Bio-printing ink, biological material three-dimensional scaffold and preparation method

Abstract

The invention provides biological printing ink, a biological material three-dimensional scaffold and a preparation method. The preparation method comprises the following steps: synthesizing a hydrogel particle precursor; performing freeze drying on the hydrogel particle precursor to obtain hydrogel dry powder; mixing the cell suspension carrier with the cell suspension to form a gap gel; and stirring and dispersing the gap glue and the hydrogel dry powder to obtain the bio-printing ink. The biological printing ink provided by the invention is a biological material which is good in compatibility and easy to print, tissues or organs of a human body or an animal can be constructed in vitro according to needs, and a three-dimensional support material beneficial to cell growth is provided. The material can be used for accurately printing high-density cells, effectively controlling the spatial distribution of the cells in a three-dimensional scaffold (cell sedimentation is avoided), improving the adhesion rate of the cells on the three-dimensional scaffold, and solving the problems of formability and biocompatibility of cell printing.

Description

Technical Field

[0001] The present application relates to the field of biomedical engineering technology, and in particular to a biological printing ink, a three-dimensional biological material scaffold and a preparation method thereof. Background Art

[0002] Three-dimensional additive manufacturing technology has become the preferred solution for constructing whole organs for therapeutic purposes. For the construction of large-scale biomimetic biological tissues in vitro, extrusion bioprinting is considered to be the most promising bioprinting technology due to its ability to quickly switch multiple components. Since this technology usually requires the extrusion of biological ink that can carry living cells from the printing needle to directly and directional construct complex tissue structures, in order to meet the growth environment required by cells, biological inks are mostly biologically related extracts, such as gelatin extracted from pig skin, alginate and hyaluronic acid extracted from brown algae, etc. Most of these materials belong to the hydrogel class, and their excellent water content and low structural strength make them extremely conducive to cell growth and development. The methods for completing the printing of such materials can be roughly divided into two categories: pre-crosslinking method and post-crosslinking method. As the name suggests, the pre-crosslinking method refers to the process of physically crosslinking the material in the barrel to form a gel state, and then using the shear-to-thinning property of the material to make the material undergo high shear stress at the needle and transform from the gel state to the liquid state, and then lose stress after being extruded from the needle and passively transform into the gel state to complete the extrusion and shaping process. The post-crosslinking method generally makes the material into a viscous fluid first, and then quickly crosslinks and solidifies it at the needle or receiving table after it is extruded from the needle. For example, light-curable gelatin (methacrylamide gelatin) can be heated to 30°C to make it fluid in the barrel and then extruded. By installing a UV lamp at the needle mouth, the extruded material is solidified into a line at the needle mouth and falls on the receiving plane. A frozen receiving platform can also be used to instantly cool the extruded liquid gelatin to gel (physical crosslinking) on ​​the receiving table and then complete the overall curing by ultraviolet irradiation. In addition, the post-crosslinking method can also make the hydrogel into droplets by microfluidics or emulsification methods and then chemically crosslink it into hydrogel balls. The hydrogel balls are purified and concentrated by filtration and washing, and then mixed with the cell suspension to form a granular ink. The capillary bridge force formed by the liquid between the particles makes the granular ink have a certain self-supporting performance. With good support performance, the curing can be completed uniformly after the structure is printed.

[0003] For pre-crosslinking technology, the barrel is not heated, and the gel-like material is directly extruded by utilizing the shear-to-thinning property of the material. In addition to the requirements for the performance of the material itself, the higher pressure required for extrusion will produce higher wall shear force at the narrow part of the needle, seriously reducing the viability of cells. For most hydrogel materials, when they have strong shear-to-thinning properties, their storage energy / elastic modulus are at a low level. The result is that as the height of the printed structure increases, the pressure on the bottom structure also increases. Since most biomaterials provide a low storage energy modulus (G') under mild physical crosslinking conditions, the bottom structure deforms and collapses, which in turn causes the upper structure to be dislocated and the needle movement to mismatch. When facing models with a print height of more than 2 cm, they often end up toppling or collapsing.

