CN116572524A

CN116572524A - Application of carrageenan in biological 3D printing

Info

Publication number: CN116572524A
Application number: CN202310554114.XA
Authority: CN
Inventors: 邵磊; 王雪萍; 蒋金洪
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2023-05-17
Filing date: 2023-05-17
Publication date: 2023-08-11

Abstract

The invention discloses application of carrageenan in biological 3D printing, wherein the biological 3D printing comprises biological ink and supporting bath, application of a combination of carrageenan and GelMA solution in the biological ink, and application of a combination of carrageenan and phosphate buffer solution in the supporting bath. The application of the carrageenan with the components in biological 3D printing can effectively control the accurate positioning printing of biological materials which cannot be printed in an extrusion mode, such as gelatin derivative GelMA.

Description

Application of carrageenan in biological 3D printing

Technical Field

The invention relates to the technical field of biological 3D printing, in particular to application of carrageenan in biological 3D printing.

Background

The high-activity biological materials commonly used for cell 3D culture mainly comprise collagen, fibrin, extracellular matrix removal (dECM), gelatin derivative GelMA and the like. The methacrylic anhydride gelatin (GelMA) is gelatin modified by olefin double bonds, is obtained by grafting Methacrylic Anhydride (MA) to replace amino groups on gelatin, can be photo-crosslinked to form a three-dimensional structure with certain strength suitable for cell growth and differentiation, has the characteristics of natural and synthetic biological materials, has reversible temperature-sensitive crosslinking characteristics and irreversible photo-crosslinking characteristics, and is widely used in the fields of tissue engineering and the like due to excellent bioactivity and adjustable physical and chemical properties, so that the GelMA gradually becomes a preferred material for cell 3D culture.

However, biological 3D printing of GelMA has always faced a great challenge. For extrusion printing which is most widely applied, as the viscosity of GelMA is extremely low and is similar to water, direct extrusion printing cannot be performed, mixed printing which influences cell compatibility is eliminated, and the current strategies for extrusion printing GelMA include condensation printing and in-situ photo-crosslinking printing. Because the degree of in situ crosslinking is difficult to control, the printing process is relatively difficult to handle, the current popularity is low, and printing by condensation thickening is mainly popular.

The condensation printing GelMA is mainly performed by utilizing the condensation characteristic pre-condensation thickening of the GelMA, but because gelatin has narrower temperature sensitivity, the gelatin starts to be hot-melted at the temperature of more than 24 ℃, the pre-condensation thickening degree of the GelMA needs to be controlled during printing, and meanwhile, the environment temperature during printing needs to be controlled, the requirement on temperature control is high, and the printing is unstable. In addition, gel formed by GelMA condensation is high in viscosity, high in elasticity and unfavorable for smooth extrusion, so that the GelMA condensation printing strategy is poor in user experience, and the user experience sense and universality of the GelMA condensation printing strategy are improved to be further optimized.

Biological functionalization is the guarantee of various subsequent tissue engineering and regenerative medicine application scenarios, the final goal of biological 3D printing is to construct functional active tissues, and compared with the printability of ink, the biological activity of the ink is the first factor to be considered. However, the high-activity biomaterial formulation ink generally has physicochemical properties of low viscosity strength, and cannot be directly extrusion printed, contrary to the high-viscosity high-strength requirement of printability. So that the extrusion type biological 3D printing with high universality and the most widely applied ink is in contradictory problems of balancing the activity and printability of biological ink for a long time.

With the development of biological 3D printing, a technology of extrusion printing in a supporting bath medium is proposed to solve the problem that low-viscosity low-strength ink is difficult to print, and suspension printing allows a needle to move to scratch a suspension supporting liquid to form gaps and simultaneously deposit ink into the gaps. The presence of the suspension support medium allows the printing filaments to avoid deformation and collapse under the force of gravity. Subsequently, research on suspension printing of various material preparation supporting baths such as microcolloidal particle type supporting baths of carbomers, agarose, alginates, gellan gum, gelatin and the like has been continuously emerging.

Researchers have concluded that a supporting bath suitable for suspension printing needs to have bingham fluid properties, i.e., when a needle-like nozzle moves in the supporting bath, high shear force makes the supporting bath fluid properties, but the shear force disappears behind the nozzle movement, the supporting bath can quickly self-heal and convert into rigidity, and extrusion ink can be instantly wrapped and fixed in place, so that layer-by-layer deposition forming is realized.

However, to prevent the ink from diffusing in the supporting bath, the real-time crosslinking environment of the ink is constructed in the supporting bath mostly for the crosslinking mechanism of the ink, so that the ink for suspension printing is mostly a material of a contact crosslinking mechanism, such as ionic crosslinking, enzymatic crosslinking and pH crosslinking, and is not suitable for a non-contact crosslinking material, such as photo crosslinking. Meanwhile, the support bath known at present has limited self-healing speed, and in printing, in order to avoid printing failure caused by free flow of ink extruded by the support bath in cracks marked by a spray head, the ink is required to have certain initial viscosity so as to slow down the fluidity.

For suspension printing of non-tacky low viscosity photo-crosslinked GelMA, however, researchers have utilized the mechanism by which sulfite can effect GelMA crosslinking, developing a sulfite-containing supporting bath for suspension printing of GelMA. Because of the slower speed of curing GelMA by sulfite, the rapid in-situ crosslinking encapsulation of GelMA cannot be realized, and the self-healing speed of the used supporting bath is limited, the concentration of GelMA (more than 10% w/v) needs to be increased to increase the viscosity of ink and the in-situ crosslinking encapsulation speed, so that the diffusion of the GelMA is prevented, but the growth of the wrapping cells is not favored by the high-concentration GelMA. At present, the supporting bath capable of being used for free suspension printing of GelMA within the full viscosity range is not reported yet.

