CN115282340A

CN115282340A - GelMA-based biological ink and preparation method and application thereof

Info

Publication number: CN115282340A
Application number: CN202210973867.XA
Authority: CN
Inventors: 岳衎; 刘祺; 王英杰; 梁钰婷; 邓可琪; 杨景周
Original assignee: Shenzhen Dazhou Medical Technology Co ltd
Current assignee: Shenzhen Dazhou Medical Technology Co ltd
Priority date: 2022-08-15
Filing date: 2022-08-15
Publication date: 2022-11-04

Abstract

The invention belongs to the technical field of biomedical high polymer materials, and particularly relates to GelMA (GelMA) -based bio-ink as well as a preparation method and application thereof.

Description

GelMA-based biological ink and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical high polymer materials, in particular to GelMA-based biological ink and a preparation method and application thereof.

Background

At present, because of the huge gap of organ transplantation and the requirement of personalized tissue repair, the engineered tissue and organ have wide application prospect. To solve the dilemma of organ shortage, a plurality of bio-fabrication techniques based on tissue engineering are being developed rapidly, and they are studied based on two major aspects of functional substitution and shape matching to produce bio-engineered structures for regenerative medicine, pharmacokinetics, and basic cell biology.

Among the numerous bio-manufacturing technologies, bio-3D printing, as an emerging processing technology, can meet the increasing demands for complex structures, has the ability to provide personalized customization, and is one of the most promising manufacturing technologies. Among them, the extrusion type biological 3D printing has the advantages of high applicability, strong processability, low cost, etc. and is widely applied to the printing of tissue engineering scaffolds containing living cells or not containing living cells.

In biological 3D printing, by virtue of good biocompatibility and characteristics similar to tissues, natural polymer hydrogel is widely applied to construction of biological tissue engineering scaffolds, and gelatin, methacrylated gelatin, hyaluronic acid, chitosan and the like are common. Among them, methacrylated gelatin (GelMA) is receiving increasing favor of researchers, firstly, because GelMA has RGD polypeptide fragment of cell adhesion site and Matrix Metalloproteinase (MMP) degradation site, it has good biocompatibility and biodegradability, and is beneficial to proliferation and differentiation of cells in the material; and secondly, after methacrylic anhydride modification, a photo-crosslinkable methacrylamide group exists, and a stable crosslinking network can be obtained after rapid ultraviolet irradiation. Meanwhile, gelMA has a certain pore structure, has the transport conditions of nutrient substances and metabolites, and provides sufficient conditions for wrapping and three-dimensional cell culture. Therefore, gelMA has the advantages of good biocompatibility, controllability of photocrosslinking, excellent cell culture carrier and the like, and becomes a good injectable biomaterial.

However, the processability of the low-concentration GelMA bio-ink is poor, the 3D printing precision is reduced, the temperature dependence is strong, the pore size of the high-concentration GelMA bio-ink is small, the material exchange is difficult, and the survival rate of the loaded cells is not high, which seriously hinders the further development of the GelMA bio-ink in biological 3D printing and limits the application prospect. How to develop a bio-ink system with good printing precision and strong applicability remains a great challenge.

Disclosure of Invention

The invention aims to provide GelMA-based bio-ink and a preparation method and application thereof. The GelMA-based biological ink is used as an ink material for extrusion type 3D printing, and can effectively solve the problems of strong temperature dependence, weak self-supporting performance, low printing precision and the like of low-concentration GelMA biological ink so as to solve the problems in the prior art.

The invention is different from the modes of a temperature crosslinking method, ion crosslinking and non-biological auxiliary additives, and takes advantage of aldehyde groups on natural saccharide/synthetic saccharide oxides with good biocompatibility and biodegradability to react with amino groups on GelMA to form a dynamic crosslinking network. The biological ink can be used for 3D printing without being influenced by temperature, has excellent printing precision and self-supporting performance, and maintains excellent biocompatibility and biodegradability. Prior to the present invention, natural/synthetic saccharide oxides were not used for GelMA bio-ink based extruded 3D printing.

In order to achieve the purpose, the invention provides the following scheme: the invention provides GelMA-based bio-ink which comprises the following components: methacrylated gelatin, saccharide oxide, chemical initiator and solvent.

As a further optimization of the invention, the mass percentage concentration of the methacrylated gelatin in the GelMA-based bio-ink is 4-20%, the degree of substitution on the methacrylated gelatin is 10-95%, namely 10-95% of amino groups on gelatin molecules are amidated.

As a further optimization of the invention, the mass percentage concentration of the saccharide oxide in the GelMA-based bio-ink is 0.5-5%, and the molecular structure of the saccharide oxide at least comprises one carbonyl and/or aldehyde group; the carbohydrate oxide structure comprises at least one of a straight chain and a branched chain; the saccharide oxide may have an oxidation degree of 0.01% to 99.99%, i.e. 0.01% to 99.99% of the saccharide monomers on the polysaccharide molecule are oxidized.

As a further optimization of the present invention, the GelMA-based bio-ink comprises the chemical initiator in a concentration of 0.01% to 0.5% by mass, wherein the chemical initiator comprises at least one of 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone (Irgacure 2959 photoinitiator), phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP photoinitiator), and phenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO photoinitiator).

As a further optimization of the invention, the GelMA-based bio-ink comprises 4-20% by mass of methacryloylated gelatin, 0.5-5% by mass of carbohydrate oxide, 0.01-0.5% by mass of chemical initiator and the balance of solvent.

As a further optimization of the invention, the solvent is deionized water, and the pH is one of physiological saline with 7.0-7.5, PBS solution, sodium acetate buffer solution, tris hydrochloride buffer solution and cell culture medium containing 0% -20% of serum.