[0004] For post-crosslinking technology, heating the barrel to reduce the viscosity and storage modulus of the ink will cause the problem of cell sedimentation in the barrel. Long-term printing will cause uneven cell distribution (dense in the front and sparse in the back). This greatly reduces the controllability of 3D printing of large-scale structures. Not only that, take the most common gelatin as an example. Because it has the characteristics of heating, liquefaction, cooling and solidification, when the heated barrel material is printed on the freezing table after it is in liquid state, the distance between the printing layer and the receiving platform increases with the increase in the number of layers. This leads to a decrease in the cooling effect. Even if an embedded freezing structure such as a freezing tank is used, it will increase the cost and cannot solve the problem of poor cooling effect on the vertical center axis of the printed object. As for adding a light-curing module to the needle, in addition to the problem of cell sedimentation, the extrusion process is prone to the phenomenon of ink cross-linking in the needle causing needle clogging and the problem of poor structural properties that cannot be cross-linked as a whole cannot be solved.

[0005] For inks based on wet hydrogel balls, their self-supporting properties are often determined by the size of the gaps between the particles. Usually, the smaller the distance between the particles, the greater the capillary bridge force that can be provided, and the greater the viscosity of the ink. However, reducing the distance between the particles will often further squeeze the living space of the cells and reduce the viability of the print. Therefore, in the results presented in the current literature reports, the method of concentrating by filtration and then mixing with the cell suspension has a very low number of printing layers, and often collapses after about 4 layers.

[0006] Therefore, due to the limitations of current biomaterials, it is difficult to achieve both printing formability and biocompatibility (or cell viability). Summary of the invention

[0007] In view of this, it is necessary to provide a bioprinting ink, a biomaterial three-dimensional scaffold and a preparation method that have good compatibility and are easy to print in order to address the defects of existing printing that are difficult to achieve both formability and biocompatibility (or cell viability).

[0008] To solve the above problems, this application adopts the following technical solutions:

[0009] One of the purposes of this application is to provide a method for preparing a bioprinting ink, comprising the following steps:

[0010] Synthesizing hydrogel particle precursors;

[0011] freeze-drying the hydrogel particle precursor to obtain a hydrogel dry powder;

[0012] mixing the cell suspension carrier with the cell suspension to form a gap glue;

[0013] The gap glue and the hydrogel dry powder are stirred and dispersed to obtain the bioprinting ink.

[0014] In some embodiments, the step of synthesizing a hydrogel particle precursor specifically includes the following steps:

[0015] Mixing and stirring liquid hydrogel particles with a photoinitiator to obtain a hydrogel precursor solution, wherein the hydrogel particles are dry powdered gelatin spheres with a porous structure, and the hydrogel particles include gelatin-based modified hydrogels, hyaluronic acid-based modified hydrogels, alginate-based modified hydrogels, or polyvinyl alcohol-based modified hydrogels;

[0016] Mixing and stirring the hydrogel precursor solution and silicone oil to form an emulsion, wherein the silicone oil contains an emulsifier;

[0017] The hydrogel particle precursor is obtained by curing the emulsion. The hydrogel particles in the hydrogel particle precursor are composed of double-bond modified chemically cross-linked gelatin particles, and the mass fraction of the chemically cross-linked gelatin particles is 2%-30%.

[0018] In some embodiments, the mass fraction of the photoinitiator is 0.1%-3% of the mass fraction of the hydrogel particles, the photoinitiator includes a blue light initiator, and the emulsifier includes SPAN 80 and TWEEN 20.

[0019] In some embodiments, the step of freeze-drying the hydrogel particle precursor to obtain a hydrogel dry powder specifically includes the following steps:

[0020] The hydrogel particle precursor is cleaned and suspended in an aqueous solution and then placed on a magnetic stirrer for stirring. It is then rapidly frozen and fixed with liquid nitrogen and freeze-dried to obtain the hydrogel dry powder, which is in a granular form.

[0021] In some embodiments, the step of mixing the cell suspension carrier with the cell suspension to form the gap glue specifically includes the following steps:

[0022] After the cell suspension carrier is dissolved in phosphate buffer or culture medium, a photoinitiator is added, and then stirred evenly and mixed with the cell suspension to form a gap glue. The concentration range of the cell suspension carrier in the gap glue is between 0.5% and 30%. The cell suspension carrier includes gelatin, hyaluronic acid, collagen, chitosan or a mixture of several thereof and their modified derivatives.

[0023] In some embodiments, the step of stirring and dispersing the gap glue and the hydrogel dry powder to obtain the bioprinting ink specifically includes the following steps:

[0024] The gap glue is added into the hydrogel dry powder with a mass fraction of 5%-40%, and stirred and dispersed to obtain the bioprinting ink.

[0025] The second purpose of the present application is to provide a bio-printing ink prepared by any of the methods for preparing the bio-printing ink described above.