Disclosure of Invention

The invention aims to provide the application of carrageenan in biological 3D printing, which can effectively control the accurate positioning printing of biological materials which cannot be printed in an extrusion mode, such as gelatin derivative GelMA.

To achieve the above object, the present invention provides an application of carrageenan in biological 3D printing, the biological 3D printing includes a biological ink and a supporting bath, an application of a combination of carrageenan and GelMA solution in the biological ink, and an application of a combination of carrageenan and phosphate buffer in the supporting bath.

Preferably, the concentration of carrageenan in the application of the bio-ink is one of 0.1% w/v, 0.3% w/v and 0.5% w/v.

Preferably, the concentration of carrageenan in the support bath application is one of 0.3% w/v, 0.4% w/v and 0.5% w/v.

Preferably, the carrageenan is one of kappa-carrageenan, iota-carrageenan and lambda-carrageenan.

Therefore, the application of the carrageenan with the components in biological 3D printing has the beneficial effects that: 1. when the kappa-carrageenan is used for preparing the biological ink, the kappa-carrageenan not only serves as a thickener to enhance the printing performance of the GelMA, but also enhances the reversible thermosensitive crosslinking stability at room temperature (25-28 ℃), and the biocompatibility of the GelMA is not affected;

2. the gel kappa-carrageenan/GelMA bio-ink can be spatially recombined in a syringe and biologically printed into a complex structure with a natural transition section and a gradient pore structure;

3. the carrageenan has a two-step gelling mechanism due to the existence of the unique sulfate ester group, a double-helix structure is formed through disordered polymer chains at low temperature, the double helices are mutually connected into a network under the action of further cooling or ions, and compared with iota type and lambda type, kappa type serving as a support bath preparation material has the characteristics of strong gelling property and typical thermosensitive property;

4. the kappa-carrageenan supporting bath can be converted from solid state property with main elasticity to liquid state property with main viscosity under the condition of smaller shearing and stress deformation degree of the needle head, so that smooth embedding and deposition of ink are facilitated, after the needle head leaves a printing area, the flowing kappa-carrageenan supporting bath with the liquid state property is quickly restored to the solid state with main elasticity, and the kappa-carrageenan supporting bath can rapidly wrap the printing filament diameter and avoid collapse and deformation of the printing filament through the supporting capacity of high modulus;

5. the extremely small low yield stress of the kappa-carrageenan supporting bath ensures that after the needle is removed, the kappa-carrageenan supporting bath can quickly yield and heal under the action of the hydrostatic pressure around the needle, so that the space divided by the needle is filled in time, and the extruded material is embedded when the needle moves, so that the printing ink is prevented from rising;

6. the printing process and the kappa-carrageenan supporting bath can not influence the activity of cells, the kappa-carrageenan supporting bath is biocompatible, and the low-viscosity GelMA biological ink has application prospect in the field of cell culture and regenerative medicine in the cell culture carrier extruded and printed in the kappa-carrageenan supporting bath.

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

FIG. 1 is a graph of the mechanical properties of kappa-carrageenan/GelMA bio-ink of the invention at different carrageenan concentrations;

FIG. 2 is a graph showing the relationship between the structural morphology, porosity and mechanical properties of the kappa-carrageenan/GelMA bio-ink according to the present invention before and after dissolution of the carrageenan;

FIG. 3 is a print parameter optimization diagram of the present invention;

FIG. 4 is a bioprinting of a complex structure of the present invention;

FIG. 5 is a graph of cell viability and cell diffusion of three cells of the invention;

FIG. 6 is a graph of a kappa-carrageenan supporting bath at three concentrations according to the present invention;

FIG. 7 is a graph of the performance of a kappa-carrageenan supporting bath of the present invention at three concentrations;

FIG. 8 is a schematic diagram of the invention printed directly in a kappa-carrageenan supporting bath;

FIG. 9 is a graph of brain, heart and branch coronary artery models printed with GelMA ink in a kappa-carrageenan supporting bath in accordance with the present invention;

FIG. 10 is a diagram of a chip with flow channels printed in a kappa-carrageenan supporting bath in accordance with the present invention;

FIG. 11 is a block diagram of a grid and bone sample printed with cell-laden GelMA in a kappa-carrageenan supporting bath according to the present invention.

Detailed Description

The technical scheme of the invention is further described below through the attached drawings and the embodiments.

The present invention will be explained in more detail by the following examples, and the purpose of the present invention is to protect all changes and modifications within the scope of the present invention, and the present invention is not limited to the following examples.

Example 1

Preparation of biological ink

S11, adding 5% GelMA solution (containing 0.25% LAP) into kappa-carrageenan, iota-carrageenan and lambda-carrageenan with the concentration of 0.1% w/v respectively, and stirring at 37 ℃ until the mixture is completely dissolved to obtain three mixed solutions.

S12, transferring the three mixed solutions obtained in the S11 into different syringes, and cooling for 5min at 4 ℃ to obtain different types of carrageenan/GelMA biological ink.

Example 2

S1, preparation of biological ink

S11, adding 5% GelMA solution (containing 0.25% LAP) into kappa-carrageenan with concentration of 0.1% w/v, 0.3% w/v and 0.5% w/v respectively, and stirring at 37 ℃ until the mixture is completely dissolved to obtain a mixed solution.

S12, transferring the mixed solution obtained in the step S11 into a syringe, and cooling for 5min at 4 ℃ to obtain the kappa-carrageenan/GelMA bio-ink with different concentrations.

S2, physicochemical properties of kappa-carrageenan/GelMA biological ink with different concentrations

S21, measuring rheological behavior: fresh kappa-carrageenan/GelMA bio-ink was added between parallel plates at 25 ℃ with a gap value of 100 μm, excess samples were removed, and the rheological behavior of the kappa-carrageenan/GelMA bio-ink was determined as shown in FIG. 1.

