WO2020103918A1 - 用于3d打印的浆料、3d结构体及其制备方法和应用 - Google Patents

用于3d打印的浆料、3d结构体及其制备方法和应用

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Publication number
WO2020103918A1
WO2020103918A1 PCT/CN2019/120103 CN2019120103W WO2020103918A1 WO 2020103918 A1 WO2020103918 A1 WO 2020103918A1 CN 2019120103 W CN2019120103 W CN 2019120103W WO 2020103918 A1 WO2020103918 A1 WO 2020103918A1
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polylysine
printing
alginate
paste
slurry
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PCT/CN2019/120103
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English (en)
French (fr)
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阮长顺
林子锋
吴明明
潘浩波
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深圳先进技术研究院
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Publication of WO2020103918A1 publication Critical patent/WO2020103918A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing

Definitions

  • the present application belongs to the technical field of three-dimensional bioprinting, and particularly relates to a slurry used for 3D printing, a 3D structure, and a preparation method and application thereof.
  • Three-dimensional (3D) bioprinting technology is derived from the three-dimensional printing technology.
  • the printing technology that loads biological cells and growth factors while printing biomaterials is very important in preparing artificial organs and tissues and drug screening models. potential.
  • Biological ink material used for three-dimensional bioprinting determines the physical and chemical properties of three-dimensional bioprinting products, and also affects the survival rate of cells and the activity of growth factors.
  • Alginate sodium
  • Alginate has shear thinning properties (the viscosity of the ink is reduced due to shear force), as well as its excellent biocompatibility and can achieve rapid ionic cross-linking, and is widely used in the construction of cell capsules and biological inks.
  • alginate scaffolds after cross-linking with calcium ions are poor. Under physiological environment, calcium ions are easily replaced with surrounding monovalent ions such as sodium ions, resulting in collapse of the scaffold.
  • alginate has excellent biocompatibility and can achieve the survival of entrapped cells, alginate has no biological activity, and the cells adhere and spread poorly on alginate scaffolds, and cannot achieve controllable growth factors. It does not have the ability to induce the differentiation of stem cells.
  • One of the purposes of the present application is to provide a paste for 3D printing, which aims to solve at least some of the above technical problems in the prior art.
  • the second object of the present application is to provide a method for preparing the above paste for 3D printing, which is simple and easy to obtain.
  • the third object of the present application is to provide a 3D structure body, which is prepared by using the slurry for the 3D printing or the slurry preparation method for the 3D printing, the 3D structure is easy to form, More stable, while improving the biological inertness of alginate.
  • the fourth purpose of the present application is to provide a method for preparing the above 3D structure, which is simple in operation and does not require a complicated chemical synthesis process.
  • the 3D structure after further chemical crosslinking has good stability and can be used for a long time Maintain its integrity.
  • the fifth object of the present application is to provide an application of the 3D structure obtained by the above 3D structure or the preparation method of the 3D structure in tissue engineering.
  • a paste for 3D printing the paste includes alginate, polylysine and water, wherein the mass of alginate is 20-40% of the mass of water, alginic acid
  • the molar ratio of the carboxyl group of the salt to the amino group of polylysine is 1: 0.1-2.
  • the technical solutions adopted in the examples of the present application further include: on the basis of the technical solutions of the present application, the molar ratio of the carboxyl group of alginate and the amino group of polylysine is 1: 0.5-1, preferably 1: 1.
  • the technical solutions adopted in the embodiments of the present application further include: on the basis of the technical solutions of the present application, the weight average molecular weight of polylysine is 750-5000, preferably 1000-4200;
  • the melting point of polylysine is 170-175 ° C, preferably 172-175 ° C.
  • the technical solution adopted in the embodiments of the present application further includes: on the basis of the technical solution of the present application, the slurry further includes an effective biological component, and the effective biological component includes nutrients, extracellular matrix, growth factors, cells or pharmaceutical active ingredients One or more.
  • a method for preparing a paste for 3D printing including the following steps:
  • the technical solution adopted in the embodiments of the present application further includes: adding alginate to the solution of polylysine and water, mixing uniformly to obtain a slurry;
  • the technical solution adopted in the embodiments of the present application further includes: the solution of polylysine and water also includes effective biological components.
  • a 3D structure body which is prepared by using the slurry prepared by the foregoing slurry for 3D printing or the foregoing method for preparing a slurry for 3D printing.
  • the 3D structure includes a tissue repair scaffold, living tissue or organoid body, preferably a tissue repair scaffold.
  • a method for preparing the above 3D structure including the following steps:
  • the technical solutions adopted in the embodiments of the present application further include: the printing pressure of 3D printing is 50-600 kPa, and the printing speed is 5-10 mm / s.
  • the technical solution adopted in the embodiments of the present application further includes: on the basis of the technical solution of the present application, the method further includes placing the obtained 3D structure in a condensing agent solution or a mixed solution of a condensing agent and a stabilizer to obtain a chemical exchange Steps for connecting 3D structures;
  • the technical solutions adopted in the examples of the present application further include: the molar ratio of the condensing agent and the stabilizer is 1-2: 1;
  • the condensing agent includes water-soluble and oil-soluble condensing agents, preferably water-soluble condensing agents, further preferably 1- (3-dimethylaminopropyl) -3-ethylcarbodicarbonate Imine hydrochloride;
  • the stabilizer includes imide or benzotriazole stabilizers, preferably N-hydroxysuccinimide or 1-hydroxybenzotriazole.
  • the technical solution adopted in the embodiments of the present application further includes: on the basis of the technical solution of the present application, the method further includes placing the obtained chemically cross-linked 3D structure in a polylysine solution to obtain an amino-modified 3D structure A step of;
  • the technical solution adopted in the embodiments of the present application further includes: the mass fraction of the polylysine solution is 2-10%, preferably 4-6%;
  • the technical solution adopted in the embodiments of the present application further includes: the method further includes placing the obtained amino-modified 3D structure in an effective biological component solution to obtain the effective biological component-loaded 3D structure.
  • the paste used for 3D printing in this application is based on alginate, and polylysine is added. Through the physical electrostatic interaction between alginate and polylysine, physical crosslinks are formed through electrostatic self-assembly
  • the three-dimensional network structure forms a hydrogel.
  • the paste can be directly formed after printing and extrusion, which improves the printing performance of alginate-based bio-ink.
  • the electrostatic effect is conducive to obtaining a more stable paste and helping to improve pure seaweed.
  • the agglomeration of salt slurry makes the slurry more uniform after formation; at the same time, the ratio of alginate carboxyl group and polylysine amino group is controlled to ensure that the network structure after physical crosslinking is in a relatively stable state.
  • the slurry can directly obtain a molded structure without curing, the structure is not easily destroyed under physiological environment, and the stability is good, which improves the instability defects of traditional calcium ion cross-linked alginate hydrogel in physiological environment.
  • the paste for 3D printing of this application can control the overall charge of the paste by controlling the ratio of alginate and polylysine, and obtain structures with different charges and potentials, which is beneficial to carry different types of substances (such as nutrients, extracellular matrix, growth factors, etc.), can achieve controlled release of substances, promote cell adhesion and proliferation, promote cell activity and function (proliferation, differentiation, migration, secretion or metabolism), improve alginic acid Salt is biologically inert.
  • the physical cross-linked structure after 3D printing is further chemically cross-linked through a carboxyl group and an amino group, and a 3D structure with higher stability can be obtained, and its integrity can be maintained for a long time.