[0026] The third purpose of the present application is to provide a biological three-dimensional printing method using the biological printing ink, including: using multi-channel three-dimensional printing, each channel carries the biological printing ink, and can print a biological material three-dimensional scaffold according to a pre-printed model.

[0027] In some of these embodiments, the bioprinting ink can carry different cell suspensions.

[0028] In some of the embodiments, in the step of using multi-channel three-dimensional printing, each channel carries the bioprinting ink, and can print a multi-cell distributed tissue structure according to the pre-printed model, specifically including the following steps:

[0029] Correcting the position of each channel so that the bottoms of all gun tips connected to the channel are on the same horizontal line;

[0030] Set up the printing process based on the pre-printed model;

[0031] A three-dimensional biomaterial scaffold is printed according to the printing process; wherein, during the printing process, the printing substrate is kept stationary, and each time a layer of cell-biomaterial composite scaffold structure is printed, all the gun tips are moved upward and the bottoms of the gun tips are kept at the same horizontal line.

[0032] In some of the embodiments, the channels are provided with material supply cavities, and the gun heads can move relatively up, down, left and right.

[0033] In some embodiments, the step of setting the printing process according to the pre-printed model specifically includes the following steps:

[0034] According to the pre-printed model, the three-dimensional space occupied by different cells is marked with different colors in the three-dimensional modeling software and imported into the software that comes with the printer to automatically generate the G code required for different barrels.

[0035] In some embodiments, during the printing process, the printing substrate is kept stationary, and each time a layer of the cell-biomaterial composite scaffold structure is printed, all the gun tips are moved upward and the bottoms of the gun tips are kept at the same horizontal line, specifically including the following steps:

[0036] During the printing process, the printing substrate is kept stationary, and the first gun tip to discharge material is used as a reference, and the positions of other gun tips on the printing platform are adjusted so that the positions of the other gun tips are moved up and the bottoms of all the gun tips are kept on the same horizontal line.

[0037] In some of the embodiments, the biomaterial three-dimensional scaffold may be a single component or may be composed of multiple channels containing the same material in different ratios or may be composed of multiple channels of different materials to print out a cell-biomaterial three-dimensional composite scaffold composed of different layers of materials.

[0038] The fourth purpose of the present application is to provide a three-dimensional biomaterial scaffold, which is printed by the biological three-dimensional printing method.

[0039] This application adopts the above technical solution, and its beneficial effects are as follows:

[0040] The bioprinting ink, biomaterial three-dimensional scaffold and preparation method provided by the present application are synthesized hydrogel particle precursor; the hydrogel particle precursor is freeze-dried to obtain hydrogel dry powder; the cell suspension carrier is mixed with the cell suspension to form a gap glue; the gap glue and the hydrogel dry powder are stirred and dispersed to obtain the bioprinting ink. The hydrogel particles provided by the present application are in the form of a porous dry powder. The dry powder particles absorb water and the polymer chains between the particles to form a chain ball structure. This structure is physically cross-linked and provides good elasticity (self-supporting performance) in a static state. This physical structure can be opened by applying a small shear force to provide good fluidity (shear-to-thinning performance); and the hydrogel porous structure is directly utilized. After the hydrogel ball is formed, it is rapidly frozen so that the water between the hydrogel molecular chains quickly freezes into ice crystals, and then freeze-dried to extract the ice crystals to form a porous structure without introducing any organic reagents, and has good biocompatibility; the prepared particle ink (before molding) has good tensile fracture performance, so that it will not tail at the needle tip, so it will not cause excess line accumulation at the starting point of the next line, and the self-supporting, shear-thinning and self-repairing properties of the biological ink make it unable to collapse or tilt when printing large-sized or high-height structures; not only that, the granular ink can evenly wrap the cells into the gaps of the particles without sedimentation, and at the same time has a lower extrusion pressure that will not affect the viability of the cells.

[0041] The bioprinting ink provided by the present application is a biomaterial with good compatibility and easy printing operation, which can construct human or animal tissues or organs in vitro as needed, and provide a three-dimensional support material that is conducive to cell growth. The material can accurately print high-density cells, effectively control the spatial distribution of cells in the three-dimensional scaffold (no cell sedimentation occurs), increase the adhesion rate of cells on the three-dimensional scaffold, and solve the problems of formability and biocompatibility of cell printing. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 A flowchart of the steps of a method for preparing a biological printing ink provided in one embodiment of the present invention;

[0044] Figure 2 A flow chart of the steps of synthesizing a hydrogel particle precursor provided in one embodiment of the present application;

[0045] Figure 3A physical picture of a granular hydrogel dry powder provided in one embodiment of the present application;