S22, measuring porosity: respectively soaking kappa-carrageenan/GelMA biological ink and GelMA in phosphate buffer solution at 37deg.C for 2 hr to obtain kappa-carrageenan with different concentrations before and after soaking

The GelMA bio-ink was frozen in liquid nitrogen and dried by a freeze dryer and then scanned using a scanning electron microscope, and the results were shown in FIG. 2.

S23, compression test: hydrogel samples of various concentrations of kappa-carrageenan/GelMA bio-ink were placed between the flow plates and compressed to a compression ratio of 50% at a rate of 0.2mm/s, and the results obtained are shown in FIG. 3.

S24, measuring Young' S modulus: the results obtained using a biological universal tester are shown in FIG. 1.

S3 analysis of FIGS. 1, 2 and 3

S31, as can be seen from FIG. 1, FIG. 1 is a graph showing the mechanical properties of kappa-carrageenan/GelMA bio-ink of the present invention at different kappa-carrageenan concentrations.

FIG. 1 (A) is a plot of storage modulus (G ') and loss modulus (G') for a bio-ink containing 0.1% w/v, 0.3% w/v, and 0.5% w/v kappa-carrageenan/GelMA. It is known that at strains less than 100%, shear induced gel-fluid transition occurs with 0.1% w/v and 0.3% w/v kappa-carrageenan/GelMA bioink, whereas shear induction is higher with 0.5% w/v kappa-carrageenan/GelMA bioink.

Fig. 1 (B) shows the compressive stress-shear rate curve of the same hydrogel.

The viscosity versus shear rate of the kappa-carrageenan/GelMA bio-ink at concentrations of 0.1% w/v, 0.3% w/v and 0.5% w/v is shown in FIG. 1 (C). It can be seen that when the shear rate is increased to 0.0001s ^-1 About, the viscosity of the 0.1% w/v kappa-carrageenan/GelMA bio-ink was significantly reduced, exhibiting shear thinning behavior. In contrast, 0.3% w/v kappa-carrageenan/GelMA bio-ink and 0.5% w/v kappa-carrageenan GelMA bio-ink require greater than 0.01s ^-1 The shear rate of (c) can cause a viscosity change.

The (D) in FIG. 1 is the creep compliance-time and recovery compliance-cure of kappa-carrageenan/GelMA bioinks at concentrations of 0.1% w/v, 0.3% w/v and 0.5% w/v. It is clear that the three groups have different creep recovery curve shapes, and that upon constant stress, a transient deformation of the 0.3% w/v kappa-carrageenan/GelMA bio-ink occurs.

The different viscosity-time curves for kappa-carrageenan/GelMA bioinks at concentrations of 0.1% w/v, 0.3% w/v and 0.5% w/v are for (E) in FIG. 1 and (F) in FIG. 1. It can be seen that after two cycles, the modulus of the three groups of bio-inks is not much different from the initial amount, indicating that they remain stable during printing, and the recovery time of the kappa-carrageenan/GelMA bio-ink gradually increases as the concentration of carrageenan increases.

The different viscosity-temperature curves for kappa-carrageenan/GelMA bioinks at concentrations of 0.1% w/v, 0.3% w/v and 0.5% w/v are shown in FIG. 1 (H). It is known that the thermosensitive reversible crosslinking temperature of 0.3% w/v kappa-carrageenan/GelMA bio-ink is in the range of 0-31.89 ℃, is close to the thermosensitive reversible crosslinking temperature of 0-32.91 ℃ of 0.5% w/v kappa-carrageenan/GelMA bio-ink, and is obviously higher than the thermosensitive reversible crosslinking temperature of 0.1% w/v kappa-carrageenan/GelMA bio-ink by 0-27.15 ℃ and the thermosensitive reversible crosslinking temperature of pure GelMA by 0-24.31 ℃.

S32, as can be seen from FIG. 2, FIG. 2 is a graph showing the relationship among the structural morphology, the porosity and the mechanical properties of the kappa-carrageenan in the kappa-carrageenan/GelMA bio-ink according to the present invention before and after dissolution.

FIG. 2 (A) is a Scanning Electron Microscope (SEM) image of kappa-carrageenan/GelMA bioink at 0.1% w/v, 0.3% w/v and 0.5% w/v concentrations, and a stent printed with the above bioink before and after soaking in phosphate buffer at 37 ℃.

Fig. 2 (B) shows the quantitative porosity of the same kappa-carrageenan/GelMA bio-ink before and after 37 ℃ phosphate buffer immersion. Compared with (a) in fig. 2, the porosity of the GelMA bio-ink scaffold in which the kappa-carrageenan is dissolved is significantly enhanced, and the porosity slightly increases with the increase of the kappa-carrageenan concentration.

FIG. 2 (C) is a graph showing the compressive stress-strain curve of kappa-carrageenan/GelMA bioink at 0.1% w/v, 0.3% w/v and 0.5% w/v concentrations prior to immersion in phosphate buffer at 37 ℃. It can be seen that the introduction of kappa-carrageenan significantly improves the mechanical properties of the GelMA bio-ink. The kappa-carrageenan/GelMA bio-ink at 0.1% w/v concentration showed excellent compressive stress at 50% strain, up to 59MPa, before kappa-carrageenan was dissolved.

FIG. 2 (D) is a graph showing the compressive stress-strain curve of kappa-carrageenan/GelMA bioink at 0.1% w/v, 0.3% w/v and 0.5% w/v after immersion in phosphate buffer at 37 ℃.

FIG. 2 (E) is the elastic modulus of the kappa-carrageenan/GelMA bioink at 0.1% w/v, 0.3% w/v and 0.5% w/v concentrations before and after phosphate buffer soaking at 37 ℃. Data are reported as mean ± standard deviation, n=3. It was found that the elastic modulus was maximum at 0.3% w/v and 0.5% w/v, respectively, of kappa-carrageenan concentration prior to dissolution, approaching 0.8kPa.