The invention provides a preparation method of GelMA-based biological ink, which comprises the following steps:

(1) Preparation of methacrylated gelatin: reacting the gelatin solution with methacrylic anhydride to obtain methacrylated gelatin;

(2) Preparing a saccharide oxide: reacting the saccharide solution with an oxidant to obtain a saccharide oxide;

(3) Weighing methacrylated gelatin, carbohydrate oxide and chemical initiator, adding the mixture into a solvent, and uniformly mixing to obtain the GelMA-based biological ink.

As a further optimization of the invention, the step of preparing the methacrylated gelatin in the step (1) is as follows: dissolving gelatin in water phase system completely at 30-70 deg.C under stirring, adding methacrylic anhydride into gelatin solution, reacting for 0.5-24 hr, dialyzing, and freeze drying to obtain methacrylated gelatin (GelMA);

as a further optimization of the invention, the dialysis is to select a dialysis bag with the molecular weight cut-off of 3500-14000Da to dialyze in deionized water for 2-7 days.

As a further optimization of the invention, in the step (1), the dosage ratio of the gelatin to the methacrylic anhydride is 10g: (0.01-20) mL.

As a further optimization of the invention, the step (2) for preparing the carbohydrate oxide comprises the following steps: dissolving saccharide in the water phase system or DMSO phase system at 20-70 deg.C under stirring to obtain saccharide solution, adding oxidant into the saccharide solution, reacting for 0.5-24 hr, dialyzing the reaction product, and freeze drying to obtain saccharide oxide;

as a further optimization of the invention, the aqueous phase system is one of deionized water, PBS with pH ranging from 7.5 to 8.5, sodium acetate buffer and Tris buffer.

As a further optimization of the present invention, in the step (2), the total molar ratio of the saccharide solution to the oxidizing agent is 1: (0.01-5), the oxidizing agent comprises at least one of sodium periodate, sodium hypochlorite, hydrogen peroxide, ozone, chlorine dioxide and potassium permanganate.

As a further optimization of the invention, the saccharides have a molecular weight above 100Da.

As a further optimization of the present invention, the saccharide comprises at least one of a natural saccharide and an artificially synthesized saccharide.

As a further optimization of the present invention, the natural saccharide and the synthetic saccharide may be the same or different.

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As a further optimization of the invention, in the step (3), the solvent is deionized water, and the pH is one of physiological saline with 7.0-7.5, PBS solution, sodium acetate buffer solution, tris hydrochloride buffer solution and cell culture medium containing 0% -20% serum.

As a further optimization of the invention, the step (3) further comprises adding a proper amount of cells, mixing and uniformly mixing, wherein the cells comprise at least one of human umbilical vein endothelial cells, mesenchymal stem cells, NIH 3T3 cells and HepG2 cells.

As a further optimization of the invention, the step (3) also comprises the step of standing for 2-120 minutes at 25 ℃ after uniformly mixing until the reaction system is in a gel state.

The invention provides application of the GelMA-based bio-ink in the aspects of tissue repair, regenerative medicine, pharmacokinetics and basic cell biology.

The invention provides application of GelMA-based bio-ink in 3D printing, which comprises the following steps.

(a) Placing the GelMA-based bio-ink in an extrusion device of a 3D printer; then setting printing parameters, and extruding the GelMA-based biological ink onto a printing platform to obtain a primarily cured tissue engineering scaffold;

(b) And carrying out ultraviolet irradiation on the tissue engineering scaffold to form a stable photocuring 3D printed product.

As a further optimization of the present invention, in the step (a), the temperature of the extrusion device and the printing platform is 0-50 ℃; the printing parameters include: the printing speed is 1-30mm/s, and the extrusion speed is 0.1-10.0mm ³ S; the wavelength of the ultraviolet light in the step (b) is 320-500nm, and the power density is 0.001-10W/cm ² The illumination time is 1-600s.

The preparation method of GelMA-based biological ink provided by the invention comprises the following steps of firstly grafting an active group with double bonds on gelatin to obtain GelMA which can be solidified by a free radical initiator; then, carrying out oxidation reaction on the natural saccharide/synthetic saccharide to obtain carbonyl-containing natural saccharide/synthetic saccharide oxide; gelMA, natural saccharide/synthetic saccharide oxide and a free radical initiator are further mixed and stood to obtain the preliminarily crosslinked room-temperature printable bio-ink, which has the characteristics of strong room-temperature printability, high printing precision, good self-supporting performance, strong anti-fatigue capability, good biocompatibility and the like, and can realize in-situ cell wrapping.

The invention discloses the following technical effects:

(1) The GelMA-based bio-ink and the preparation method thereof provided by the invention take methacrylic acylated gelatin (GelMA) and natural saccharide/synthetic saccharide oxide as raw materials, the amino on the GelMA and the carbonyl of the natural saccharide/synthetic saccharide oxide are subjected to Schiff base reaction to form the preliminary cross-linked bio-ink, and the bio-ink is ready for printing in an extrusion device, so that the GelMA bio-ink capable of being printed at room temperature is obtained; and then carrying out photocuring crosslinking on the 3D printing structure to obtain the stable tissue engineering scaffold. The obtained tissue engineering scaffold has good biocompatibility, room-temperature printability and photocrosslinking glue forming property, can be suitable for a 3D printer without a temperature auxiliary device, and improves the applicability and application universality of a biological 3D printing technology.

(2) The GelMA-based bio-ink and the preparation method thereof provided by the invention can realize in-situ encapsulation of cells and 3D printing of cell compatibility, and show good printing precision, biocompatibility and cell activity, thereby showing good application prospects in the fields of cell-containing tissue engineering scaffolds, customized tissue repair, regenerative medicine, pharmacokinetics, basic cell biology and the like.