  • the chemically cross-linked 3D structure can be further processed by polylysine solution to obtain a surface-modified 3D structure, which not only can further complete the reaction of the carboxyl group, to form a denser cross-linked network, and further improve stability And the charge of the structure can be adjusted.
  • This application can be used for various tissue repair applications, further expanding the application value of alginate in the field of tissue engineering.
  • FIG. 1 is a schematic diagram of a paste provided by an embodiment of the present application and a process of using it for 3D bioprinting;
  • Example 2 is a printed bone model photo provided by an embodiment of the present application (the left side is the bone model photo printed in Example 1, and the right side is the bone model photo printed in Example 2);
  • Example 3 is a graph showing the stability test results of the bone model printed in Example 1 and Example 2 of the present application and the bone model printed in Comparative Example 1 in water;
  • Example 4 is a surface morphology diagram of the chemically cross-linked stent obtained in Example 9 of the present application.
  • Example 5 is a surface morphology diagram of the chemically cross-linked scaffold obtained in Example 11 of the present application.
  • Example 6 is a zeta potential diagram of the surface of the stent obtained in Example 11, Example 15 and Example 17 of the present application;
  • Example 7 is a graph showing the stability test results of the physical cross-linked scaffold printed in Example 3 of the present application and the scaffold obtained in Comparative Example 2 under a physiological environment;
  • Example 8 is a graph of the adsorption and release of chondroitin sulfate obtained by the scaffolds obtained in Example 11, Example 15, Example 17, and Comparative Example 2 of the present application (the adsorption figure on the left and the release diagram on the right);
  • Example 9 is a graph of the adsorption and release of vascular endothelial growth factor by the scaffolds obtained in Example 11, Example 15, Example 17 and Comparative Example 2 of the present application (the left side is the adsorption map and the right side is the release graph);
  • FIG. 10 is a graph of the life-and-death staining of the scaffold of human bone marrow mesenchymal stem cells mounted in Example 20 of the present application after printing and after 7 days of culture.
  • a paste for 3D printing including alginate, polylysine and water, wherein the quality of the alginate is 20-40% of the water quality.
  • the molar ratio of the carboxyl group of the acid salt to the amino group of the polylysine is 1: 0.1-2.
  • Paste also known as ink, is a material used for 3D printing.
  • the biological ink used for three-dimensional bioprinting needs to have the following properties: a) shear thinning performance, the viscosity of the ink is reduced by shear force to facilitate extrusion; b) biological activity, achieving high cell survival rate and biological activity; c) Rapid prototyping performance, rapid prototyping after printing and extrusion to build complex-shaped stents; d) Controllable degradation performance, which can controllably maintain the stability of the printed stents.
  • sodium alginate is widely used as a bio-ink, but the stability of the stent after sodium alginate is cross-linked with calcium ions is poor.
  • sodium alginate does not have biological activity, and the cells adhere and spread poorly on the sodium alginate scaffold, nor does it have the ability to induce differentiation of stem cells.
  • This application proposes an alginate and polylysine slurry for the problems of poor alginate stability and biological inertness.
  • alginate Typical but non-limiting examples of alginate are sodium alginate or potassium alginate, preferably sodium alginate.
  • Polylysine ( ⁇ -polylysine ( ⁇ -PL)) is a homopolymer monomer containing 25-30 lysine residues.
  • Polylysine is a natural polyamino acid with Good biocompatibility, and has a certain antibacterial ability; polylysine is a small molecule peptide, the main material of alginate and polylysine slurry is alginate, so polylysine is different from other poly
  • the electrolyte does not affect the properties of alginate shear thinning, and thus does not affect the printing performance of the ink.
  • the quality of the alginate is controlled to be 20-40% of the water quality, including but not limited to 20%, 22%, 24%, 25%, 26%, 28%, 30%, 32 %, 35%, 38% or 40%.
  • the molar ratio of the carboxyl group of alginate to the amino group of polylysine is 1: 0.1-2, for example, 1: 0.1, 1: 0.2, 1: 0.5, 1: 0.6, 1: 0.8, 1: 1 , 1: 1.2, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.8 or 1: 2.
  • the slurry for 3D printing of the present application may also include effective biological components, such as growth factors and / or extracellular matrix components.
  • the paste for 3D printing of the present application is based on alginate, and polylysine is added.
  • the physical electrostatic interaction between alginate and polylysine forms a physically cross-linked three-dimensional network gel
  • the structure and electrostatic effect keep the paste in a more stable state, which helps to improve the agglomeration of pure alginate paste, makes the paste more uniform after formation, and improves the printing performance of alginate-based biological ink; Control the ratio of alginate carboxyl groups and polylysine amino groups to ensure that the network structure after physical cross-linking is in a relatively stable state.
  • the structure of the structure printed with this paste is good, and it can be directly molded without curing
  • the structure is not easy to be destroyed in the physiological environment, and has good stability, which improves the instability defects of the traditional calcium ion-crosslinked alginate hydrogel in the physiological environment.
  • the use of the paste of the present application enables printing of complex models.
  • the paste for 3D printing of the present application can control the overall charge of the paste by controlling the ratio of alginate and polylysine, and obtain structures with different charges and potentials, which is beneficial for carrying different types of substances (such as nutrients) , Extracellular matrix, growth factors, etc.), can achieve controlled release of substances, promote cell adhesion and proliferation, promote cell activity and function (proliferation, differentiation, migration, secretion or metabolism), improve the alginate organism Inert.
  • This application can be used for various tissue repair applications, further expanding the application value of alginate in the field of tissue engineering.
  • the molar ratio of the carboxyl group of alginate to the amino group of polylysine is 1: 0.5-1, preferably 1: 1.
  • the structure formed by this ratio of alginate and polylysine is more stable, especially when 1: 1, the charge balance in the structure, the electrostatic network structure is the most stable.
  • the polylysine has a weight average molecular weight of 750-5000, preferably 1000-4200; and / or the melting point of polylysine is 170-175 ° C, preferably 172-175 ° C.
  • the weight average molecular weight of polylysine includes but is not limited to 880, 1030, 1175, 1800, 2000, 3000, 4200 or 4700.
  • the melting point of polylysine includes but is not limited to 170 ° C, 171 ° C, 172 ° C, 173 ° C, 174 ° C or 175 ° C.
  • the structure formed by polylysine and alginate within this parameter range is more stable.
  • the slurry further includes an effective biological ingredient, and the effective biological ingredient includes one or more of nutrients, extracellular matrix, growth factors, cells, or pharmaceutical active ingredients.
  • Nutrients include but are not limited to nucleotides, polypeptides, amino acids, lipids, carbohydrates (such as monosaccharides, oligosaccharides or polysaccharides) or vitamins;
  • Extracellular matrix includes but is not limited to proteoglycans, glycosaminoglycans, structural proteins (such as collagen and elastin) or adhesion proteins (such as fibronectin and laminin), etc .;
  • Growth factors include but are not limited to insulin, insulin-like growth factors (such as IGF-I, IGF-II), transforming growth factors (such as TGF ⁇ and TGF ⁇ ), vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor, platelet origin Growth factor, osteosarcoma-derived growth factor, growth hormone release inhibitory factor, nerve growth factor, interleukin (such as IL-1, IL-2, IL-3), erythropoietin, colony stimulating factor, cortisol or thyroxine Wait;
  • Cells include but are not limited to bacteria, yeast, plant cells or animal cells;
  • Pharmaceutical active ingredients include, but are not limited to, rhIL-11, rhIL-2, rhEPO, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , G-CSF, GM-CSF, rhTNF- ⁇ , sTNF-R1 or rHuEPO, etc.