As can be seen from fig. 2 (C) and fig. 2 (E), as the concentration of kappa-carrageenan increases, the compressive modulus and toughness of the kappa-carrageenan/GelMA bio-ink increase, and the elastic modulus and stress strain at break increase.

As can be seen from fig. 2 (D) and fig. 2 (E), as the kappa-carrageenan dissolves, the stress strain and elastic modulus at break decrease to a state similar to GelMA, while the stress strain and elastic modulus of the kappa-carrageenan/GelMA bio-ink remain slightly higher.

Example 3

S1, as can be seen from FIG. 3, FIG. 3 is a printing parameter optimization chart of the present invention.

Fig. 3 (a) shows macroscopic changes in thermal sensitivity of 5% GelMA and 0.5% w/v concentration kappa-carrageenan/GelMA bio-ink. It is known that the 0.5% w/v concentration kappa-carrageenan/GelMA bio-ink extruded from the needle is more uniform and smooth after cooling at 4 ℃ for 10min, and the solid colloid is still stable even if rewarming at 28 ℃ for 10-60 min. However, gelMA cooling induced gelation exhibited significant particle size during printing and extrusion. Meanwhile, after rewarming for 0.5h at 28 ℃, the kappa-carrageenan/GelMA bio-ink with the concentration of 0.5% w/v begins to melt and cannot be used for printing.

The effect of kappa-carrageenan/GelMA bio-ink flow and different on-axis nozzles (outer/inner: 17G/25G,17G/26G, 17G/27G) on fiber diameter for 5% GelMA and 0.5% w/v concentration in FIG. 3 (B). It is known that the diameter of the printed fiber can be adjusted by adjusting the flow rate of the material or the size and height of the outer/inner needle.

Fig. 3 (C) shows quantitative analysis of fiber diameter (mean value, error bar indicates Standard Deviation (SD) of independent repetition). 37 ℃. Data are reported as mean ± standard deviation, n=3. It was found that a series of fibres could be printed by varying the inner needle (25G-27G) with a fixed outer needle (17G), by varying the flow rate of 0.5% w/v kappa-carrageenan/GelMA bio-ink (0.01-0.07 mL/min) or the speed of movement of the needle. When the coaxial needle size and material flow rate were determined, the fiber diameter increased as the needle movement speed increased, and the channel diameter was quantitatively analyzed.

S2, through automatic slicing programming of 3D bio-printer software, kappa-carrageenan/GelMA bio-ink can be continuously extruded and successfully deposited, so that a large-scale complex structure with a required shape and structure is created.

As shown in fig. 4, fig. 4 is a bioprinting view of the complex structure of the present invention.

Fig. 4 (a) is a bioprinting stent, fig. 4 (B) is a bifurcated vessel, fig. 4 (C) is a solid nose, and fig. 4 (D) is a solid ear, and it is known that kappa-carrageenan/GelMA bio-ink can stably construct a complex large organ structure at 28 ℃.

Fig. 4 (E) is a topological cube based on three-cycle minimum curved surface printed with 0.3% w/v kappa-carrageenan/GelMA bio-ink.

Fig. 4 (F) shows heterogeneous bioprinting structures with heterogeneous 0.3% w/v kappa-carrageenan/GelMA bio-ink in different ratios and spatial distribution patterns. It is known that gel-like kappa-carrageenan/GelMA bio-ink can be spatially reconstituted in a syringe.

Fig. 4 (G) shows two different layered scaffolds printed with 0.3% w/v kappa-carrageenan/GelMA bio-ink. It is known that the structure has a spontaneous interface section and a gradient pore structure, as shown in the vertical section view.

Example 4

Biocompatibility of kappa-carrageenan/GelMA biological ink after dissolution

S1 osteoblasts (MC-3T3,1.0X 10) ⁶ personal/mL), human umbilical vein endothelial cells (HUVECs, 1.0X10) ⁶ individual/mL) and mouse bone marrow mesenchymal stem cells (BMSCs, 1.0x10) ⁶ individual/mL) was introduced into kappa-carrageenan/GelMA bio-ink and bioprinting cell-loaded constructs for in vitro culture.

S2, carrying out live/dead cell staining on the 1 st day, and showing that cells are uniformly distributed in the biological printing block, wherein the calcein positive living cells account for more than 85%.

S3, staining F-actin and cell nucleus respectively by TRITC phalloidin and DAPI, monitoring the diffusion of the packed cells in the construct, and gradually expanding the cells packed in the construct into long strips after culturing for 5 days.

As shown in FIG. 5, FIG. 5 is a graph of cell viability and cell expansion of three cells of the invention.

Live/dead stained fluorescence images of BMSCs, HUVECs and MC-3T3 were encapsulated in scaffolds for 1 day. Immunofluorescence images of encapsulated BMSCs, HUVECs and MC-3T3 morphology within the scaffolds were bioprinted after 3 and 5 days of incubation. It is known that the use of kappa-carrageenan does not affect the biocompatibility of GelMA.

Example 5

S1, preparation of kappa-carrageenan supporting bath: the kappa-carrageenan is added into 100mL of phosphate buffer solution and stirred for 30min at 70 ℃ to obtain kappa-carrageenan solution. Placing in a refrigerator for at least 2h to completely gel. The kappa-carrageenan gel was crushed into particles using an electric stirrer at 1000 rpm/min. The supporting bath composed of kappa-carrageenan microgel particles is subpackaged into 50mL centrifuge tubes, and air bubbles are removed by centrifugation at 1000rpm/min, so that kappa-carrageenan supporting bath is obtained.

Preparation of S2, iota-carrageenan supporting bath: and adding the iota-carrageenan into 100mL of phosphate buffer solution, and stirring for 30min at 70 ℃ to obtain the iota-carrageenan solution. Placing in a refrigerator for at least 2h to completely gel. The iota-carrageenan gel was mashed into particles using an electric stirrer at 1000 rpm/min. And subpackaging the support bath consisting of iota-carrageenan microgel particles into a 50mL centrifuge tube, and centrifuging at 1000rpm/min to remove bubbles to obtain the iota-carrageenan support bath.