(3) The GelMA-based bio-ink provided by the invention has excellent structural self-supporting performance and strength adjustability, and provides powerful technical support for realizing 3D printing of large-scale biological structures. Because the biological ink has certain mechanical strength, the times and time of external photocuring can be reduced, and the aims of reducing cell death and improving the survival rate and activity of cells are further fulfilled.

(4) The GelMA-based biological ink printed three-dimensional tissue engineering scaffold provided by the invention has excellent anti-fatigue property. The tissue engineering scaffold can still maintain structural integrity and high mechanical strength after being subjected to continuous cyclic compression for hundreds of times. Therefore, the tissue engineering scaffold, the tissue repair material and the human body implant obtained by the preparation method can bear the biological stress caused by the human body activity after being implanted, keep the integrity of the material, avoid the medical problems of incomplete wound healing, secondary operation and the like, and are suitable for popularization and use in the field of biological medicine.

(5) The invention provides a preparation method of a material for improving a hydrogel network structure and promoting cell migration. In the bio-ink system of the present invention, the cross-linked structure is composed of two parts: schiff base reactions dominate the dynamic cross-linked network and free radical reactions dominate the permanent cross-linked network. Has a Schiff base reaction of dynamic balance, provides space for the migration and movement of cells, is beneficial to the proliferation and the extension of the cells, and has wide application prospect in the field of biomedicine.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 Infrared Spectroscopy of example 7 ¹ H NMR spectrum;

FIG. 2 shows GelMA-based bio-ink gelation results at room temperature, wherein the results of example 30 are shown in FIG. 2A, the results of example 33 are shown in FIG. 2B, the results of example 36 are shown in FIG. 2C, the results of example 39 are shown in FIG. 2D, the results of examples 28 and 32 are shown in FIG. 2E, and the results of example 57 are shown in FIG. 2F;

FIG. 3 is rheological data of GelMA-based bio-ink at room temperature, wherein the results of example 33 are shown in FIGS. 3A-D, and wherein the results of example 57 are shown in FIGS. 3E-F;

FIG. 4 shows the precision results of room temperature 3D printing of GelMA-based bio-ink prepared in example 69;

FIG. 5 is an example of GelMA-based bio-ink 3D printing infrastructure at room temperature, where the results of example 69 are shown in FIG. 5A, example 73 is shown in FIG. 5B, example 83 is shown in FIG. 5C, and example 86 is shown in FIG. 5D;

FIG. 6 is an example of a GelMA-based bio-ink 3D printed biomimetic structure at room temperature prepared in example 69;

FIG. 7 is a graph of modulus data for GelMA-based bio-inks prepared in examples 95 and 119;

FIG. 8 shows the results of self-healing performance tests on GelMA-based bio-inks prepared in examples 123 and 128;

FIG. 9 shows the results of the cyclic compression test for fatigue resistance of GelMA-based bio-ink prepared in example 123;

FIG. 10 is a graph showing the observation of cell viability in three-dimensional culture of HUVEC cells for 1 day, 7 days and 14 days in GelMA-based bio-ink prepared in example 130;

FIG. 11 is a graph showing the observation of cell viability in three-dimensional culture of HUVEC cells for 1 day, 7 days and 14 days by GelMA-based bio-ink prepared in example 131;

FIG. 12 is a graph of cell viability observations for 1 day, 7 days, and 14 days of 3D printing of GelMA-based bio-ink loaded HUVEC cells prepared in example 136;

FIG. 13 is a graph of cell viability observations for 1 day, 7 days, and 14 days of 3D printing of GelMA-based bio-ink loaded HUVEC cells prepared in example 137;

FIG. 14 is a graph of cell viability data for HUVEC cells loaded with GelMA-based bio-ink prepared in examples 130, 131, 136 and 137.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.

In the present invention, the terms "methacrylated gelatin" and "gelMA" generally refer to methacrylic acid modified gelatin, also known as gelatin metacryloyl, gelatin metacrylate, metacrylated gelatin, metacrylamide modified gelatin, gelatin metacrylamide. In the present invention, the degree of substitution of the methacrylated gelatin may be 0.01% to 99.9% (i.e., 0.01% to 99.9% of the amino groups on the gelatin molecule are amidated). The gelatin is derived from skin and bone of animals such as Corii Sus Domestica, fish skin, corii bovis Seu Bubali, and Os bovis Seu Bubali, and waste materials of leather industry, and can be produced by alkali gelatin, acid gelatin, and enzyme gelatin.

In the present invention, the term "natural saccharide/synthetic saccharide oxide" generally refers to various kinds of saccharides oxidized by an oxidizing agent, and may be also called oxidized dextran (oxidized dextran), aldehyde dextran (aldehyde dextran), etc., taking dextran oxide as an example. In the present invention, the degree of oxidation of the natural saccharide/synthetic saccharide oxide may be in the range of 0.01% to 99.99% (i.e., 0.01% to 99.99% of the saccharide monomers on the polysaccharide molecule are oxidized). The molecular weight of the natural saccharide/synthetic saccharide oxide may be 100-1000Da,1-10kDa,10-250kDa,250-1000kDa,1-10MDa, or more than 10MDa. The natural saccharide/synthetic saccharide oxide has a viscosity of 0.01-10 mPas, 10-50 mPas, 50-200 mPas, 200-500 mPas, 500-1000 mPas, or more than 1000 mPas.