[0046] Figure 4 A schematic diagram of the principle of a granular dry powder biological ink provided in one embodiment of the present application;

[0047] Figure 5 A schematic diagram of the rheology of a granular dry powder biological ink provided in one embodiment of the present application;

[0048] Figure 6 A schematic diagram of the recovery performance of a granular dry powder biological ink after extrusion provided in one embodiment of the present application;

[0049] Figure 7 A physical picture of the printing performance of a granular dry powder biological ink provided in one embodiment of the present application;

[0050] Figure 8 A schematic diagram of the principle verification of the granular dry powder biological ink provided in one embodiment of the present application;

[0051] Fig. 9 A schematic diagram of the printing viability and biocompatibility of a granular dry powder bio-ink provided in one embodiment of the present application. DETAILED DESCRIPTION

[0052] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0053] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0054] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0055] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0056] See also Figure 1 , a flow chart of the steps of the method for preparing the biological printing ink provided in an embodiment of the present application includes the following steps S110 to S140, and the implementation method of each step is described in detail below.

[0057] Step S110: synthesizing hydrogel particle precursors.

[0058] See also Figure 2 In the step of synthesizing the hydrogel particle precursor, the following steps S111 to S113 are specifically included, and the implementation method of each step is described in detail below.

[0059] Step S111: mixing liquid hydrogel particles with a photoinitiator to obtain a hydrogel precursor solution, wherein the hydrogel particles are dry powdered gelatin balls with a porous structure.

[0060] In this embodiment, the dispersed phase used is a porous structure of dry powdered gelatin spheres with hydrogel particles. The main component of the porous powder of hydrogel particles can be other macromolecules, such as gelatin modification, hyaluronic acid modification, alginate modification, polyvinyl alcohol modification or a mixture of several macromolecules.

[0061] It can be understood that in practice, the hydrogel particles are added into water, heated at 40° C.-60° C. to dissolve into liquid state, stirred evenly, and then a proper amount of photoinitiator is added to obtain a hydrogel precursor solution.

[0062] Furthermore, the mass fraction of the photoinitiator is 0.1%-3% of the mass fraction of the hydrogel particles, and the photoinitiator includes a blue light initiator.

[0063] It should be noted that the hydrogel particles of this embodiment are in the form of dry powder with a porous structure. The dry powder particles absorb water and the polymer chains between the particles form a chain ball structure. This structure is physically cross-linked, providing good elasticity (self-supporting performance) when static. By applying a small shear force, this physical structure can be opened to provide good fluidity (shear-to-thinning performance). And because the cross-linking method of the microspheres is photocross-linking, this method is mild and has little damage to cells, and other polymerization methods can also be used.

[0064] Step S112: mixing and stirring the hydrogel precursor solution and silicone oil to form an emulsion, wherein the silicone oil contains an emulsifier.

[0065] In this embodiment, the emulsifiers include SPAN 80 and TWEEN 20.

[0066] It can be understood that the silicone oil contains an emulsifier, has good biological inertness, and will not affect cells.

[0067] Step S113: curing the emulsion to obtain the hydrogel particle precursor, wherein the hydrogel particles in the hydrogel particle precursor are composed of double-bond modified chemically cross-linked gelatin particles, and the mass fraction of the chemically cross-linked gelatin particles is 2%-30%.

[0068] Specifically, the curing treatment used in this embodiment is ultraviolet light curing, which is mild and causes little damage to cells. Other polymerization methods can also be used, such as thermal polymerization, ionic crosslinking, and chemical crosslinking.

[0069] Step S120: freeze-drying the hydrogel particle precursor to obtain hydrogel dry powder.

[0070] Specifically, the hydrogel particle precursor is cleaned and suspended in an aqueous solution and then stirred on a magnetic stirrer, and then rapidly frozen and fixed with liquid nitrogen and freeze-dried to obtain the hydrogel dry powder, which is in granular form. Figure 3 , is a physical picture of the granular hydrogel dry powder provided in this embodiment.

[0071] It can be understood that the hydrogel porous structure is directly utilized. After the hydrogel ball is formed, it is rapidly frozen with liquid nitrogen to make the water between the hydrogel molecular chains quickly form ice crystals, and then freeze-dried to extract the ice crystals to form porous. This method does not introduce any organic reagents and has good biocompatibility.

[0072] S130: mixing the cell suspension carrier with the cell suspension to form a gap glue.