S3, preparing a lambda-carrageenan supporting bath: the lambda-carrageenan is added into 100mL of phosphate buffer solution and stirred for 30min at 70 ℃ to obtain lambda-carrageenan solution. Placing in a refrigerator for at least 2h to completely gel. The lambda-carrageenan gel was crushed into particles using an electric stirrer at 1000 rpm/min. And subpackaging the support bath consisting of the lambda-carrageenan microgel particles into a 50mL centrifuge tube, and centrifuging at 1000rpm/min to remove bubbles to obtain the lambda-carrageenan support bath.

Example 6

S1, preparation of kappa-carrageenan supporting baths with different concentrations: 100mL of phosphate buffer solution is added to 0.3% w/v, 0.4% w/v and 0.5% w/v kappa-carrageenan respectively, and the mixture is stirred for 30min at 70 ℃ to obtain kappa-carrageenan solutions with different concentrations.

Placing in a refrigerator for at least 2h to completely gel. The kappa-carrageenan gels of varying concentrations were crushed into particles using an electric stirrer at 1000 rpm/min. And subpackaging the supporting bath consisting of kappa-carrageenan microgel particles with different concentrations into a 50mL centrifuge tube, and centrifuging at 1000rpm/min to remove bubbles to obtain kappa-carrageenan supporting bath with different concentrations.

The kappa-carrageenan supporting bath which is not used immediately can be stored in a refrigerator (4 ℃) for 1 month or more (the room temperature is polluted by microorganisms when being placed for a long time), and can be used without being restored to the room temperature when being used.

S11, as shown in FIG. 6, FIG. 6 is a graph of a kappa-carrageenan supporting bath at three concentrations according to the present invention.

As can be seen from FIG. 6 (below), the prepared kappa-carrageenan supporting baths of 0.3% w/v and 0.4% w/v had better liquid-like flow properties, whereas the kappa-carrageenan supporting baths of 0.5% w/v had poorer flow properties. Therefore, the low-concentration carrageenan has better flowability, higher self-healing speed in theory, lower turbidity and lower influence on photopolymerization of GelMA.

S2, preparation of a sterile kappa-carrageenan supporting bath: firstly, the kappa-carrageenan powder is irradiated for 30min by an ultraviolet lamp, then soaked in 75% alcohol for 30min and then prepared. The rest steps are the same as the preparation process of the kappa-carrageenan supporting bath, and the prepared kappa-carrageenan supporting bath should avoid the irradiation of an ultraviolet lamp. The addition of 0.1% w/v diabody solution to the sterile kappa-carrageenan supporting bath further inhibited microbial contamination.

S3, preparing biological ink: 0.05g of GelMA, 0.025% photoinitiator LAP and 5mL of phosphate buffer solution were placed in a 15mL centrifuge tube and heated at 40℃for 30min to give 5% w/vGelMA.5% w/v GelMA was heated at 50℃for 5min and sterilized by filtration using a sterile 0.2 μm sterilizer. Mixing sterilized 5% w/v GelMA to collect cells in advance, mixing, filling into sterile syringe, and loading cell inkThe number of water-borne cells was 1X 10 ⁷ /mL. In order that the printing process can be more easily observed, a very small amount of ink with a neutral pen is added when printing in the ink.

S4, physicochemical properties of kappa-carrageenan supporting baths with different concentrations

S41, kappa-carrageenan supporting baths with the concentration of 0.3% w/v, 0.4% w/v and 0.5% w/v are prepared according to the steps in S1.

S42, rheological measurement: measurements were made using a rheometer (HAAKE) with a 20mm parallel jig, 1mm gap, all at a detection frequency of 10Hz. Except Flow temperature, the other experimental measurements were 25 ℃. The experimental temperature of the Flow temperature was increased from 10 ℃ to 40 ℃ at a constant rate (Ramp mode).

S43, an electron microscope: electron microscopy was performed on 0.3% w/v, 0.4% w/v, 0.5% w/v kappa-carrageenan supporting baths. As shown in FIG. 6 (above), the particles in the kappa-carrageenan supporting bath were less than 1 μm at all three concentrations.

S5, analyzing the figure 7: as shown in fig. 7, fig. 7 is a graph of the performance of a kappa-carrageenan supporting bath of the present invention at three concentrations.

A in FIG. 7 is a spin test, shear rates of 0.001-100mm/s, observing the change in viscosity of the kappa-carrageenan supporting bath at different shear rates. Yield stress at each concentration: 0.3% w/v, τ0=0.46 Pa;0.4% w/v, τ0=0.47 Pa;0.5% w/v, τ0=0.92 Pa.

B in FIG. 7 is a strain oscillation scan of the kappa-carrageenan supporting bath, the strain range is 0.025% -10%, and the process of converting high elasticity into high fluidity (viscosity) after the kappa-carrageenan supporting bath changes along with the stress is observed.

As can be seen from a in fig. 7 and B in fig. 7, the extremely low yield stress of the kappa-carrageenan supporting bath ensures a rapid yielding and healing under the influence of the ambient hydrostatic pressure after removal of the needle.

C in fig. 7 is the ability to simulate repeated switching of the kappa-carrageenan supporting bath from liquid to solid using alternating oscillating sweeps of low strain (1%) and high strain (100%) continuously. It can be seen that when the continuous recovery capacity and stability of the kappa-carrageenan supporting bath were tested using the low strain and high strain alternating oscillation method, the elastic modulus of the kappa-carrageenan supporting bath did not change after two cycle tests, indicating that it can maintain a stable supporting effect during printing.

D in FIG. 7 is the time to recover to low strain observed viscosity after 0.01% action for 1s and then 100% action for 0.1 s.

