linking with the acellular matrix modified with methacrylic anhydride of other materials, as well as cell suspensions.

The methacrylic acid-modified acellular matrix is obtained by adding methacrylic anhydride to the pepsin solution of the acellular matrix for amidation reaction.

The initiator is a photocrosslinking initiator or a thermal initiator.

The photocrosslinking initiators include: 2-hydroxy-2-methyl-1-phenylacetone, 1-hydroxycyclohexyl phenyl ketone, 2-methyl-2-(4-morpholinyl)-1 -[4-(Methylthio)phenyl]-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2,4,6-trimethylbenzene Formyl-diphenylphosphine oxide or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

The thermal initiators include azo initiators, organic peroxide initiators or inorganic peroxide initiators.

The other material that can be chemically cross-linked with the methacrylic acid-modified acellular matrix is a compound that can undergo a Michael addition reaction or an addition reaction with the methacrylic anhydride-modified acellular matrix.

The compounds that undergo Michael addition reaction or addition reaction with the decellularized matrix modified with methacrylic anhydride include amino acids containing sulfhydryl groups or disulfide bonds, proteins or polymers containing unsaturated bonds.

The polymers containing unsaturated bonds include: thiol-terminated polyethylene glycol (4-ARM-PEG-SH, 6-ARM-PEG-SH, or 8-ARM-PEG-SH), thiolated chitosan , PEG or PEGMA modified by thiolated hyaluronic acid, maleic anhydride or its derivatives, proteins involving one or more of methionine, cysteine, and cystine, etc.

The cells include: one or more of stem cells, chondrocytes, skin cells, IPS cells, and cardiomyocytes.

The bio-ink can be used with natural materials or synthetic materials to prepare composite bio-inks.

The present invention also provides a method for preparing a bioink that can be cross-linked by multiple methods, comprising the following steps:

(1) Add the acellular matrix powder to the solution of pepsin, stir until dissolved, adjust the pH value to 7.0-8.0, dropwise add methacrylic anhydride, amidation reaction, dialysis, freeze, freeze-dry to obtain methacrylic acid modification acellular matrix;

(2) Dissolving the methacrylic anhydride-modified acellular matrix in step (1) in a solution of pepsin, adding an initiator or other materials and cell suspensions that can be chemically cross-linked with the methacrylic anhydride-modified acellular matrix liquid to obtain bio-ink.

The method for preparing the acellular matrix powder in the step (1) includes: (1) obtaining fresh animal tissue materials and storing them at low temperature at 4°C; (2) decellularizing the animal tissue materials to prepare an acellular matrix; (3) The acellular matrix was frozen and ground at -80°C, and the above operation was repeated until the acellular matrix powder was obtained.

The acellular matrix is derived from mammals or fish.

The animal tissue includes skin, cartilage, lung, valve, small intestine, large intestine, ligament or adipose tissue.

The pH value of the pepsin solution in the step (1) is 3.0-4.0.

Stirring to dissolve in the step (1) is: stirring slowly at room temperature to dissolve completely.

In the step (1), the amidation reaction temperature is room temperature, and the amidation reaction time is 12h-24h.

The dialysis in the step (1) is as follows: using a dialysis bag with a molecular weight cut-off of 3500 in deionized water for three days, and changing the water three times a day.

The freezing temperature is -80°C.

In the step (1), the gel-forming methods used in the bioprinting of the methacrylic anhydride-modified acellular matrix include photo-crosslinking, thermal cross-linking, and chemical cross-linking with other materials.

beneficial effect

(1) The present invention can impart the properties of photocrosslinking and chemical crosslinking to the decellularized extracellular matrix on the basis of the original thermal crosslinking.

(2) Enrich the application scenarios of acellular extracellular matrix bioinks.

Description of drawings

FIG. 1 is a graph of the acellular matrix bioink before and after photocrosslinking in Example 1. FIG.

FIG. 2 is a graph of the acellular matrix bioink before and after thermal crosslinking in Example 1. FIG.

FIG. 3 is (a) image and (b) scanning electron microscope image of the acellular matrix bioink hydrogel in Example 1 after lyophilization.

4 is an H-NMR chart of the methacrylic anhydride-modified acellular matrix and the acellular matrix in Example 1. FIG.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

A novel bio-ink that can be cross-linked by multiple methods is composed of an acellular matrix modified with methacrylic anhydride, thiolated chitosan, cells and a solvent. The preparation method is as follows:

(1) Weigh pepsin and dissolve it in a 0.01 mol/L HCl solution with a concentration of 1 mg/ml. The acellular matrix was weighed and dissolved in the above solution at a concentration of 15 mg/ml, stirred at room temperature for 18 h, and the stirring rate was 300 r/min.

(2) Adjust the pH of the acellular matrix solution (15 mg/ml) to 7.5, and then dropwise add methacrylic anhydride (the weight ratio of methacrylic anhydride to the acellular matrix is 0.5 ml/g); react at room temperature for 12 hours, and then use A dialysis bag with a molecular weight cut-off of 3500 was dialyzed in deionized water for three days, and the water was changed three times a day; the solution after dialysis was frozen at -80°C, and then freeze-dried to obtain methacrylic acid-modified acellular matrix powder.

(3) Add concentrated hydrochloric acid to PBS buffer to prepare a hydrochloric acid solution with a concentration of 0.01 mol/L, then add pepsin to the hydrochloric acid solution to make the pepsin concentration 1 mg/ml, and prepare methacrylic acid modified at a concentration of 15 mg/ml The hydrochloric acid solution of pepsin of the acellular matrix powder was added, and sodium hydroxide was added to adjust the pH to 7.5. The cells ^were digested and centrifuged. According to 1.5mg/ml thiolated chitosan, 5*10 ⁵ cell/ml L929 cell culture medium was dissolved in the pH-adjusted acellular matrix solution to prepare the bioink precursor.