  • the loading method of effective biological ingredients includes one or two of the following ways: (a) directly add effective biological ingredients to the slurry for loading; (b) print the finished product and put it in the effective biological ingredient solution To carry the load.
  • a method for preparing a paste for 3D printing including the following steps:
  • the preparation method of the slurry is simple and easy to obtain.
  • the method for preparing a slurry for 3D printing includes the following steps: adding alginate to a solution of polylysine and water, and mixing to obtain a slurry.
  • the method includes the following steps: dissolve the formulated amount of polylysine in water and mix evenly; add alginate to the aqueous polylysine solution and mix evenly to obtain a 3D printed slurry.
  • the solution of polylysine and water also includes effective biological ingredients.
  • Effective biological ingredients include one or more of nutrients, extracellular matrix, growth factors, cells, or pharmaceutical active ingredients.
  • the growth factor When preparing the slurry, the growth factor can be mixed in the aqueous solution of polylysine; the load of the cell can dissolve the polylysine in the medium solution containing the cells, (the amount of water at this time is the The amount is correct) Then add alginate and mix well to obtain a 3D printed paste.
  • a 3D structure body obtained by using the slurry prepared by the above-mentioned slurry for 3D printing or the above-mentioned slurry preparation method.
  • the structure is not easy to be destroyed in the physiological environment, the stability is better, and the traditional calcium ion cross-linked alginate hydrogel is improved in the physiological environment Instability defects, at the same time, the overall charge of the obtained structure is controllable, which is beneficial to carry different types of substances and has good biological activity.
  • the paste can be directly obtained by 3D printing to obtain a physically cross-linked 3D structure, or can be further cured by means of photo-curing to obtain a chemically cross-linked 3D structure.
  • the type and shape of the 3D structure are not limited.
  • the 3D structure includes but is not limited to a tissue repair scaffold, and may also be a living tissue or an organoid body.
  • 3D printing is carried out using the slurry for 3D printing described above to obtain a 3D structure.
  • 3D printing is typical but not limited to 3D bioprinting.
  • the preparation method of the 3D structure is simple in operation, does not require a complicated chemical synthesis process, and can be directly printed to obtain the 3D structure.
  • the printing pressure depends on the ratio of sodium alginate / polylysine and their mass fraction in the slurry.
  • the minimum pressure can be adjusted to 50kPa, which is suitable for three-dimensional biological printing with cells.
  • the upper limit depends on the printer's own parameters.
  • the printing pressure for 3D bioprinting is 50-600 kPa, and the printing speed is 5-10 mm / s.
  • the method for preparing a 3D structure further includes the step of placing the obtained 3D structure in a mixed solution of a condensing agent and a stabilizer to obtain a chemically cross-linked 3D structure.
  • Condensing agents include water-soluble or oil-soluble condensing agents, preferably water-soluble condensing agents, including but not limited to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC).
  • water-soluble condensing agents including but not limited to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC).
  • Stabilizer refers to an agent capable of forming a stable activated intermediate, including but not limited to imide or benzotriazole stabilizers, such as N-hydroxysuccinimide (NHS) or 1- Hydroxybenzotriazole (HOBt).
  • imide or benzotriazole stabilizers such as N-hydroxysuccinimide (NHS) or 1- Hydroxybenzotriazole (HOBt).
  • the molar ratio of the condensation agent to the stabilizer is 1-2: 1, for example 1.5: 1 or 2: 1.
  • the chemical cross-linking method is a condensation reaction between an amino group and a carboxyl group.
  • An exemplary method is to activate the carboxyl group, and then add an amino-containing compound to condense the amino group with the carboxyl group.
  • the 3D structure cross-linked by electrostatic physical is further cured through the chemical cross-linking of the amino group and the carboxyl group to obtain a 3D structure with higher stability, and the stability is further controlled.
  • the 3D structure can be maintained in water or physiological environment. High stability.
  • the method for preparing the 3D structure further includes the step of placing the obtained chemically cross-linked 3D structure in a polylysine solution to obtain an amino-modified 3D structure.
  • Chemically cross-linked 3D structures have excess activated carboxyl groups that are not chemically cross-linked. Putting the structure in a polylysine solution (soaking) can further complete the reaction of the carboxyl groups to form a denser cross-linking network, thereby further improving stability.
  • the mass fraction of the polylysine solution is 2-10%, preferably 4-6%.
  • the mass fraction of the polylysine solution is typically, but not limited to, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.
  • the structure is placed in a polylysine solution immersed in a certain mass fraction.
  • a large amount of amino groups can be introduced on the surface of the structure to obtain an amino-modified 3D structure to achieve The charge of the structure is adjustable.
  • the method for preparing the 3D structure further includes placing the obtained amino-modified 3D structure in an effective biological component solution to obtain the effective biological component-loaded 3D structure.
  • Effective biological ingredients include one or more of nutrients, extracellular matrix, growth factors, cells, or pharmaceutical active ingredients.
  • the above-mentioned polylysine solution regulates the charge of the structure, so as to realize the loading of different effective biological components, and can regulate the proliferation, differentiation, migration and metabolism of cells.
  • An exemplary method for preparing a 3D stent includes the following steps:
  • Preparation of sodium alginate / polylysine slurry dissolve polylysine in water and mix evenly; add sodium alginate to the aqueous solution of polylysine, the mass percentage of sodium alginate in water is 20 -40%, the molar ratio of the carboxyl group of sodium alginate and the amino group of polylysine is 1: 0.1-2, and the sodium alginate / polylysine slurry is evenly mixed, and effective biological ingredients can be added to the slurry;
  • An exemplary method for preparing a 3D stent includes the following steps:
  • Preparation of sodium alginate / polylysine slurry dissolve polylysine in water and mix evenly; add sodium alginate to the aqueous solution of polylysine, the mass percentage of sodium alginate in water is 20 -40%, the molar ratio of the carboxyl group of sodium alginate and the amino group of polylysine is 1: 0.1-2, and the sodium alginate / polylysine slurry is evenly mixed, and effective biological ingredients can be added to the slurry
  • Bracket printing install the sodium alginate / polylysine slurry into a printing cylinder, and use a three-dimensional bioprinter to perform three-dimensional printing at room temperature (see Figure 1);
  • An exemplary method for preparing a 3D stent includes the following steps:
  • Preparation of sodium alginate / polylysine slurry dissolve polylysine in water and mix evenly; add sodium alginate to the aqueous solution of polylysine, the mass percentage of sodium alginate in water is 20 -40%, the molar ratio of the carboxyl group of sodium alginate and the amino group of polylysine is 1: 0.1-2, and the sodium alginate / polylysine slurry is evenly mixed, and effective biological ingredients can be added to the slurry;
  • Bracket printing install the sodium alginate / polylysine slurry into a printing cylinder, and use a three-dimensional bioprinter to perform three-dimensional printing at room temperature (see Figure 1);
  • An exemplary method for preparing a 3D stent includes the following steps:
  • Preparation of sodium alginate / polylysine slurry dissolve polylysine in water and mix evenly; add sodium alginate to the aqueous solution of polylysine, the mass percentage of sodium alginate in water is 20 -40%, the molar ratio of the carboxyl group of sodium alginate and the amino group of polylysine is 1: 0.1-2, and the sodium alginate / polylysine slurry is evenly mixed, and effective biological ingredients can be added to the slurry;
  • Bracket printing install the sodium alginate / polylysine slurry into a printing cylinder, and use a three-dimensional bioprinter to perform three-dimensional printing at room temperature (see Figure 1);
  • the scaffold with effective biological components can be directly put into the culture medium for cultivation;
  • Adsorption of effective biological components After washing the stent several times with water, soaking in an aqueous solution containing different effective biological components, the charge of the stent is adjusted by the previous method, so as to achieve the loading of different effective biological components.