[0073] In this embodiment, the step of mixing the cell suspension carrier with the cell suspension to form a gap glue specifically includes the following steps: dissolving the cell suspension carrier in a phosphate buffer or a culture medium, adding a photoinitiator, and then stirring evenly and mixing with the cell suspension to form a gap glue, wherein the concentration range of the cell suspension carrier in the gap glue is between 0.5% and 30%, and the cell suspension carrier includes one of hyaluronic acid, collagen, chitosan or a mixture of several thereof, and the hyaluronic acid includes methacrylamide gelatin.

[0074] Step S140: stirring and dispersing the gap glue and the hydrogel dry powder to obtain the bioprinting ink.

[0075] In this embodiment, the step of stirring and dispersing the gap glue and the hydrogel dry powder to obtain the bioprinting ink specifically includes the following steps: adding the gap glue to the hydrogel dry powder with a mass fraction of 5%-40% and stirring and dispersing to obtain the bioprinting ink.

[0076] Specifically, the gap glue is added into the freeze-dried block dry powder aggregate with a mass fraction of 5%-40% by a pipette and stirred until uniformly dispersed to obtain the bioprinting ink.

[0077] Furthermore, the cell density of the gap glue used in this embodiment is as high as 2*10^6 / ml, which is related to the cell type and can also be adjusted according to the bionic tissue similarity of the scaffold. The cells used in this embodiment can be osteoblasts, stem cells, osteosarcoma cells and other cells.

[0078] It is understood that in subsequent printing, the ink can be transferred to the printing barrel and used after centrifugation. When the granular dry powder meets the gap glue, it absorbs the water in the glue, but cannot absorb the long chain of gelatin. The gelatin chain is adsorbed on the surface of the gelatin ball to form a chain ball model ( Figure 4 ). Due to this physical cross-linking method, the chains are pulled and released under high shear force, and are re-adsorbed back to the ball surface under low shear.

[0079] Furthermore, rheological tests have shown that granular dry powder materials can provide higher initial viscosity (viscosity at low shear rate) and similar post-shear viscosity (viscosity at high shear rate) under the same solid content conditions as homogenized hydrogels ( Figure 5 This indicates that the granular dry powder bio-ink has better shear-thinning properties and is more conducive to material extrusion. Moreover, by testing the storage modulus (G', representing material elasticity) and loss modulus (G", representing material viscosity) of the material under different strains, it was found that the granular dry powder bio-ink has better elasticity than ordinary hydrogels, confirming that the material has better self-supporting properties after extrusion ( Figure 5 In the variable temperature test, the granular dry powder ink can maintain the gel state in a wider printing range, which greatly increases the printing window period ( Figure 5 In the cyclic oscillation test, compared with the homogeneous hydrogel, which will cause unstable modulus after many oscillations, the granular dry powder ink can complete rapid recovery and constant modulus value in more oscillation cycles, making its lines more stable in long-term large-scale printing ( Figure 5 Finally, in the shear recovery experiment, the granular dry powder ink achieved a faster viscosity recovery, which means that the extruded lines quickly recovered high viscosity and set more quickly, resulting in higher line fidelity ( Figure 6 ).

[0080] The hydrogel particles provided in the above embodiments of the present application are in the form of dry powder with a porous structure. The dry powder particles absorb water and the polymer chains between the particles to form a chain ball structure. This structure is physically cross-linked and provides good elasticity (self-supporting performance) when static. This physical structure can be opened by applying a small shear force to provide good fluidity (shear-to-thinning performance); and the hydrogel porous structure is directly used to rapidly freeze the hydrogel balls after forming them so that the water between the hydrogel molecular chains quickly forms ice crystals, and then freeze-drying is performed to extract the ice crystals to form a porous structure without introducing any organic reagents, and has good biocompatibility; the prepared particle ink (before molding) has good tensile fracture performance, so that it will not tail at the needle, so it will not cause excess line accumulation at the starting point of the next line, and the self-supporting, shear-thinning and self-repairing properties of the biological ink make it not collapse or tilt when printing large-size or high-height structures; not only that, the granular ink can evenly wrap the cells into the gaps of the particles without sedimentation, and at the same time has a low extrusion pressure that will not affect the viability of the cells.

[0081] In addition, the bioprinting ink provided by the present application has a biomaterial with good compatibility and easy printing operation, which can construct human or animal tissues or organs in vitro as needed, and provide a three-dimensional support material that is conducive to cell growth. The material can accurately print high-density cells, effectively control the spatial distribution of cells in the three-dimensional scaffold (no cell sedimentation occurs), increase the adhesion rate of cells on the three-dimensional scaffold, and solve the problems of cell printing formability and biocompatibility.