Examples 1 to 6: synthesis of methacrylated gelatin with different degrees of substitution

Adding 10g of gelatin into 100mL of deionized water, and magnetically stirring at 50 ℃ until the gelatin is completely dissolved; methacrylic anhydride was added dropwise to the gelatin solution, and the addition amounts of methacrylic anhydride were 0.3mL, 0.6mL, 1.2mL, 2.0mL, 3.6mL, and 6.0mL in the order of example 1-6; the reaction was continued at 50 ℃ for three hours, during which time the pH was adjusted by addition of saturated sodium bicarbonate solution; transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 3500Da after the reaction is finished, and dialyzing for 5 days; followed by lyophilization, the methacrylated gelatin is obtained.

The methacrylated gelatins prepared in examples 1-6 were dissolved in heavy water containing 3- (trimethylsilyl) propanesulfonic acid sodium salt nuclear magnetic internal standard and tested by liquid nuclear magnetic resonance spectrometer (JEOL, JNM-ECZ500R,500 MHz) to obtain the degree of substitution of the methacrylated gelatins prepared in examples 1-6 in the order of 15. + -. 5%, 30. + -. 5%, 45. + -. 5%, 60. + -. 5%, 75. + -. 5%, 90. + -. 5% from nuclear magnetic resonance hydrogen spectra.

Examples 7 to 26: synthesis of carbohydrate oxides

10g of saccharide was dissolved in 100mL of deionized water or DMSO at 50 ℃; after mixing to a homogeneous solution, 0.1M H is used ₂ SO ₄ Solution the solution was adjusted to pH =3; then adding sodium periodate with different molar proportions, and reacting for 5 hours in a dark place; after the reaction is finished, adding 20mL of glycol to terminate the reaction, and stirring for 30 minutes; transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 7000Da, and dialyzing for 5 days; followed by lyophilization to yield different saccharide oxides.

The specific examples are as follows:

examples 7-10 the dextran was selected for addition, and the molar ratio of dextran to sodium periodate in examples 7-10 was 1:0.50, 1:0.65, 1: 80. 1:0.95, and 0.5x, 0.65x, 0.8x and 0.95x according to the oxidation degree.

Examples 11-14 selected starch additions, examples 11-14 having a starch to sodium periodate molar ratio of 1:0.50, 1:0.65, 1: 80. 1:0.95, and 0.5x, 0.65x, 0.8x and 0.95x according to the oxidation degree.

Examples 15-18 select to add sodium alginate, and the molar ratio of the sodium alginate to the sodium periodate in examples 15-18 is 1:0.50, 1:0.65, 1: 80. 1:0.95, and 0.5x, 0.65x, 0.8x and 0.95x according to the oxidation degree.

Examples 19-22 were selected for the addition of galactooligosaccharides, and examples 19-22 had a mole ratio of galactooligosaccharide to sodium periodate of 1:0.50, 1:0.65, 1: 80. 1:0.95, and 0.5x, 0.65x, 0.8x and 0.95x according to the oxidation degree.

Examples 23-26 with the option of adding maltose, the molar ratio of maltose to sodium periodate in examples 23-26 is, in order, 1:0.50, 1:0.65, 1: 80. 1:0.95, and 0.5x, 0.65x, 0.8x and 0.95x according to the oxidation degree.

The IR spectrum (obtained using a Nicolet iS5 instrument, thermo Scientific, USA) of the final product of the oxide of the sugar prepared in examples 7 to 26 iS 1730cm ^-1 Compared with the raw material, obvious absorption peaks appear. The oxidation degree of the final product is determined by a titration method, 0.10g of the final product is dissolved in hydroxylamine hydrochloride solution to react for 2 hours, 1mol of aldehyde group in the final product in the solution reacts with hydroxylamine hydrochloride to release 1mol of HCl, then methyl orange solution is added, 1M sodium hydroxide solution is used for titration, and the oxidation degree is calculated and measured.

Infrared Spectrum of example 7 and ¹ the H NMR spectrum is shown in FIG. 1. From FIGS. 1A-B, it can be seen that the infrared spectrum is 1730cm ^-1 Compared with the raw material, the absorption peak is obvious at ¹ An obvious absorption peak is at a chemical shift of 5.6 in an H NMR spectrum.

Examples 27 to 66; comparison of gel Effect of different kinds of saccharide oxides on methacrylated gelatin (GelMA) Bio-ink

Example 27: weighing a proper amount of methacrylated gelatin and an LAP photoinitiator, dissolving the methacrylated gelatin and the LAP photoinitiator in a PBS solution with the pH value of 7.4, and uniformly mixing to obtain a final product, wherein the substitution degree of the methacrylated gelatin is 60%, and the mass percentage concentration of the LAP photoinitiator is 0.2%.

Example 27 the effect of various variables on bio-inks was investigated according to the above preparation method, wherein the mass percentage concentration of the methacrylated gelatin was varied by 4%, 5%, 6%, 7%, and the final product prepared in this example was observed at 25 ℃ or tested for the change of G' and G "with time by a rheometer (ARES-G2 rheometer, TA Instruments, usa) to obtain the gel times of different bio-inks.

Examples 28 to 66:

weighing a proper amount of methacrylated gelatin, carbohydrate oxide and LAP photoinitiator, dissolving the mixture in PBS solution with the pH value of 7.4, and uniformly mixing to obtain the GelMA-based biological ink, wherein the mass percentage concentration of the LAP photoinitiator is 0.2%.

Examples 28 to 66 the effect of various variables on bio-ink was investigated according to the above preparation method, wherein the degree of substitution of methacrylated gelatin was varied to 30%, 60%, 90%, the degree of mass percentage concentration of methacrylated gelatin was varied to 4%, 5%, 6%, 7%, 10%, 20%, the type of saccharide oxide was varied to dextran oxide, starch oxide, sodium alginate oxide, galacto-oligosaccharide oxide, maltose oxide, the degree of oxidation of saccharide oxide was varied to 0.5x, 0.65x, 0.8x, 0.95x, and the mass percentage concentration of saccharide oxide was varied to 0.5%, 1%, 2%, 2.5%, 3%, 4%, 5%. Gel times for different bio-inks were obtained by observing the GelMA-based bio-inks prepared in examples 27-54 at 25 ℃ on standing or by measuring the changes in G 'and G' with time using a rheometer (ARES-G2 rheometer, TA Instruments, USA).