  • an application of the 3D structure or the 3D structure prepared by the method for preparing the 3D structure in tissue engineering is provided.
  • Typical but non-limiting applications are, for example, bone-cartilage integrated repair scaffolds, vascularized tissue repair scaffolds, and 3D structures can be used in cell behavior research models or drug screening models.
  • the 3D structure has the advantages of good stability and good biological activity, which can realize the loading of different types of substances (nutrients, extracellular matrix, growth factors, cells or active pharmaceutical ingredients), and can be used for a variety of tissue repair applications , Further expanding the application value of sodium alginate in the field of tissue engineering.
  • Polylysine was purchased from Maclean's P832586, M w ⁇ 5000.
  • a preparation method of sodium alginate / polylysine bio-ink and three-dimensional bio-printing bone model includes the following steps:
  • the bio-ink is loaded into a printing cartridge, and a three-dimensional bio-printer is used to print a bone model.
  • the printing pressure is 600 kPa and the printing speed is 10 mm / s.
  • Figure 2 is a photo of the bone model printed by the paste of Example 1 and Example 2.
  • the sodium alginate / polylysine bio-ink has the self-supporting ability, which can realize the printing of complex models and the moldability better.
  • a preparation method of sodium alginate biological ink and its three-dimensional biological printing bone model includes the following steps:
  • the bio-ink is loaded into a printing cartridge, and a three-dimensional bio-printer is used to print a bone model.
  • the printing pressure is 600 kPa and the printing speed is 10 mm / s.
  • Example 1 The bone models printed in Example 1, Example 2 and Comparative Example 1 were soaked in water and subjected to stability tests. The results are shown in FIG. 3.
  • Example 1 The bone model printed by sodium alginate / polylysine bio-ink was soaked in water for 30 minutes and then maintained its integrity, with good stability, and directly printed by sodium alginate bio-ink The structure of the bone model was almost destroyed after being immersed in water for 30 minutes. It can be seen that the structure printed by sodium alginate / polylysine bio-ink is better than the structure printed directly by sodium alginate.
  • a preparation method of sodium alginate / polylysine bio-ink and three-dimensional bio-printing stent includes the following steps:
  • Example 4-7 The difference between Examples 4-7 and Example 3 is that the amount of polylysine added is changed to make the molar ratio of the carboxyl group of sodium alginate and the amino group of polylysine different, as shown in Table 1.
  • the difference between this example and example 3 is that the molecular weight M w of the polylysine used is greater than 6000.
  • the scaffolds obtained by the three-dimensional bioprinting of Examples 3-8 are independently carried out as follows:
  • the surface morphology of the chemical cross-linked scaffold of Example 9 is shown in FIG. 4, and the surface morphology of the chemically cross-linked scaffold of Example 11 is shown in FIG. 5.
  • the surface of the scaffold with carboxyl group of sodium alginate and amino group of polylysine is 1: 1, and the surface of the scaffold with carboxyl group of sodium alginate and amino group of polylysine is 2: 1.
  • the fibrous structure at the nanometer level is closer to the natural extracellular matrix structure.
  • the carboxyl group of sodium alginate and the amino group of polylysine can be completely cross-linked, while the 2: 1 sodium alginate part cannot be cross-linked with the amino group of polylysine.
  • Sodium sulfate dissolves in water and forms a fibrous morphology.
  • a preparation method of sodium alginate hydrogel stent includes the following steps:
  • Example 9 The difference between this comparative example and Example 9 is that the molar ratio of the carboxyl group of sodium alginate and the amino group of polylysine is 1: 5.
  • the chemically cross-linked scaffold was immersed in a 5% mass fraction polylysine aqueous solution for 30 minutes.
  • the stent was washed several times with double distilled water, and then freeze-dried to obtain the amino-modified stent of Examples 15-17.
  • the process in the polylysine solution did not destroy the fibrous morphology of the scaffold surface.
  • the zeta potential diagrams of the surfaces of the scaffolds obtained in Example 11, Example 15 and Example 17 are shown in FIG. 6, and it can be seen from the figure that the ratio of the carboxyl group of sodium alginate to the amino group of polylysine and the immersed poly
  • the concentration of lysine solution has an effect on the surface charge of the stent.
  • the zeta potential on the surface of the stent can be adjusted by adjusting the ratio of carboxyl group to amino group and the mass fraction of the immersed polylysine solution.
  • Example 3 The physical cross-linked scaffold printed in Example 3 and the scaffold obtained in Comparative Example 2 were soaked in PBS solution for 1 and 7 days. The results are shown in FIG. 7.
  • Example 3 It can be seen from the figure that the physical cross-linked scaffold printed in Example 3 can keep the structure stable.
  • the scaffold obtained in Comparative Example 2 was almost destroyed on the surface of the scaffold in 1 day, and became a flocculent in about 7 days.
  • the stents obtained in Examples 9-19 and Comparative Examples 2-3 were subjected to stability testing.
  • the stability of the chemically cross-linked scaffold in water is significantly higher than that of the sodium alginate hydrogel scaffold.
  • the ratio of the carboxyl group of sodium alginate to the amino group of polylysine has an important influence on the stability of the stent. As the amount of polylysine in the sodium alginate ink increases, the stability of the stent will increase. When the ratio is 1: 1, the stent will be more stable, but too much polylysine will have a negative impact on the stability of the stent.
  • the molecular weight of the polylysine used will also affect the stability of the stent.
  • Test Example 3 Active scaffold loaded with growth factor or extracellular matrix
  • the stent was immersed in 100ng / mL vascular endothelial cell growth factor (VEGF) solution or 0.5mg / mL, 1.0mg / mL, 5.0mg / mL chondroitin sulfate (CS) solution for 12 hours.
  • VEGF vascular endothelial cell growth factor
  • CS chondroitin sulfate
  • the active scaffold was placed in a 24-well plate, and 1 mL of PBS solution was added to each well and placed in a 37 ° C shaker at a shaking speed of 60 rpm. After a fixed interval time, 100 ⁇ L of the solution was taken out for detection, and 100 ⁇ L of PBS solution was added to the original well. The results are shown in Figures 8 and 9.
  • chondroitin sulfate is negatively charged, vascular endothelial growth factor is positively charged, and the amount of adsorption on different stents is different.
  • the stent of this application can effectively fix CS and continuously release VEGF Taking these two as examples, it was proved that the scaffold with adjustable charge can effectively carry the extracellular matrix and growth factors, and the biological activity of the sodium alginate scaffold was improved.