E in FIG. 7 is a detailed graph of observed viscosity recovery time after 0.01% action for 1s and then 100% action for 0.1 s.

As can be seen from D in fig. 7 and E in fig. 7, the kappa-carrageenan supporting bath exhibits extremely low yield stress (around 0.46 pa), ensuring that after the needle is removed, it can yield and heal quickly under the effect of the ambient hydrostatic pressure, filling the space delimited by the needle in time, embedding the extruded material as the needle is moved, avoiding the rise of printing ink. After the needle leaves the printing zone, the flowing liquid kappa-carrageenan supporting bath quickly returns to a solid state with a dominant elasticity, which allows the kappa-carrageenan supporting bath to quickly wrap the printing filament diameter and avoid collapse deformation of the printing filament by the high modulus supporting capability.

The kappa-carrageenan supporting bath has high self-healing speed, and the kappa-carrageenan supporting bath with three concentrations only needs about 0.03s to be converted from a liquid sample to a solid sample.

F in fig. 7 is a graph of the change in viscosity of the test kappa-carrageenan supporting bath from 10-40 c, the temperature increasing at a constant rate from 10 c to 40 c, the rate of change being 5 c/min. The kappa-carrageenan supporting baths of three concentrations all show a stable state at 15-25 ℃ and are beneficial to the stability of printing at room temperature.

At around 37 ℃, the viscosities of the 0.3% w/v kappa-carrageenan supporting bath and the 0.4% w/v kappa-carrageenan supporting bath were significantly reduced by four orders of magnitude, indicating that the kappa-carrageenan supporting bath was highly heat sensitive, facilitating removal of carrageenan particles trapped in the print enclosed cavity or in the fine narrow gap.

G in fig. 7 is a stress-shear rate plot showing the fitting of three concentrations of kappa-carrageenan supporting bath and 5% w/v GelMA to a Herschel-Bukley fluid model with a degree of fitting of: 0.3% w/v,0.91;0.4% w/v,0.89;0.5% w/v,0.91. When both the printed ink and the supporting bath are in the form of a Herschel-Bukley fluid, the printed wire diameter is more likely to have a cylindrical wire diameter.

H in fig. 7 is a printed and photo-crosslinked hollow sphere suspended in a kappa-carrageenan supporting bath. The hollowed-out spheres were printed by GelMA in 0.4% w/v kappa-carrageenan supporting bath for observation of thermal dissolution at 37℃of the 0.4% w/v kappa-carrageenan supporting bath. After the printed hollowed-out ball is cured by 405nm light, the printed whole container is placed in a water bath kettle at 37 ℃.

I in FIG. 7 is that the kappa-carrageenan supporting bath was allowed to grow in a 37℃water bath and the hollow spheres gradually dropped and collapsed. It is known that with increasing heating time, the edge of the kappa-carrageenan supporting bath is subject to rapid thermal motion and gradually dissolves, after 8min, a part of the hollow ball starts to fall down, after 10min, the hollow ball completely loses the support of the kappa-carrageenan supporting bath, and the whole hollow ball collapses to the bottom of the container.

In summary, the 0.3% w/v kappa-carrageenan supporting bath and the 0.4% w/v kappa-carrageenan supporting bath are soluble in a short time at a temperature of 37 ℃. The kappa-carrageenan supporting bath with various concentrations is comprehensively compared, and the 0.5% w/v kappa-carrageenan supporting bath has poor hot melting effect at 37 ℃. The 0.3% w/v kappa-carrageenan supporting bath has a narrow temperature stabilizing range, can be thermally dissolved at about 27 ℃, and has low viscosity and good fluidity, thus leading to shaking in the printing process. Thus, a 0.4% w/v kappa-carrageenan supporting bath was chosen to verify the possibility of 5% GelMA printing.

Example 7

S1, printing a simple model: the straight lines, spiral lines and simple grid structures were printed in a kappa-carrageenan supporting bath using 5% GelMA ink grafted with rhodamine B.

S2, printing of a complex model:

s21, downloading a left brain model with a complex groove back structure from an NIH website (https:// 3d.nih.gov /), then reducing to 0.4 times, slicing by using a repeater Host, and printing by using a needle with the inner diameter of 0.06nm and the outer diameter of 0.25nm.

S22, a complete heart model is found on a 3D body database (http:// life science db. Jp/bp3D /), the wall thickness of the heart is thickened by using Rhino software (https:// www.rhino3d.com /), and most of the vascular structures are deleted so that slicing printing can be performed. The left and right blood vessels are reserved and bolded for observing the accuracy of the printing. The modified heart model has obvious cavity structure and blood vessel, and is reduced to 0.5 times for slice printing.

S23, selecting a thin-wall multi-branch coronary artery vascular structure for printing except for a solid structure and a solid structure containing a small amount of cavities.

S24, drawing a length of less than 1cm ³ And (3) designing and printing a blood vessel chip structure with a spiral runner, designing and printing a chip with two branches and four branches, and designing and printing a space runner chip with three branches.

The chip models are all drawn by using Blender (https:// www.blender.org /), the runner part draws paths firstly and then materializes, and multiple branches use Booler operation to combine the separately drawn parts. And subtracting the runner drawn in advance by using a rectangular structure to obtain the chip structure containing the runner. All model sections were preceded by netfab (https:// www.autodesk.com/products/netfab /) for detection and repair of fluid structures.

S3, analyzing the printing model in the S2

S31, as shown in FIG. 8, FIG. 8 is a diagram of the printing structure of the invention directly in a kappa-carrageenan supporting bath.

Printing needle specification: the inner diameter is 0.06mm and the outer diameter is 0.25mm. And shooting by a split microscope, and carrying out 10x.