The variables in examples 27-66 are shown in Table 1, and the gel times of the final products prepared in examples 27-66 are shown in Table 1.

TABLE 1

Wherein the GelMA-based bio-ink room temperature gel results obtained in example 30 are shown in fig. 2A, the GelMA-based bio-ink room temperature gel results obtained in example 33 are shown in fig. 2B, the GelMA-based bio-ink room temperature gel results obtained in example 36 are shown in fig. 2C, the GelMA-based bio-ink room temperature gel results obtained in example 39 are shown in fig. 2D, and the GelMA-based bio-ink room temperature gel results obtained in examples 28 and 32 are shown in fig. 2E, wherein the first panel at the top of fig. 2E is example 28, the second panel at the bottom of fig. 2E is example 32, and the GelMA-based bio-ink room temperature gel results obtained in example 57 are shown in fig. 2F.

Wherein the rheological data at room temperature for the GelMA-based bioink prepared in example 33 are shown in FIGS. 3A-D, and wherein the rheological data at room temperature for the GelMA-based bioink prepared in example 57 are shown in FIGS. 3E-F.

The concentrations of the saccharide oxide used in the above experiments were all 4%.

As can be seen from the results in table 1 and fig. 2 and 3: the addition of natural saccharide/synthetic saccharide oxide can obviously raise gelMA gelling effect at room temperature, and can obviously raise printing performance and printing accuracy. The higher the oxidation degree and the higher the concentration of the oxide, the better the gelling effect of the oxide by standing at room temperature.

Examples 67 to 87

Weighing a proper amount of methacrylated gelatin, carbohydrate oxide and LAP photoinitiator, dissolving the mixture in PBS solution with the pH value of 7.4, uniformly mixing, and standing the mixture at 25 ℃ for 30 minutes to obtain the GelMA-based biological ink, wherein the mass percentage concentration of the LAP photoinitiator is 0.2%.

3D printing is carried out on the prepared GelMA-based biological ink, and the temperature of an extrusion device and a printing platform is set by using a common extrusion type 3D printer, a biological 3D printer and a glue dispenser on the marketIt was 25 ℃. The printing parameters of the 3D printer are that the printing speed is 5mm/s, and the extrusion speed can be 0.8mm ³ Printing was carried out at a wavelength of 405nm and an intensity of 25mW/cm ² The structure is irradiated by the light source (2) for 20 seconds to obtain a permanently cross-linked stable three-dimensional structure.

Examples 67-87 the effect of various variables on the printing effect of bio-ink was investigated according to the above preparation method, wherein the degree of substitution of methacrylated gelatin was 60%, the variation of the mass percentage concentration of methacrylated gelatin was 4%, 5%, 6%, 7%, 10%, the kind variation of saccharide oxide was dextran oxide, starch oxide, sodium alginate oxide, galacto-oligosaccharide oxide, maltose oxide, the degree variation of oxidation of saccharide oxide was 0.5x, 0.65x, 0.8x, 0.95x, and the mass percentage concentration variation of saccharide oxide was 0%, 0.5%, 2%, 2.5%, 3%, 4%, 5%.

The printing accuracy of examples 67-87 was tested, observed through an optical microscope and recorded to obtain the evaluation results of the printing effects. Wherein the 'excellent' is that the biological ink forms smooth microfilaments after being extruded, and the shape is stably maintained on a platform for 15 minutes; "good" is that the biological ink forms smooth microfilaments after being extruded, but the photo-curing crosslinking needs to be carried out within 15 minutes to keep the shape stable; "poor" is the inability of the bio-ink to form smooth micro-filaments after extrusion. The bio-ink in the relevant embodiment is then selected and printed to obtain various shapes, such as grid shape, hollow cylinder shape, branched blood vessel shape, "south China university" Logo, "continent" Logo, and other different shapes, wherein an example of the 3D printing infrastructure of the bio-ink prepared in embodiments 69, 73, 83, and 86 is shown in fig. 5.

The variables in examples 67-87 are shown in Table 2, and the results of 3D printing evaluation in examples 67-87 are shown in Table 2.

TABLE 2

As can be seen from the results of table 2, the GelMA-based bio-ink was effective in realizing extrusion 3D printing at room temperature after adding the natural saccharide/synthetic saccharide oxide. In the following experimental examples 70 and 75, the concentration of the saccharide oxide used was 4%, and in examples 84 and 87, the concentration of the saccharide oxide used was 2.5%.

The GelMA-based bio ink with a dextran oxide concentration of 4% at 0.5x prepared in example 69 was subjected to 3D printing at room temperature under different nozzle moving speeds and different extrusion rates, and the 3D printing precision data under different printing conditions are shown in fig. 4. In this example, the bio-ink moves at 5, 6, 7mm/s and the extrusion rate is 0.8, 1.2, 1.6mm ³ The printing accuracy was tested under the conditions of/s, and it can be seen in FIGS. 4A-F that the average diameter of the microwires was less than 500 μm. In addition, as can be seen in the summary of fig. 4G, the average diameter of the microfilaments increases with increasing extrusion rate and nozzle travel speed. Fig. 4 thus shows the printing accuracy of the GelMA-based bio-ink test prepared in example 70, and the results show that the GelMA-based bio-ink has excellent printing properties.