  • hBMSCs human bone marrow mesenchymal stem cells

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Abstract

一种用于3D打印的浆料、3D结构体及制备方法和应用。用于3D打印的浆料包括海藻酸盐、聚赖氨酸和水，其中海藻酸盐的质量是水质量的20-40%，海藻酸盐的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2。浆料成型性好，可直接3D打印得到生理环境下较为稳定的3D结构体，同时改善了海藻酸盐的生物惰性，通过控制海藻酸盐和聚赖氨酸的比例能够调控支架的整体电荷，利于搭载不同类型的有效生物成分，促进细胞的粘附和增殖。打印后的3D结构体经过进一步化学交联稳定性更好，能长时间保持完整性。

Description

用于3D打印的浆料、3D结构体及其制备方法和应用 技术领域
本申请属于三维生物打印技术领域，特别涉及用于3D打印的浆料、3D结构体及其制备方法和应用。
背景技术
三维(Three Dimensions，3D)生物打印技术是在三维打印技术基础上衍生出的，在打印生物材料的同时负载细胞或者生长因子的打印技术，在制备人造器官与组织和药物筛选模型上具有很大的潜力。生物墨水(用于三维生物打印的材料)则决定了三维生物打印制品的物理化学性能，同时影响细胞的存活率以及生长因子的活性等。海藻酸盐(钠)具有剪切稀化性能(受到剪切力作用墨水粘度降低)，以及其优异的生物相容性和能够实现快速离子交联，被广泛应用于构建细胞胶囊和生物墨水。但是使用钙离子交联后的海藻酸盐支架的稳定性较差，在生理环境下钙离子容易与周围的单价离子如钠离子发生置换，从而导致支架的坍塌。虽然海藻酸盐具有优异的生物相容性，能够实现包载细胞的存活，但是海藻酸盐不具备生物活性，细胞在海藻酸盐支架上粘附与铺展较差，无法实现生长因子等可控释放，也不具备诱导干细胞分化的能力。
目前为了改进海藻酸盐支架的生物惰性问题，有研究者在海藻酸钠中的羧基上接枝精氨酸-甘氨酸-天门冬氨酸序列(RGD)，从而促进细胞在海藻酸钠支架上的粘附[J.Yu,Y.Gu,K.T.Du,S.Mihardja,R.E.Sievers,R.J.Lee,Biomaterials 2009,30,751]。但是该海藻酸钠支架依然使用钙离子进行交联，稳 定性仍然不足。针对海藻酸钠支架稳定性的问题，研究者在海藻酸钠羧基上接枝甲基丙烯酸基团，使用光交联替代钙离子交联方式，从而提高了海藻酸钠支架的稳定性，但是这样的支架缺乏生物活性。[O.Jeon,K.H.Bouhadir,J.M.Mansour,E.Alsberg,Biomaterials 2009,30,2724.]。
发明内容
本申请的目的之一在于提供一种用于3D打印的浆料，旨在至少在一定程度上解决现有技术中的上述技术问题之一。
本申请的目的之二在于提供一种上述用于3D打印的浆料的制备方法，该方法简单易得。
本申请的目的之三在于提供一种3D结构体，采用上述用于3D打印的浆料或上述用于3D打印的浆料的制备方法制得的浆料制得，该3D结构体易成型、更稳定，同时改善了海藻酸盐的生物惰性。
本申请的目的之四在于提供一种上述3D结构体的制备方法，操作简单，不需要复杂的化学合成过程，此外，优选经过进一步化学交联后的3D结构体稳定性很好，能长时间保持其完整性。
本申请的目的之五在于提供一种上述3D结构体或上述3D结构体的制备方法制得的3D结构体在组织工程中的应用。
为了解决上述问题，本申请提供了如下技术方案：
第一方面，提供了一种用于3D打印的浆料，所述浆料包括海藻酸盐、聚赖氨酸和水，其中海藻酸盐的质量是水质量的为20-40％，海藻酸盐的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2。
本申请实施例采取的技术方案还包括：在本申请技术方案的基础上，海藻酸盐的羧基和聚赖氨酸的氨基的摩尔比为1:0.5-1，优选为1:1。
本申请实施例采取的技术方案还包括：在本申请技术方案的基础上，聚赖氨酸的重均分子量为750-5000，优选为1000-4200；
和/或，聚赖氨酸的熔点为170-175℃，优选为172-175℃。
本申请实施例采取的技术方案还包括：在本申请技术方案的基础上，所述浆料还包括有效生物成分，有效生物成分包括营养物质、细胞外基质、生长因子、细胞或药物活性成分中的一种或几种。
第二方面，提供了一种用于3D打印的浆料的制备方法，包括以下步骤：
将海藻酸盐、聚赖氨酸和水混合均匀，得到用于3D打印的浆料；
本申请实施例采取的技术方案还包括：将海藻酸盐加入聚赖氨酸和水的溶液中，混合均匀得到浆料；
本申请实施例采取的技术方案还包括：聚赖氨酸和水的溶液中还包括有效生物成分。
第三方面，提供了一种3D结构体，采用上述用于3D打印的浆料或上述用于3D打印的浆料的制备方法制得的浆料制得。
本申请实施例采取的技术方案还包括：所述3D结构体包括组织修复支架、活组织或类器官体，优选为组织修复支架。
第四方面，提供了一种上述3D结构体的制备方法，包括以下步骤：
使用所述用于3D打印的浆料进行3D打印，得到3D结构体；
本申请实施例采取的技术方案还包括：3D打印的打印压力为50-600kPa，打印速度为5-10mm/s。
本申请实施例采取的技术方案还包括：在本申请技术方案的基础上，所述 方法还包括将得到的3D结构体置于缩合剂溶液或缩合剂和稳定剂的混合溶液中，得到化学交联3D结构体的步骤；
本申请实施例采取的技术方案还包括：缩合剂和稳定剂的摩尔比为1-2:1；
本申请实施例采取的技术方案还包括：缩合剂包括水溶性和油溶性缩合剂，优选为水溶性缩合剂，进一步优选为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；
本申请实施例采取的技术方案还包括：稳定剂包括酰亚胺或苯并三唑类稳定剂，优选为N-羟基丁二酰亚胺或1-羟基苯并三唑。
本申请实施例采取的技术方案还包括：在本申请技术方案的基础上，所述方法还包括将得到的化学交联3D结构体置于聚赖氨酸溶液中，得到氨基修饰的3D结构体的步骤；
本申请实施例采取的技术方案还包括：聚赖氨酸溶液的质量分数为2-10％，优选为4-6％；
本申请实施例采取的技术方案还包括：所述方法还包括将得到的氨基修饰的3D结构体置于有效生物成分溶液中，得到负载有效生物成分的3D结构体。
第五方面，提供了一种上述3D结构体或上述3D结构体的制备方法制得的3D结构体在组织工程中的应用。
相对于现有技术，本申请实施例产生的有益效果在于：
(1)本申请用于3D打印的浆料以海藻酸盐为基础，增加了聚赖氨酸，通过海藻酸盐与聚赖氨酸之间的物理静电作用，通过静电自组装形成物理交联的三维网状结构，形成水凝胶，浆料打印挤出后可直接成型，提高了海藻酸盐基生物墨水的打印性能，静电作用有利于获得更加稳定的浆料，有助于改善单纯海藻酸盐浆料的结块现象，使浆料形成后更加均匀；同时通过控制海藻酸盐 的羧基和聚赖氨酸的氨基的比例，保证物理交联后的网络结构处于较为稳定的状态，使用该浆料无需固化就能直接获得成型结构体，在生理环境下结构不容易被破坏，稳定性较好，改善了传统钙离子交联海藻酸盐水凝胶生理环境下的不稳定性缺陷。