[0082] The present application also provides a biological three-dimensional printing method using the biological printing ink, comprising: using multi-channel three-dimensional printing, each channel carrying the biological printing ink, and being able to print out a biological material three-dimensional scaffold according to a pre-printed model.

[0083] In this embodiment, the bioprinting ink can carry different cell suspensions.

[0084] It can be understood that in actual operation, each channel is responsible for printing ink carrying different cell suspensions, and filling and printing are performed according to the distribution of corresponding colors in the model; if a filling structure without cells is encountered, it can be printed without adding cell suspension; if a suspended structure is encountered, commonly used support materials (such as nanoclay, F127) can be used to print in the suspended area, and then sacrificed after printing is completed, or this ink without photoinitiator and cell suspension can be used for printing, which can be dissolved in water after printing.

[0085] Furthermore, in the step of adopting multi-channel three-dimensional printing, each channel carries the bioprinting ink, and printing a multi-cell distributed tissue structure according to the pre-printed model, the following steps are specifically included:

[0086] Step S210: Correcting the position of each channel so that the bottoms of all gun tips connected to the channel are on the same horizontal line.

[0087] In this embodiment, the channels are all provided with a material supply cavity, and the gun head can move up, down, left and right relatively.

[0088] Step S220: setting a printing process according to the pre-printing model.

[0089] In this embodiment, according to the pre-printed model, the three-dimensional space occupied by different cells is marked with different colors in the three-dimensional modeling software and imported into the software provided by the printer to automatically generate the G codes required for different barrels.

[0090] Step S230: Printing a three-dimensional biomaterial scaffold according to the printing process; wherein, during the printing process, the printing substrate is kept stationary, and each time a layer of cell-biomaterial composite scaffold structure is printed, all the gun tips are moved up and the bottoms of the gun tips are kept at the same horizontal line.

[0091] In this embodiment, the biomaterial three-dimensional scaffold can be a single component or a cell-biomaterial three-dimensional composite scaffold composed of materials of different levels printed out from multiple channels containing the same material in different proportions or multiple channels containing different materials.

[0092] It can be understood that the shape of the cell-biomaterial composite three-dimensional scaffold is related to the program design. The program can be designed according to different needs to print out three-dimensional cell scaffolds with different morphologies.

[0093] The biological three-dimensional printing method provided in the above embodiments of the present application can construct human or animal tissues or organs in vitro, can accurately control the distribution of cells in the three-dimensional scaffold, and the printed cells can have a very high concentration and can contact each other, effectively solving the problems of low cell adhesion rate on the three-dimensional scaffold and slow growth into the scaffold.

[0094] The above technical solution of the present application is described in detail below in conjunction with specific embodiments.

[0095] Embodiment 1:

[0096] A. Gelatin-based hydrogel is used as the main structure of the cell-biomaterial composite bio-ink. The components of the particle hydrogel precursor are 10% by mass fraction of double-bond modified gelatin macromolecules, 0.5% by mass fraction of ultraviolet light initiator, and the remaining components are water.

[0097] B. Double-bond modified gelatin liquid is used as the carrier of cell suspension, and the mass fraction of gelatin is 5%.

[0098] C. Add 5 ml of 5% gap glue into 10% (0.5 g) of freeze-dried block dry powder aggregates with a pipette and stir until evenly dispersed.

[0099] D. Use single-channel and multi-channel 3D printing methods.

[0100] E. Single-channel printing of large-size bones (and comparison with pure gelatin ink printing), the scaffold size is a bone with an outer contour length, width and height of 24mm×18mm×40mm.

[0101] F. Double-channel printing layered structure, the size of the bracket is a cube with a length, width and height of 10mm×10mm×15mm (lower layer is blue, upper layer is red) ( Figure 7 ).

[0102] G. After the scaffold is printed, it can be placed in a UV cross-linking instrument for light curing for 120 seconds to make the gel plastic.

[0103] See also Figure 7 , which is a physical display diagram of the printing performance of the granular dry powder biological ink provided in this embodiment. After the cell-biomaterial composite scaffold is printed, it can be placed in a UV cross-linking instrument for photocuring to make the gel plastic.

[0104] Embodiment 2:

[0105] A. Gelatin-based hydrogel is used as the main structure of the cell-biomaterial composite bio-ink. The components of the particle hydrogel precursor are 10% by mass fraction of double-bond modified gelatin macromolecules, 0.5% by mass fraction of ultraviolet light initiator, and the remaining components are water.