The printing results of the embodiment 69 are shown in FIG. 5A, the printing results of the embodiment 73 are shown in FIG. 5B, the printing results of the embodiment 83 are shown in FIG. 5C, and the printing results of the embodiment 86 are shown in FIG. 5D. Fig. 5 shows that the GelMA-based bio-ink containing saccharide oxide can 3D print a desired three-dimensional structure with high precision. In fig. 5, (i) is a front view of the structure, and (ii) is a top view of the structure.

To further characterize the 3D printing performance of the GelMA-based bio-ink at room temperature, the GelMA-based bio-ink with a 0.5x dextran oxide concentration of 4% prepared in example 69 printed more complex biomimetic structures, the results of which are shown in fig. 6. Figure 6A shows that after the hollow branched blood vessel can be accurately printed, liquid can be smoothly perfused into the lumen of the blood vessel without leakage. Fig. 6B shows a chip 1:1 equi-scaled biomimetic teeth, which exhibit excellent precision and stability. Fig. 6 is a front view of the structure (i) and a side view of the structure (ii).

Examples 88 to 120: compression modulus and self-supporting Performance of saccharide oxide versus Methacryloylated gelatin (GelMA) Bio-ink of varying degrees of substitution

Weighing a proper amount of methacrylated gelatin, carbohydrate oxide and LAP photoinitiator, dissolving the mixture in PBS solution with the pH value of 7.4, uniformly mixing, and standing at 25 ℃ for 60 minutes to obtain the GelMA-based biological ink, wherein the mass percentage concentration of the LAP photoinitiator is 0.2%.

3D printing is carried out on the prepared GelMA-based biological ink, and an extrusion type 3D printer, a biological 3D printer and a dispenser which are commonly available on the market are used, wherein the temperature of an extrusion device and the temperature of a printing platform are set to be 25 ℃. The printing parameters of the 3D printer are 6mm/s in printing speed and 1.6mm in extrusion speed ³ Printing was carried out at a wavelength of 405nm and an intensity of 25mW/cm ² The structure was irradiated with light for 20 seconds to produce a cylindrical sample having a diameter of 8mm and a height of 2 mm.

Examples 88 to 120 the effects of various variables on the compression modulus and self-supporting property of bio-ink printing were investigated according to the above preparation methods, wherein the degree of substitution of methacrylated gelatin was varied at 30%, 60%, 90%, the degree of mass percentage concentration of methacrylated gelatin was varied at 4%, 5%, 6%, 7%, 10%, 15%, 20%, the type variable of saccharide oxide was dextran oxide, starch oxide, the degree of oxidation variable of saccharide oxide was 0.5x, 0.65x, 0.8x, 0.95x, and the mass percentage concentration variable of saccharide oxide was 0%, 0.5%, 2%, 2.5%, 3%, 4%, 5%. The variables in examples 88-120 are shown in Table 3.

TABLE 3

In examples 91-120, the mechanical properties of the gelma-based bio-ink were improved compared to examples 88-90, and the gelma-based bio-ink was self-supporting, which was not found in examples 88-90 of the control group. The saccharide oxide concentrations used in the following experiments were all 4%.

The data in table 3 and fig. 6 and 7 show that the mechanical strength and self-supporting performance of the GelMA-based bio-ink are significantly improved after the addition of the saccharide oxide, and the strength is improved along with the increase of the concentration of the added oxide. Wherein fig. 7A-B are graphs of modulus data for GelMA-based bio-inks prepared in example 95 and fig. 7C for example 119. The data in fig. 7 show that the GelMA-based bio-ink can be printed in an equal-proportion bionic structure without collapse, shows good self-supporting performance, and is expected to be applied to the field of biomedicine. It is evident from FIGS. 7A-B that the mechanical strength of the GelMA-based bio-ink is significantly improved after addition of the saccharide oxide, and that the Young's modulus is improved from 0.8545 + -0.1938 kPa (5% GelMA) to 11.0059 + -1.6173 kPa (5%/3% GelMA/OD), 13.3926 + -1.2622 kPa (5%/4% GelMA/OD), 15.1188 + -1.3428 kPa (5%/5% GelMA/OD). The change in strength of the GelMA-based bio-ink with dextran oxide and starch oxide added can be seen from the mechanical curves of fig. 7A and 7C.

Examples 121 to 129: comparison of self-repairability and fatigue resistance of saccharide oxides to methacrylated gelatin (GelMA) bio-inks of different degrees of substitution

3D printing is carried out on the prepared GelMA-based biological ink, and an extrusion type 3D printer, a biological 3D printer and a dispenser which are commonly available on the market are used, wherein the temperature of an extrusion device and the temperature of a printing platform are set to be 25 ℃. The printing parameters of the 3D printer are that the printing speed is 7mm/s, and the extrusion speed can be 0.8mm ³ Printing at a wavelength of 500nm and an intensity of 25mW/cm ² The structure was irradiated with light for 20 seconds to produce a cylindrical sample having a diameter of 8mm and a height of 2 mm.

Examples 121 to 129 the effect of various variables on the printing compression modulus and self-supporting property of bio-ink was investigated according to the above preparation methods, wherein the degree of substitution of methacrylated gelatin was varied to 30%, 60%, 90%, the variation of mass percentage concentration of methacrylated gelatin was varied to 5%, 10%, 15%, the kind of saccharide oxide was dextran oxide, the degree of oxidation of saccharide oxide was 0.5x, and the variation of mass percentage concentration of saccharide oxide was 0%, 2%, 3%, 4%, 5%.

The prepared cylindrical samples of examples 121 to 129 were left standing at 25 ℃ for 60 minutes, and then the samples after standing were subjected to a step-strain test using a rheometer (ARES-G2 rheometer, TA Instruments, USA) to analyze the self-healing properties of the samples.