(2)本申请的用于3D打印的浆料通过控制海藻酸盐和聚赖氨酸的比例能够调控浆料的整体电荷，获得不同电荷和电位的结构体，有利于搭载不同类型的物质(例如营养物质、细胞外基质、生长因子等)，能够实现物质的可控释放，促进细胞的粘附和增殖，促进细胞活性和功能(增殖、分化、迁移、分泌或新陈代谢)，改善了海藻酸盐的生物惰性。
(3)作为优选方案，3D打印后的物理交联结构体通过羧基与氨基的进一步化学交联，能得到稳定性更高的3D结构体，能长时间保持其完整性。
(4)作为优选方案，化学交联3D结构体通过聚赖氨酸溶液进一步处理能获得表面氨基修饰的3D结构体，不仅能进一步将羧基反应完全，形成更致密的交联网络，进一步提高稳定性，而且实现了结构体的电荷可调。
(5)由于浆料的稳定性，使用本申请的浆料能够实现复杂模型的打印。
(6)本申请能够用于多种组织修复应用，进一步扩大了海藻酸盐在组织工程领域的应用价值。
附图说明
图1是本申请实施例提供的浆料及使用其进行3D生物打印的过程示意图；
图2是本申请实施例提供的打印出的骨头模型照片(左侧为实施例1打印出的骨头模型照片，右侧为实施例2打印出的骨头模型照片)；
图3是本申请实施例1和实施例2打印出的骨头模型与对比例1打印出的骨头模型在水中的稳定性试验结果图；
图4是本申请实施例9得到的化学交联支架表面形貌图；
图5是本申请实施例11得到的化学交联支架表面形貌图；
图6是本申请实施例11、实施例15和实施例17得到的支架的表面zeta电位图；
图7是本申请实施例3打印出的物理交联支架与对比例2得到的支架在生理环境下的稳定性试验结果图；
图8是本申请实施例11、实施例15、实施例17以及对比例2得到的支架对硫酸软骨素的吸附和释放图(左侧为吸附图，右侧为释放图)；
图9是本申请实施例11、实施例15、实施例17以及对比例2得到的支架对血管内皮生长因子的吸附和释放图(左侧为吸附图，右侧为释放图)；
图10是本申请实施例20搭载的人骨髓间充质干细胞的支架打印后和培养7天后的活死染色图。
具体实施方式
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本申请，并不用于限定本申请。
根据本申请的第一个方面，提供了一种用于3D打印的浆料，包括海藻酸盐、聚赖氨酸和水，其中海藻酸盐的质量是水质量的为20-40％，海藻酸盐的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2。
浆料也可称为墨水，是用于3D打印的材料。
用于三维生物打印的生物墨水需具备以下性能：a)剪切稀化性能，受到剪切力作用墨水粘度降低利于挤出；b)生物活性，实现高的细胞存活率和生物活性；c)快速成型性能，打印挤出后能够快速成型从而能够构建复杂形状支架；d)可控的降解性能，能够可控地维持打印得到的支架的稳定性。目前广泛使用海藻酸钠作为生物墨水，但是海藻酸钠使用钙离子交联后支架的稳定性较差，在生理环境下钙离子容易与周围的单价离子如钠离子发生置换，从而导致支架的坍塌。且海藻酸钠不具备生物活性，细胞在海藻酸钠支架上粘附与铺展较差，也不具备诱导干细胞分化的能力。
本申请针对海藻酸盐稳定性差和生物惰性等问题，提出了一种海藻酸盐和聚赖氨酸浆料。
海藻酸盐典型但非限制性的例如为海藻酸钠或海藻酸钾，优选是海藻酸钠。
聚赖氨酸(ε-多聚赖氨酸(ε-PL))是一种含有25-30个赖氨酸残基的同型单体聚合物，聚赖氨酸是天然的聚氨基酸，具有很好的生物相容性，而且具有一定的抗菌能力；聚赖氨酸是小分子多肽，海藻酸盐和聚赖氨酸浆料的主体材料还是海藻酸盐，因此聚赖氨酸不同于其他聚电解质，不会影响海藻酸盐剪切稀化的属性，从而不影响该墨水的打印性能。
海藻酸盐和聚赖氨酸之间存在静电作用，通过静电自组装可形成三维网状结构，实现物理性交联。
为了保证墨水的可打印性，控制海藻酸盐的质量是水质量的为20-40％，包括但不限于20％、22％、24％、25％、26％、28％、30％、32％、35％、38％或40％。
海藻酸盐的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2，示例性的例如为 1:0.1、1:0.2、1:0.5、1:0.6、1:0.8、1:1、1:1.2、1:1.4、1:1.5、1:1.6、1:1.8或1:2。
随着聚赖氨酸比例的提高，电荷相互作用逐渐增强，水凝胶结构在溶剂(例如水、缓冲液或培养基)中更加稳定，但聚赖氨酸比例过大，海藻酸盐作为主体结构相对量少，会影响其结构的稳定性。
需要注意的是，本申请的用于3D打印的浆料除了包括海藻酸盐和聚赖氨酸外，还可以包括有效生物成分，例如生长因子和/或细胞外基质成分等。
本申请的用于3D打印的浆料以海藻酸盐为基础，增加了聚赖氨酸，通过海藻酸盐与聚赖氨酸之间的物理静电作用，形成物理交联的三维网状凝胶结构，静电作用使浆料保持较为稳定的状态，有助于改善单纯海藻酸盐浆料的结块现象，使浆料形成后更加均匀，提高了海藻酸盐基生物墨水的打印性能；同时通过控制海藻酸盐的羧基和聚赖氨酸的氨基的比例，保证物理交联后的网络结构处于较为稳定的状态，使用该浆料打印后的结构体成型性好，无需固化就能直接获得成型结构体，在生理环境下结构不容易被破坏，具有很好的稳定性，改善了传统钙离子交联海藻酸盐水凝胶生理环境下的不稳定性缺陷。
由于浆料易于成型以及其稳定性，使用本申请的浆料能够实现复杂模型的打印。
本申请的用于3D打印的浆料通过控制海藻酸盐和聚赖氨酸的比例能够调控浆料的整体电荷，获得不同电荷和电位的结构体，有利于搭载不同类型的物质(例如营养物质、细胞外基质、生长因子等)，能够实现物质的可控释放，促进细胞的粘附和增殖，促进细胞活性和功能(增殖、分化、迁移、分泌或新陈代谢)，改善了海藻酸盐的生物惰性。本申请能够用于多种组织修复应用，进一步扩大了海藻酸盐在组织工程领域的应用价值。
在一种实施方式中，海藻酸盐的羧基和聚赖氨酸的氨基的摩尔比为1:0.5-1， 优选为1:1。
该比例的海藻酸盐和聚赖氨酸形成的结构更加稳定，尤其当1:1时，结构中电荷平衡，静电网络结构最为稳定。
在一种实施方式中，聚赖氨酸的重均分子量为750-5000，优选为1000-4200；和/或，聚赖氨酸的熔点为170-175℃，优选为172-175℃。
聚赖氨酸的重均分子量包括但不限于880、1030、1175、1800、2000、3000、4200或4700。
聚赖氨酸的熔点包括但不限于170℃、171℃、172℃、173℃、174℃或175℃。
该参数范围内的聚赖氨酸与海藻酸盐形成的结构更加稳定。
在一种实施方式中，浆料还包括有效生物成分，有效生物成分包括营养物质、细胞外基质、生长因子、细胞或药物活性成分中的一种或几种。
营养物质包括但不限于核苷酸、多肽、氨基酸、脂质、碳水化合物(例如单糖、寡糖或多糖)或维生素等；
细胞外基质包括但不限于蛋白聚糖、糖胺聚糖、结构蛋白(例如胶原和弹性蛋白)或粘着蛋白(例如纤粘连蛋白和层粘连蛋白)等；