A in FIG. 8, 200mm/min, 400mm/min, 600mm/min and 800mm/min of printed straight strips immersed in a kappa-carrageenan supporting bath, and confocal microscopy layer scanning observed the thickness, morphology and roundness of the cross-section of the printed wire diameter. It is known that the kappa-carrageenan supporting bath supports 5% GelMA printing of approximately cylindrical filament diameters. The filament diameter can be regulated by changing the printing speed and the extrusion flow, and meanwhile, due to the low viscosity of GelMA, a very fine needle (inner diameter: 0.18 mu m; outer diameter: 0.22 mu m) can be adopted to print out high-precision filaments (about 200 mu m), so that the method is very beneficial to the construction of an ultra-high-precision structure.

B in fig. 8, a relation chart of printing speed and printing wire diameter thickness. The relationship between the printing speed and the thickness of the printing wire diameter was a negative correlation, and the correlation was 97%. The figure is a projection of a GelMA print silk confocal layer scan image immersed in a kappa-carrageenan supporting bath, n=3.

C in fig. 8, presentation supporting spiral path printing in bath. It is known that the spiral-rising print path is smooth, and the wire diameter is free of significant irregularities and deformations, indicating that the kappa-carrageenan supporting bath supports free movement of the print head in three dimensions.

D in FIG. 8, a GelMA printing wire was photographed at a time delay, and captured every 10min for 1h. After 6 hours, an image was taken. The widths of the printing filaments at different times are calculated separately. n=3, each wire diameter is measured at least three times. It was found that there was no statistical difference between the width of the printed filaments after 0min and 70min immersed in the kappa-carrageenan supporting bath after printing (p=0.27 > 0.5), and that the average increase in the printed filaments after 6h was 60 μm (about 1/5 of the original width).

E in fig. 8, the printed grid structure is used for confocal layer scanning after soaking rhodamine B. 10X; na=0.95. The fine grid structure of printing wires and the printing wire spacing was 200 μm.

F in FIG. 8, the grid structure printed with 5% GelMA was cured and then removed in phosphate buffer, and the printed grid was immersed in diluted neutral ink to reveal the cross section (5 mm) of the printed grid structure.

The grid structure printed with 5% GelMA at G in fig. 8 was cured and then removed in phosphate buffer, and the printed grid was immersed in diluted neutral ink to reveal the cross section (1 mm) of the printed grid structure.

As is clear from E in fig. 8, F in fig. 8, and G in fig. 8, the top view of the printed grid structure and the microscopic image of the mirror of the selected portion indicate a morphology in which the printed wire diameters are not twisted and piled up, and the printed wire stably exhibits a cylindrical shape. The kappa-carrageenan supporting bath can support printing a single layer of grid structure with more than 100 apertures in a coin scale with the diameter of 25mm, and is taken out after being solidified by 405nm light, so that the effect of the 0.4% w/v kappa-carrageenan supporting bath on the photocrosslinking of GelMA is little.

In summary, the 0.4% w/v kappa-carrageenan supporting bath supports free and stable construction of a high precision structure with 5% GelMA.

S32, as shown in FIG. 9, FIG. 9 is a graph of brain, heart and branch coronary artery models printed with GelMA ink in a kappa-carrageenan supporting bath according to the present invention.

Printing needle specification: an inner diameter of 0.06nm and an outer diameter of 0.25nm.

The medial (left) and right (right) sides of the a, left brain model in fig. 9.

The B, 5% GelMA printed left brain in fig. 9 was immersed in the phosphate buffer on the inside (left) and right (right) sides.

The C, printed left brain in fig. 9 was left in air and after staining with diluted neutral pen ink, the sulcus structure of the left brain was shown. It is known that the printed left brain can still exhibit a microscopic sulcus structure.

D in fig. 9, schematic of the heart (left), right side (middle) and left side (right) of the heart model are printed.

5% GelMA printed heart structures in E, kappa-carrageenan supporting baths in FIG. 9.

F in fig. 9, the printed heart structure was removed and immersed in phosphate buffer, and the right and left chambers were filled with diluted neutral pen ink, respectively. It is known that dye injected at the superior vena cava (position 1) can pass through to the right atrium and right ventricle in turn, and flow out from the pulmonary artery (position 2), red dye from the aorta (position 4), from the left ventricle, through the left atrium and out from the pulmonary vein (position 3).

Fig. 9 is a sagittal sectional view of a 5% GelMA-printed heart with G mixed with a small amount of neutral pen ink, with the pulmonary veins at the solid triangle.

H in fig. 9, the inner (left) and right (right) sides of the heart printed with ink of the ink-containing ink, respectively show the vascular-like structure designed in the model.

I in fig. 9, model of coronary arteries of heart (top), in kappa-carrageenan supporting bath (middle) and take out printed structures in phosphate buffer (bottom).

The fidelity of the printing is measured by comparing the differences in included angles between the branches of the printing model and the branches of the object, and the differences in included angles between the selected model and the branches of the blood vessel between the printing object are no more than 6 degrees. Although each included angle is measured three times to reduce errors caused by manual hooking during measurement, errors caused by angles of the lens during shooting still exist. In summary, the kappa-carrageenan supporting bath supports printing of complex structures with millimeter-sized structures, internal chambers, multiple branches, and the like.

S33, as shown in FIG. 10, FIG. 10 is a diagram of a chip with flow channels printed in a kappa-carrageenan supporting bath according to the present invention.

Schematic of a single-screw chip (left one) in fig. 10, a single-screw channel-containing chip structure printed by 5% GelMA in phosphate buffer (left two) and diluted neutral ink-filled chip immersed in phosphate buffer (right two) and standing in air (right one). It is known that the single spiral flow channel chip is vertically arranged in the air, and the dye added at the top drops along the flow channel in a spiral manner, so that the filling process is very smooth.

Schematic of B, double helix-containing chip in fig. 10 (left), double helix channel-containing chip structure printed with 5% GelMA in phosphate buffer (middle), standing on air diluted neutral ink-filled chip (right). The ink is used for filling respectively, and the independent ink shows that two space spiral flow channels in the millimeter-sized rectangular gel structure are not mutually interfered.