The prepared cylindrical samples of examples 121 to 129 were left standing at 25 ℃ for 60 minutes, and then subjected to a 50% strain cyclic compression test using a force test (Shanghai) universal tensile tester to analyze fatigue resistance.

The variables in examples 121-129 are shown in Table 4, and the results of the self-healing and anti-fatigue effects of the 3D printed samples of examples 121-129 are shown in Table 4.

TABLE 4

In experimental example 123, the concentration of the saccharide oxide used was 4%, and in example 128, the concentration of the saccharide oxide used was 2.5%.

The results of the self-healing performance test of the GelMA-based bio-ink prepared in examples 123 and 128 are shown in fig. 8.

The results of the fatigue cycle compression test on the GelMA-based bio-ink prepared in example 123 are shown in fig. 9.

The data in table 4 and fig. 8 and 9 show that the GelMA-based bio-ink has self-healing and fatigue resistance properties that are not present in the pure GelMA sample after saccharide oxide addition. The yield strength of the saccharide oxide-containing GelMA-based bio-ink is shown in fig. 8A, and the bio-ink can be rapidly restored from a liquid state to a gel state when the strain is varied between 1%, 1500% and 0.5%, 500% in fig. 8B-D, showing excellent self-healing performance. In fig. 8E, the GelMA-based bio-ink containing saccharide oxide was all self-repairing intact within minutes and resisted the effects of gravity without signs of damage. In fig. 9, the GelMA-based bio-ink containing saccharide oxide recovered after multiple multidirectional pressings. And it can be seen in fig. 9 (vi) that by injecting the pigment-containing aqueous solution into the hollow structure, the aqueous solution did not leak, further illustrating the good stability of the hollow structure. Fig. 9B shows experimental data of cycle compression, in which the GelMA-based bio-ink containing saccharide oxide can be maintained after 400 times of 50% cycle compression, and the original sample still has 80% strength after 50 times of cycle compression.

Examples 130 to 177: comparison of cell viability of carbohydrate oxide versus methacrylated gelatin (GelMA) bio-ink of varying degrees of substitution on Human Umbilical Vein Endothelial Cells (HUVEC), mesenchymal Stem Cells (MSC), NIH 3T3 cells, hepG2 cells (Human hepatocyte carcinomas)

Weighing a proper amount of methacrylated gelatin, carbohydrate oxide, cell suspension and a LAP photoinitiator, dissolving the mixture in PBS (phosphate buffer solution) with the pH value of 7.4, uniformly mixing, and standing at 25 ℃ for 60 minutes to obtain the GelMA-based biological ink, wherein the mass percentage concentration of the LAP photoinitiator is 0.2%. The concentration of the cell suspension is 500000-1500000/mL.

And (4) performing three-dimensional culture on the GelMA-based biological ink, and detecting the survival rate of cells in the biological ink.

3D printing is carried out on the prepared GelMA-based biological ink, and an extrusion type 3D printer, a biological 3D printer and a dispenser which are commonly available on the market are used, wherein the temperature of an extrusion device and the temperature of a printing platform are set to be 25 ℃. The printing parameters of the 3D printer are that the printing speed is 7mm/s, and the extrusion speed can be 0.8mm ³ Printing was carried out at a wavelength of 500nm and an intensity of 25mW/cm ² The structure was irradiated with the light source for 20 seconds to produce a grid-like sample.

The grid samples were transferred to 12-well plates for culture, and the medium was changed every two days. After 14 days of further culture, the material was treated with Calcein/PI cell viability assay kit (i.e. live-dead staining) using a two-photon confocal microscope (LSM 880NLO, zeiss, germany) to determine the viability of cells in 3, 7, and 14 days of culture of the 3D-printed samples.

Examples 130 to 177 comparative cell viability in Human Umbilical Vein Endothelial Cells (HUVEC), mesenchymal Stem Cells (MSC), NIH 3T3 cells, hepG2 cells (Human hepatocyte carcinomas) was investigated according to the above preparation methods, wherein the degree of substitution of methacrylated gelatin was varied at 30%, 60%, 90%, the percentage concentration by mass of methacrylated gelatin was varied at 5%, 10%, 15%, the type of saccharide oxide was varied at dextran oxide, starch oxide, sodium alginate oxide, maltose oxide, the degree of oxidation of saccharide oxide was varied at 0.5x, 0.65x, 0.8x, 0.95x, and the percentage concentration by mass of saccharide oxide was varied at 0%, 2%, 3%, 4%, 5%.

Wherein: examples 130-141 the results of cell three-dimensional culture cell survival and 3D-printed sample cell survival using Human Umbilical Vein Endothelial Cells (HUVECs) are shown in tables 5 and 6, respectively; examples 142-153 the results of cell three-dimensional culture cell survival and 3D printed sample cell survival using Mesenchymal Stem Cells (MSCs) are shown in tables 7 and 8, respectively; examples 154 to 165 results of cell survival in three-dimensional culture and 3D-printed samples using NIH 3T3 cells are shown in tables 9 and 10, respectively; examples 166-177 the results of cell three-dimensional culture cell viability and cell viability of 3D printed samples using HepG2 cells correspond to tables 11 and 12, respectively.

The variables in examples 130-135 are shown in Table 5, and the cell viability of the bio-ink three-dimensional culture in examples 130-135 is shown in Table 5.

TABLE 5

The relationship between the variables in examples 136-141 is shown in Table 6, and the results of the cell viability of the 3D-printed samples of examples 136-141 are shown in Table 6.

TABLE 6

In the following experimental examples 130 and 136, the GelMA concentration was 5%, and the saccharide oxide concentration was 4% in examples 131 and 137.

The observation of cell viability of HUVEC cells cultured in three dimensions for 1 day, 7 days and 14 days using GelMA-based bio-ink prepared in example 130 is shown in FIG. 10.