生长因子包括但不限于胰岛素、类胰岛素生长因子(如IGF-Ⅰ、IGF-Ⅱ)、转化生长因子(如TGFα和TGFβ)、血管内皮生长因子、表皮生长因子、成纤细胞生长因子、血小板来源生长因子、骨肉瘤来源生长因子、生长激素释放抑制因子、神经生长因子、白细胞介素(如IL-1、IL-2、IL-3)、红细胞生长素、集落刺激因子、皮质醇或甲状腺素等；
细胞包括但不限于细菌、酵母、植物细胞或动物细胞；
药物活性成分包括但不限于rhIL-11、rhIL-2、rhEPO、IFN-α、IFN-β、IFN-γ、G-CSF、GM-CSF、rhTNF-α、sTNF-R1或rHuEPO等。
将营养物质、细胞外基质、生长因子、细胞或药物活性成分等有效生物成分加入浆料中，可实现上述物质的负载。
需要注意的是，有效生物成分的负载方式包括以下方式中的一种或两种：(a)在浆料中直接加入有效生物成分进行负载；(b)打印出成品后置于有效生物成分溶液中进行负载。
根据本申请的第二个方面，提供了一种用于3D打印的浆料的制备方法，包括以下步骤：
将海藻酸盐、聚赖氨酸和水混合均匀，得到用于3D打印的浆料。
该浆料的制备方法简单易得。
优选地，用于3D打印的浆料的制备方法，包括以下步骤：将海藻酸盐加入聚赖氨酸和水的溶液中，混合均匀得到浆料。
具体地，方法包括以下步骤：将配方量的聚赖氨酸溶解于水中，混合均匀；将海藻酸盐加入聚赖氨酸水溶液中，混合均匀得到3D打印的浆料。
优选地，聚赖氨酸和水的溶液中还包括有效生物成分。
有效生物成分包括营养物质、细胞外基质、生长因子、细胞或药物活性成分中的一种或几种。
在制备浆料时，生长因子可混合于聚赖氨酸水溶液中；细胞的负载可将聚赖氨酸溶解于含有细胞的培养基溶液中，(这时水的量以培养基溶液中水的量为准)随后加入海藻酸盐混合均匀，得到3D打印的浆料。
根据本申请的第三个方面，提供了一种3D结构体，采用上述用于3D打印的浆料或上述用于3D打印的浆料的制备方法制得的浆料制得。
使用本申请的3D打印浆料可直接形成成型结构体，该结构体在生理环境下结构不容易被破坏，稳定性较好，改善了传统钙离子交联海藻酸盐水凝胶生 理环境下的不稳定性缺陷，同时该获得的结构体的整体电荷可控，有利于搭载不同类型的物质，生物活性好。
需要注意的是，浆料可直接通过3D打印获得物理交联的3D结构体，也可以进一步进行光固化等固化方式获得化学交联的3D结构体。
对3D结构体的类型和形状不作限定，3D结构体包括但不限于组织修复支架，还可以是活组织或类器官体等。
根据本申请的第四个方面，提供了一种上述3D结构体的制备方法，包括以下步骤：
使用上述用于3D打印的浆料进行3D打印，得到3D结构体。
3D打印典型但非限制性的是3D生物打印。
该3D结构体的制备方法操作简单，不需要复杂的化学合成过程，可直接打印获得3D结构体。
打印的压力取决于海藻酸钠/聚赖氨酸的比例以及它们在浆料中的质量分数，压力最小可调节至50kPa，适用于含细胞的三维生物打印，上限则取决于打印机的本身参数。
优选地，3D生物打印的打印压力为50-600kPa，打印速度为5-10mm/s。
在一种实施方式中，3D结构体的制备方法还包括将得到的3D结构体置于缩合剂和稳定剂的混合溶液中，得到化学交联3D结构体的步骤。
缩合剂包括水溶性或油溶性缩合剂，优选为水溶性缩合剂，包括但不限于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)。
稳定剂指的是能够形成稳定的活化中间体的试剂，包括但不限于酰亚胺或苯并三唑类稳定剂，示例性的例如为N-羟基丁二酰亚胺(NHS)或1-羟基苯并三唑(HOBt)。
优选地，缩合剂和稳定剂的摩尔比为1-2:1，例如1.5:1或2:1。
化学交联方法为氨基和羧基缩合反应，示例性的方式是将羧基活化，再加入含氨基化合物，使氨基与羧基缩合。
通过静电物理交联的3D结构体通过氨基与羧基的化学交联进一步固化，得到稳定性更高的3D结构体，稳定性进一步可控，该3D结构体在水或生理环境中都能保持很高的稳定性。
在一种实施方式中，3D结构体的制备方法还包括将得到的化学交联3D结构体置于聚赖氨酸溶液中，得到氨基修饰的3D结构体的步骤。
化学交联3D结构体中存在未化学交联的多余活化羧基，将结构体置于聚赖氨酸溶液中(浸泡)，能进一步将羧基反应完全，形成更致密的交联网络，从而进一步提高稳定性。
优选地，聚赖氨酸溶液的质量分数为2-10％，优选为4-6％。
聚赖氨酸溶液的质量分数典型但非限制性的例如为2％、3％、4％、5％、6％、7％、8％、9％或10％。
随着聚赖氨酸溶液质量分数的提升，结构体的稳定性上升，但当大于10％后，聚赖氨酸已经过量，对稳定性没有显著性的提升。
同时，将结构体置于浸泡于一定质量分数的聚赖氨酸溶液中，在聚赖氨酸过量的情况下，可以在结构体表面引入大量的氨基，得到氨基修饰的3D结构体，从而实现结构体的电荷可调。
在一种实施方式中，3D结构体的制备方法还包括将得到的氨基修饰的3D结构体置于有效生物成分溶液中，得到负载有效生物成分的3D结构体。有效生物成分包括营养物质、细胞外基质、生长因子、细胞或药物活性成分中的一种或几种。
通过上述聚赖氨酸溶液调节结构体的电荷情况，从而实现不同有效生物成分的负载，能对细胞的增殖、分化、迁移、新陈代谢进行调控。
一种示例性的3D支架的制备方法，包括以下步骤：
a)制备海藻酸钠/聚赖氨酸浆料：将聚赖氨酸溶解于水中，混合均匀；将海藻酸钠加入聚赖氨酸水溶液中，海藻酸钠在水中的质量百分含量为20-40％，海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2，混合均匀得到海藻酸钠/聚赖氨酸浆料，浆料中可加入有效生物成分；
b)支架打印：将该海藻酸钠/聚赖氨酸浆料装进打印料筒中，在常温下使用三维生物打印机进行三维打印(如图1)。
一种示例性的3D支架的制备方法，包括以下步骤：
a)制备海藻酸钠/聚赖氨酸浆料：将聚赖氨酸溶解于水中，混合均匀；将海藻酸钠加入聚赖氨酸水溶液中，海藻酸钠在水中的质量百分含量为20-40％，海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2，混合均匀得到海藻酸钠/聚赖氨酸浆料，浆料中可加入有效生物成分；
b)支架打印：将该海藻酸钠/聚赖氨酸浆料装进打印料筒中，在常温下使用三维生物打印机进行三维打印(如图1)；
c)将打印出的支架浸泡于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基丁二酰亚胺的水溶液中30-40min，得到的化学交联支架。
一种示例性的3D支架的制备方法，包括以下步骤：
a)制备海藻酸钠/聚赖氨酸浆料：将聚赖氨酸溶解于水中，混合均匀；将海藻酸钠加入聚赖氨酸水溶液中，海藻酸钠在水中的质量百分含量为20-40％，海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2，混合均匀得到海藻酸钠/聚赖氨酸浆料，浆料中可加入有效生物成分；
b)支架打印：将该海藻酸钠/聚赖氨酸浆料装进打印料筒中，在常温下使用三维生物打印机进行三维打印(如图1)；
c)将打印出的支架浸泡于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基丁二酰亚胺的水溶液中30-40min，得到的化学交联支架；