C in fig. 10, schematic view (top), cross section (middle) and top view (bottom) of the chip with three-dimensional fluidic channels after dye infusion. The section shows the space runner of the chip, and the runner diameter is about 600 mu m, which proves that the kappa-carrageenan supporting bath has application prospect when the branched runner chip is printed.

D in fig. 10, chip with bifurcated flow channel (left), printed structure in phosphate buffer (middle), dye-infused chip (right).

Schematic of E, chip with four branched flow channels (left) in fig. 10, printed structure in phosphate buffer (middle) and state of dye perfusion (right).

As can be seen from D in FIG. 10 and E in FIG. 10, the result shows that the measurement is performed at 1cm ³ The flow channel structure with the diameter of about 600 μm can be successfully printed in the left and right volumes, and the accuracy of the kappa-carrageenan supporting bath printed pipeline structure is close to that of some sacrificial printing strategies.

In conclusion, the kappa-carrageenan supporting bath has wide application prospect for manufacturing the gel structure containing the simulated vessel.

Example 8

Verification of cell Activity after kappa-carrageenan supporting bath printing

S1, kappa-carrageenan supporting bath printing

S11, carrying out ultraviolet irradiation on kappa-carrageenan powder for more than 30min, then soaking the kappa-carrageenan powder in 75% sterile alcohol for more than 30min, adding the kappa-carrageenan powder into 100mL of phosphate buffer solution, and stirring the mixture at 70 ℃ for 30min to obtain kappa-carrageenan solution. Placing in a refrigerator for at least 2h to completely gel. The kappa-carrageenan gel was crushed into particles using an electric stirrer at 1000 rpm/min. The supporting bath composed of kappa-carrageenan microgel particles is subpackaged into 50mL centrifuge tubes, and air bubbles are removed by centrifugation at 1000rpm/min, so that kappa-carrageenan supporting bath is obtained.

S12, selecting three cells of HUVECs, BMSC and MC-3T3, printing a grid structure (avoiding the interference of insufficient nutrition permeation) in a kappa-carrageenan supporting bath by using 5% GelMA carrier cells, directly taking out the printed grid structure, immersing the printed grid structure in a culture medium, culturing the printed grid structure in a constant temperature oven at 37 ℃ for 30min, and then replacing the culture medium and a culture dish. After 2 hours of further culture, the culture medium and the culture dish are replaced again, so that the kappa-carrageenan is ensured to be sufficiently dissolved and removed.

And S13, taking out and cleaning the cells within 24 hours, observing the conditions of living cells and dead cells by using a living/dead staining experiment, culturing the cells for one week, performing a cytoskeletal living/dead staining experiment, and judging the activity of the cells by observing the morphology of the cells and the stretching condition of the cells.

S2, as shown in FIG. 11, FIG. 11 is a structural diagram of a grid and bone sample printed with cell-laden GelMA in a kappa-carrageenan supporting bath according to the present invention.

Schematic of the printed grid structure in the a, k-carrageenan supporting bath in fig. 11, the supporting bath dissolved with incubation at 37 ℃.

B in FIG. 11, 5% GelMA printed grid structure macroscopic top view (left) and side view (right), wire spacing 0.6mm, layer height 0.4mm.

After printing of the C, HUVEC-loaded cells in fig. 11, viability of the cells was checked in 24h using a live/dead staining experiment. It was found that the viability of the cells was above 90%, indicating that the printing process and kappa-carrageenan supporting bath did not affect cell viability.

The D, HUVEC-loaded grid structure in FIG. 11 was cultured for one week and the first, fourth and sixth day of selection of the light-microscopic morphology of HUVECs. On day 1, HUVECs can be seen to form distinct vacuoles, white arrows (left). Day 4 and day 6 cells were spread to each other (black arrows, middle and right). The vacuole structure is the basic unit of spontaneous assembly capillary blood vessel of endothelial cells, and by the time of culturing, many HUVECs show epithelium-like, extend out of amoeba-like pseudopodia, are connected with each other between cells, and the visible partial area appears in the vacuole-like structure by the time of day 6.

Immunofluorescence of E in FIG. 11, HUVECs-loaded cultured for 6 days and project-loaded BMSCs grid structure cultured for 11 days.

Immunofluorescence of the combination of global (left) and local (right) of g, MC-3T3 cell loaded bone morphogenetic structures in fig. 11.

FIG. 11 is a combined immunofluorescence of H, cultured for 7 days with MC-3T 3-loaded grid structures. It was found that BMSCs cultured for 11 days and MC-3T3 cells cultured for 7 days had the cytoskeleton stretched and cells connected to each other. After printing of the MC-3T 3-loaded larger bone-like structures in the kappa-carrageenan supporting bath, cells and cells spread and interconnect to form a network after 7 days of culture, demonstrating that MC-3T3 cells exhibit good activity.

In summary, the kappa-carrageenan supporting bath is biocompatible.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.

Claims

1. The application of carrageenan in biological 3D prints, biological 3D prints including biological ink and support bath, its characterized in that: the use of a combination of carrageenan and GelMA solution in a bio-ink, and the use of a combination of carrageenan and phosphate buffer in a supporting bath.

2. Use of carrageenan according to claim 1 in biological 3D printing, characterized in that: the concentration of carrageenan in the application of the bio-ink is one of 0.1% w/v, 0.3% w/v and 0.5% w/v.

3. Use of carrageenan according to claim 1 in biological 3D printing, characterized in that: the concentration of carrageenan in the supporting bath application is one of 0.3% w/v, 0.4% w/v and 0.5% w/v.

4. Use of carrageenan according to claim 1 in biological 3D printing, characterized in that: the carrageenan is one of kappa-carrageenan, iota-carrageenan and lambda-carrageenan.