(4) Take 5ml of the above bio-ink precursor and place it in a 37°C incubator for 2 hours to form thermal cross-linking and curing; take 5ml of the above-mentioned bio-ink precursor, add 25mg of photoinitiator I2959 to it, stir evenly, and use a wavelength of 365nm. The UV light source with a power of 10w can be irradiated for 30s to form photocrosslinking and curing.

Figure 3 shows that the solidified hydrogel was frozen at -80°C and then freeze-dried. It can be found that the freeze-dried hydrogel has a loose and porous structure. Further scanning electron microscope pictures show that the freeze-dried hydrogel scaffold has nanofibers. structure.

Figure 4 shows that the methacrylic anhydride-modified acellular matrix map was compared with the acellular matrix map at 6.2 ppm (b) and 5.7 ppm (a) by comparison of H-NMR nuclear magnetic map (solvent is D ₂ O). The new peak, which was generated by the binding of methacrylic anhydride to collagen in the acellular matrix, indicated that the methacrylic anhydride-modified acellular matrix was successfully prepared.

Example 2

A novel bio-ink that can be cross-linked by multiple methods and a preparation method thereof are composed of an acellular matrix modified with methacrylic anhydride, methacrylated gelatin, a cross-linking initiator, cells and a solvent. The preparation method is as follows:

(1) Prepare hydrochloric acid solution with PBS buffer, prepare hydrochloric acid solution of pepsin hydrochloric acid anhydride modified acellular matrix powder, methacrylic anhydride modified acellular matrix powder and pepsin hydrochloric acid solution according to the mass fraction of hydrochloric acid solution 1.5% The preparation method of the hydrochloric acid solution is the same as in Example 1, adding sodium hydroxide to adjust the pH to 7.5, taking the mass fraction of hydrochloric acid solution 2% methacrylated gelatin, the mass fraction of hydrochloric acid solution 0.05% photoinitiator I2959, 5*10 ⁵ cell/ ml L929 cell culture medium was dissolved in the pH-adjusted acellular matrix solution to prepare bioink.

(2) Take 5ml of the above bio-ink and place it in a 37°C incubator for 2 hours to thermally cross-link and solidify; take 5ml of the above-mentioned bio-ink, stir evenly, and irradiate it with an ultraviolet light source with a wavelength of 365nm and a power of 10w for 30s to photo-crosslink and solidify forming.

The bio-ink is manufactured in the prior art, and the manufacturing materials include 0.01-5wt% nano laponite, 5-40wt% methacrylated gelatin, 0.01-0.5wt% photoinitiator and 55-90wt% % cell culture medium, and the photoinitiator is 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

The difference between the present invention and the prior art is that the main material of the hydrogel is different, the present invention is an acellular matrix modified with methacrylic anhydride, and the prior art is methacrylated gelatin and nano-hectorite.

Claims (10)

1. A multi-process crosslinkable bio-ink, comprising: methacrylic anhydride modified acellular matrix, pepsin, an initiator or other material that can chemically cross-link with methacrylic anhydride modified acellular matrix, and a cell suspension.

2. The bio-ink according to claim 1, wherein the methacrylic anhydride modified acellular matrix is obtained by adding methacrylic anhydride to a pepsin solution of the acellular matrix for amidation reaction.

3. The bio-ink according to claim 1, wherein the initiator is a photo-crosslinking initiator or a thermal initiator.

4. The bio-ink according to claim 3, wherein the photo-crosslinking initiator comprises: 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexylphenylmethanone, 2-methyl-2- (4-morpholinyl) -1- [4- (methylthio) phenyl ] -1-propanone, 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone, 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide or 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone; the thermal initiator comprises an azo initiator, an organic peroxide initiator or an inorganic peroxide initiator.

5. The bio-ink according to claim 1, wherein the other material chemically crosslinkable with the methacrylic anhydride-modified acellular matrix is a compound capable of undergoing a michael addition reaction or an addition reaction with the methacrylic anhydride-modified acellular matrix.

6. The bio-ink according to claim 5, wherein the compound undergoing Michael addition reaction or addition reaction with methacrylic anhydride-modified acellular matrix comprises amino acids containing thiol or disulfide bonds, proteins, thiol-terminated polyethylene glycol, thiolated chitosan, thiolated hyaluronic acid, PEG or PEGMA modified with maleic anhydride or its derivatives.

7. The bio-ink according to claim 1, wherein the cells comprise: stem cells, cartilage cells, skin cells, IPS cells and cardiac muscle cells.

8. A preparation method of bio-ink capable of being crosslinked by a multiple method comprises the following steps:

(1) adding acellular matrix powder into a solution of pepsin, stirring until the acellular matrix powder is dissolved, adjusting the pH value to 7.0-8.0, dropwise adding methacrylic anhydride, performing amidation reaction, dialyzing, freezing, and freeze-drying to obtain an acellular matrix modified by methacrylic anhydride;

(2) and (2) dissolving the methacrylic anhydride modified acellular matrix in the step (1) in a solution of pepsin, and adding an initiator or other materials capable of chemically crosslinking with the methacrylic anhydride modified acellular matrix and a cell suspension to obtain the bio-ink.

9. The method according to claim 8, wherein the acellular matrix powder of step (1) is prepared by a method comprising: (1) obtaining fresh animal tissue material, and storing at low temperature of 4 deg.C; (2) decellularizing the animal tissue material to form a decellularized matrix; (3) the acellular matrix was frozen at-80 ℃ and ground, and the above procedure was repeated until acellular matrix powder was obtained.

10. The method of claim 8, wherein the amidation reaction temperature in step (1) is room temperature, and the amidation reaction time is 12h-24 h.