[0106] B. Use double-bond modified gelatin liquid as gap glue with a mass fraction of 1%, 5% or 10%.

[0107] C. 3 ml of gap glue with a mass fraction of 1% was divided into three equal parts of 1 ml each, and added to the freeze-dried block dry powder aggregates with a mass fraction of 1%, 5% or 10% (0.01, 0.05 or 0.1 g) by a pipette and stirred until uniformly dispersed; 3 ml of gap glue with a mass fraction of 5% was divided into three equal parts of 1 ml each, and added to the freeze-dried block dry powder aggregates with a mass fraction of 1%, 5% or 10% (0.01, 0.05 or 0.1 g) by a pipette and stirred until uniformly dispersed; 3 ml of gap glue with a mass fraction of 10% was divided into three equal parts of 1 ml each, and added to the freeze-dried block dry powder aggregates with a mass fraction of 1%, 5% or 10% (0.01, 0.05 or 0.1 g) by a pipette and stirred until uniformly dispersed. A total of 9 groups of samples were obtained.

[0108] D. Add 9 groups of samples to the well plate, add 5 ml of water around the samples to immerse the samples. Use a camera to take initial photos of the samples.

[0109] E. Then place it on a shaker (80 r / min, 37°C) and collect sample images at 1 h, 4 h, 12 h and 24 h.

[0110] F. Analysis and comparison proved that the group containing gap gel and higher gelatin concentration had a more stable overall structure (derived from porous adsorption).

[0111] See also Figure 8 , Schematic diagram of the viability and biocompatibility of granular dry powder bio-ink printing. After the cell-biomaterial composite scaffold is printed, culture medium is added and placed in a 37°C incubator for culture, so that the cells wrapped in the gaps of the granular dry powder ink slowly spread and adhere to the inside and surface of the scaffold lines, thus obtaining a true cell-biomaterial three-dimensional composite scaffold.

[0112] Embodiment 3:

[0113] A. Gelatin-based hydrogel is used as the main structure of the cell-biomaterial composite bio-ink. The components of the particle hydrogel precursor are 10% by mass fraction of double-bond modified gelatin macromolecules, 0.5% by mass fraction of ultraviolet light initiator, and the remaining components are water.

[0114] B. Double-bond modified gelatin liquid is used as the carrier of cell suspension, and the mass fraction of gelatin is 5% or 10%.

[0115] C. The cell concentration of the cell suspension is 2*10^6 / ml.

[0116] D. Use a pipette to add 5 ml of 5% or 10% gap glue to 10% (0.5 g) or 5% (0.25 g) freeze-dried block dry powder aggregates and stir until evenly dispersed (the solid content of the two groups of experiments is consistent).

[0117] E. Use single-channel 3D printing method.

[0118] F. Single-channel printing of cell-biomaterial composite scaffolds, the size of the scaffold is length and width

[0119] 10mm×10mm×4mm cross grid bracket ( Figure 7 ).

[0120] G. After the cell-biomaterial composite scaffold is printed, it can be placed in a UV cross-linking instrument for light curing for 120 seconds to make the gel plastic.

[0121] F. Finally, the three-dimensional composite scaffold was taken out, α-MEM culture medium was added, and it was placed in a 37°C incubator for culture to increase the number of cells in the ink, thus obtaining a true cell-biomaterial three-dimensional composite scaffold ( Fig. 9 ).

[0122] It can be understood that the technical features of the above-described embodiments can be arbitrarily combined. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0123] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

1. A method for preparing a bioprinting ink, It is characterized in that The steps include: Synthesizing hydrogel particle precursors; freeze-drying the hydrogel particle precursor to obtain a hydrogel dry powder; mixing the cell suspension carrier with the cell suspension to form a gap glue; The gap glue and the hydrogel dry powder are stirred and dispersed to obtain the bioprinting ink.

2. The method for preparing the bioprinting ink according to claim 1, It is characterized in that The step of synthesizing the hydrogel particle precursor specifically includes the following steps: Mixing and stirring liquid hydrogel particles with a photoinitiator to obtain a hydrogel precursor solution, wherein the hydrogel particles are dry powdered gelatin spheres with a porous structure, and the hydrogel particles include gelatin-based modified hydrogels, hyaluronic acid-based modified hydrogels, alginate-based modified hydrogels, or polyvinyl alcohol-based modified hydrogels; Mixing and stirring the hydrogel precursor solution and silicone oil to form an emulsion, wherein the silicone oil contains an emulsifier; The hydrogel particle precursor is obtained by curing the emulsion. The hydrogel particles in the hydrogel particle precursor are composed of double-bond modified chemically cross-linked gelatin particles, and the mass fraction of the chemically cross-linked gelatin particles is 2%-30%.