The observation of cell viability of HUVEC cells cultured in three dimensions for 1 day, 7 days and 14 days using GelMA-based bio-ink prepared in example 131 is shown in FIG. 11.

The 3D-printed cells loaded with HUVEC cells with GelMA-based bio-ink prepared in example 136 were visualized for 1 day, 7 days, and 14 days survival as shown in fig. 12.

The 3D-printed cells of HUVEC cells loaded with GelMA-based bio-ink prepared in example 137 were observed for 1 day, 7 days and 14 days survival as shown in fig. 13.

The cell viability data for the GelMA-based bio-ink-loaded HUVEC cells prepared in examples 130, 131, 136 and 137 in fig. 10-13 are summarized in fig. 14.

The results in table 5 and fig. 10 and 11 show that: human Umbilical Vein Endothelial Cells (HUVEC) were cultured in bio-ink in three dimensions, and the cells were found to have high viability in each example.

The results in table 6 and fig. 12, 13, 14 show that: human Umbilical Vein Endothelial Cells (HUVEC) can be damaged in the 3D printing process, and the addition of natural saccharides/synthetic saccharide oxides has an obvious relieving effect on the shearing damage of the cells, thereby being beneficial to maintaining the cell activity.

The corresponding relationship between the variables in examples 142-147 is shown in Table 7, and the cell viability of the three-dimensional culture of bio-ink in examples 142-147 is shown in Table 7.

TABLE 7

The relationship between the variables in examples 148-153 is shown in Table 8, and the cell viability of the 3D-printed samples of examples 148-153 is shown in Table 8.

TABLE 8

The results of tables 7-8 show that: mesenchymal Stem Cells (MSCs) have high survival rates in various examples when cultured in three dimensions in bio-ink. Mesenchymal Stem Cells (MSC) can be damaged in the 3D printing process, and the addition of natural saccharides/synthetic saccharide oxides has an obvious relieving effect on the shearing damage of cells, thereby being beneficial to maintaining the cell vitality.

The relationship between variables in examples 154-159 is shown in Table 9, and the results of cell viability in three-dimensional culture of bio-ink in examples 154-159 are shown in Table 9.

TABLE 9

The variables in examples 160-165 are shown in Table 10, and the results of cell viability in the 3D-printed samples of examples 160-165 are shown in Table 10.

Watch 10

The results in tables 9-10 show that: when NIH 3T3 cells were cultured in bio-ink in three dimensions, the cells showed high survival in each example. NIH 3T3 cells can be damaged in the 3D printing process, and the addition of natural sugar/synthetic sugar oxide has an obvious relieving effect on the shearing damage of the cells, thereby being beneficial to maintaining the cell activity.

The variable relationship in examples 166-171 is shown in Table 11, and the cell viability in the three-dimensional culture of bio-ink in examples 166-171 is shown in Table 11.

TABLE 11

The variables in examples 172-177 are shown in Table 12, and the cell viability of the 3D-printed samples in examples 172-177 is shown in Table 12.

TABLE 12

The results in tables 11-12 show that: when HepG2 cells were cultured in bio-ink in three dimensions, the cells showed high survival rates in each example. HepG2 cells can be damaged in the 3D printing process, and the addition of natural saccharides/synthetic saccharide oxides has an obvious relieving effect on the shearing damage of the cells, thereby being beneficial to maintaining the cell activity.

In the description of the present invention, it is to be understood that the terms "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on those shown in the drawings, are merely for convenience of description of the present invention, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A GelMA-based bio-ink is characterized by comprising the following components: methacrylated gelatin, saccharide oxide, chemical initiator and solvent.

2. A GelMA-based bio-ink according to claim 1, wherein the concentration of the methacrylated gelatin is 4% to 20% by weight.

3. The GelMA-based bio-ink according to claim 1, wherein the saccharide oxide is present at a concentration of 0.5% to 5% by weight.

4. The GelMA-based bio-ink according to claim 1, wherein the chemical initiator is present in an amount of 0.01 to 0.5 percent by weight.

5. A GelMA-based bio-ink according to claim 1, comprising 4-20% by mass of said methacrylated gelatin, 0.5-5% by mass of said saccharide oxide, 0.01-0.5% by mass of said chemical initiator, and the balance of said solvent.

6. A method of making a GelMA-based bio-ink according to any of claims 1 to 5, comprising the steps of:

(2) Preparing a carbohydrate oxide: reacting the saccharide solution with an oxidant to obtain a saccharide oxide;

7. The method for preparing GelMA-based bio-ink according to claim 6, wherein in the step (1), the addition ratio of the gelatin to the methacrylic anhydride is 10g: (0.01-20) mL;

in the step (2), the total molar ratio of the saccharide solution to the oxidizing agent is 1: (0.01-5), the oxidizing agent comprises at least one of sodium periodate, sodium hypochlorite, hydrogen peroxide, ozone, chlorine dioxide and potassium permanganate;

in the step (3), the solvent is deionized water, and the pH is 7.0-7.5, and is selected from one of normal saline, PBS solution, sodium acetate buffer solution, tris hydrochloride buffer solution and cell culture medium containing 0% -20% serum.

8. The method of claim 6, wherein the step (3) further comprises adding a proper amount of cells, and mixing the cells uniformly, wherein the cells comprise at least one of human umbilical vein endothelial cells, mesenchymal stem cells, NIH 3T3 cells and HepG2 cells.

9. Use of the GelMA-based bio-ink according to any one of claims 1-5 for tissue repair, regenerative medicine, pharmacokinetics and basic cell biology.

10. Use of a GelMA-based bio-ink according to any of claims 1 to 5 in 3D printing, characterized by the following steps.