3. The method for preparing the bioprinting ink according to claim 2, It is characterized in that The mass fraction of the photoinitiator is 0.1%-3% of the mass fraction of the hydrogel particles. The photoinitiator includes a blue light initiator. The emulsifier includes SPAN 80 and TWEEN 20.

4. The method for preparing the bioprinting ink according to claim 1, It is characterized in that The step of freeze-drying the hydrogel particle precursor to obtain a hydrogel dry powder specifically includes the following steps: The hydrogel particle precursor is cleaned and suspended in an aqueous solution and then placed on a magnetic stirrer for stirring. It is then rapidly frozen and fixed with liquid nitrogen and freeze-dried to obtain the hydrogel dry powder, which is in a granular form.

5. The method for preparing the bioprinting ink according to claim 1, It is characterized in that The step of mixing the cell suspension carrier with the cell suspension to form the gap glue specifically includes the following steps: After the cell suspension carrier is dissolved in phosphate buffer or culture medium, a photoinitiator is added, and then stirred evenly and mixed with the cell suspension to form a gap glue. The concentration range of the cell suspension carrier in the gap glue is between 0.5% and 30%. The cell suspension carrier includes gelatin, hyaluronic acid, collagen, chitosan or a mixture of several thereof and their modified derivatives.

6. The method for preparing the bioprinting ink according to claim 1, It is characterized in that The step of stirring and dispersing the gap glue and the hydrogel dry powder to obtain the bioprinting ink specifically includes the following steps: The gap glue is added into the hydrogel dry powder with a mass fraction of 5%-40%, and stirred and dispersed to obtain the bioprinting ink.

7. A bioprinting ink, It is characterized in that The bioprinting ink is prepared by the preparation method of any one of claims 1 to 6.

8. A biological three-dimensional printing method based on the biological printing ink according to claim 1 or 7, It is characterized in that include: Multi-channel three-dimensional printing is adopted, each channel carries the bio-printing ink, and a three-dimensional scaffold of biomaterial can be printed according to a pre-printed model.

9. The biological three-dimensional printing method according to claim 8, It is characterized in that The bioprinting ink can carry different cell suspensions.

10. The biological three-dimensional printing method according to claim 8, It is characterized in that In the step of adopting multi-channel three-dimensional printing, each channel carries the bioprinting ink, and can print a multi-cell distributed tissue structure according to the pre-printed model, the following steps are specifically included: Correcting the position of each channel so that the bottoms of all gun tips connected to the channel are on the same horizontal line; Set up the printing process based on the pre-printed model; A three-dimensional biomaterial scaffold is printed according to the printing process; wherein, during the printing process, the printing substrate is kept stationary, and each time a layer of cell-biomaterial composite scaffold structure is printed, all the gun tips are moved upward and the bottoms of the gun tips are kept at the same horizontal line.

11. The biological three-dimensional printing method according to claim 10, It is characterized in that The channels are all provided with material supply cavities, and the gun heads can move relatively up, down, left and right.

12. The biological three-dimensional printing method according to claim 10, It is characterized in that In the step of setting up the printing process according to the pre-printed model, the following steps are specifically included: According to the pre-printed model, the three-dimensional space occupied by different cells is marked with different colors in the three-dimensional modeling software and imported into the software that comes with the printer to automatically generate the G code required for different barrels.

13. The biological three-dimensional printing method according to claim 10, It is characterized in that During the printing process, the printing substrate is kept stationary, and each time a layer of the cell-biomaterial composite scaffold structure is printed, all the gun tips are moved upward and the bottoms of the gun tips are kept at the same horizontal line, specifically including the following steps: During the printing process, the printing substrate is kept stationary, and the first gun tip to discharge material is used as a reference, and the positions of other gun tips on the printing platform are adjusted so that the positions of the other gun tips are moved up and the bottoms of all the gun tips are kept on the same horizontal line.

14. The biological three-dimensional printing method according to claim 10, It is characterized in that The biomaterial three-dimensional scaffold can be a single component or a cell-biomaterial three-dimensional composite scaffold composed of materials of different levels printed out from multiple channels containing the same material in different proportions or from multiple channels containing different materials.

15. A three-dimensional biomaterial scaffold, It is characterized in that Printed by the biological three-dimensional printing method described in claim 8.

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