d)除去交联试剂后，将支架浸泡于质量分数为2-10％的聚赖氨酸水溶液中30-40min，得到氨基修饰的支架。
一种示例性的3D支架的制备方法，包括以下步骤：
a)制备海藻酸钠/聚赖氨酸浆料：将聚赖氨酸溶解于水中，混合均匀；将海藻酸钠加入聚赖氨酸水溶液中，海藻酸钠在水中的质量百分含量为20-40％，海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2，混合均匀得到海藻酸钠/聚赖氨酸浆料，浆料中可加入有效生物成分；
b)支架打印：将该海藻酸钠/聚赖氨酸浆料装进打印料筒中，在常温下使用三维生物打印机进行三维打印(如图1)；
c)后处理：搭载有效生物成分的支架可直接放入培养基中培养；
d)将打印出的支架浸泡于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基丁二酰亚胺的水溶液中30-40min，得到的化学交联支架；
e)除去交联试剂后，将支架浸泡于质量分数为2-10％的聚赖氨酸水溶液中30-40min，得到氨基修饰的支架；
f)有效生物成分的吸附：将支架用水清洗数次后，浸泡于含有不同有效生物成分的水溶液中，通过上一步方法调节支架的电荷情况，从而实现不同有效生物成分的负载。
根据本申请的第五个方面，提供了一种上述3D结构体或上述3D结构体的制备方法制得的3D结构体在组织工程中的应用。
典型但非限制性的应用例如为骨-软骨一体化修复支架、血管化组织修复支架，3D结构体可以用于细胞行为研究模型或药物筛选模型。
由于3D结构体具有稳定性较好，生物活性好的优点，可以实现不同类型的物质(营养物质、细胞外基质、生长因子、细胞或药物活性成分)的搭载，能够用于多种组织修复应用，进一步扩大了海藻酸钠在组织工程领域的应用价值。
下面通过具体的实施例和对比例进一步说明本申请，但是，应当理解为，这些实施例仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本申请。本申请涉及的各原料均可通过商购获取。
聚赖氨酸购自麦克林的P832586，M w<5000。
实施例1海藻酸钠/聚赖氨酸生物墨水的制备及其三维生物打印骨头模型
一种海藻酸钠/聚赖氨酸生物墨水及其三维生物打印骨头模型的制备方法，包括以下步骤：
(1)将2.924g聚赖氨酸溶解于10mL双蒸水中搅拌均匀，称取4g海藻酸钠并加入到聚赖氨酸溶液中搅拌至均匀，得到海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比为1:1的海藻酸钠/聚赖氨酸生物墨水；
(2)将该生物墨水装进打印料筒中，使用三维生物打印机进行打印骨头模型，打印的压力为600kPa，打印速度为10mm/s。
实施例2
本实施例与实施例1的区别在于，海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比为2:1。
图2为实施例1和实施例2浆料打印出的骨头模型照片，图2可以看出， 海藻酸钠/聚赖氨酸生物墨水具有自支撑的能力，可以实现复杂模型的打印，成型性较好。
对比例1海藻酸钠生物墨水的制备及其三维生物打印骨头模型
一种海藻酸钠生物墨水及其三维生物打印骨头模型的制备方法，包括以下步骤：
(1)称取4g海藻酸钠并加入到10mL双蒸水中搅拌至均匀，得到海藻酸钠生物墨水；
(2)将该生物墨水装进打印料筒中，使用三维生物打印机进行打印骨头模型，打印的压力为600kPa，打印速度为10mm/s。
将实施例1、实施例2和对比例1打印出的骨头模型浸泡水中，进行稳定性试验，结果如图3所示。
实施例1、实施例2海藻酸钠/聚赖氨酸生物墨水打印出的骨头模型浸泡水中30min后然能保持其完整性，具有较好的稳定性，而直接用海藻酸钠生物墨水打印出的骨头模型浸泡水中30min后结构几乎被破坏，可见，海藻酸钠/聚赖氨酸生物墨水打印出的结构体在水中的稳定性优于海藻酸钠直接打印出的结构体。
实施例3海藻酸钠/聚赖氨酸生物墨水的制备及其三维生物打印支架
一种海藻酸钠/聚赖氨酸生物墨水及其三维生物打印支架的制备方法，包括以下步骤：
(1)将2.924g聚赖氨酸溶解于10mL双蒸水中搅拌均匀，称取4g海藻酸钠并加入到聚赖氨酸溶液中搅拌至均匀，得到海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比为1:1的海藻酸钠/聚赖氨酸生物墨水；
(2)将该生物墨水装进打印料筒中，使用三维生物打印机进行打印支架模 型，打印的压力为600kPa，打印速度为10mm/s。
实施例4-7
实施例4-7与实施例3的不同之处在于改变聚赖氨酸加入量，使海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比不同，具体如表1所示。
表1
  海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比
实施例4 4:1
实施例5 2:1
实施例6 1:1.5
实施例7 1:2
实施例8
本实施例与实施例3的区别在于，使用的聚赖氨酸的分子量M w大于6000。
实施例9-14化学交联支架的制备
各自独立地将实施例3-8三维生物打印得到的支架进行如下步骤：
将支架浸泡于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基丁二酰亚胺(NHS)比例为2:1的水溶液中，时间为30分钟。使用双蒸水冲洗支架数次，随后冷冻干燥，得到实施例9-14化学交联支架。
实施例9化学交联支架的表面形貌图如图4所示，实施例11化学交联支架的表面形貌图如图5所示。
从扫描电子显微镜图可以看出海藻酸钠的羧基与聚赖氨酸的氨基为1:1的支架表面光滑，海藻酸钠的羧基与聚赖氨酸的氨基为2:1的支架表面具有微纳米级别的纤维状结构，更加接近天然的细胞外基质结构。这时由于1:1配比使海藻酸钠的羧基和聚赖氨酸的氨基理论能完全交联，而2:1海藻酸钠有部分不 能与聚赖氨酸的氨基交联，多余的海藻酸钠溶于水中，形成纤维状的形貌。
对比例2
一种海藻酸钠水凝胶支架的制备方法，包括以下步骤：
(1)称取4g海藻酸钠并加入到10mL双蒸水中搅拌至均匀，得到海藻酸钠生物墨水；
(2)将该生物墨水装进打印料筒中，使用三维生物打印机进行打印支架模型，打印的压力为600kPa，打印速度为10mm/s；
(3)将打印出来的坯体浸泡于1M CaCl 2溶液中30min，得到海藻酸钠水凝胶支架。
对比例3
本对比例和实施例9的区别在于，海藻酸钠的羧基和聚赖氨酸的氨基的摩尔比为1:5。
实施例15-17氨基修饰支架的制备
各自独立地将实施例9-11得到的化学交联支架进行如下步骤：
将化学交联支架浸泡于5％质量分数的聚赖氨酸水溶液中，时间为30分钟。使用双蒸水冲洗支架数次，随后冷冻干燥，得到实施例15-17氨基修饰支架。聚赖氨酸溶液中的过程没有破坏支架的表面纤维状形貌。
实施例11、实施例15和实施例17得到的支架的表面zeta电位图如图6所示，从图中可以看出，海藻酸钠的羧基与聚赖氨酸的氨基的比例以及浸泡的聚赖氨酸溶液的浓度均对支架表面电荷产生影响，支架的表面zeta电位可以通过调节羧基与氨基的比例以及浸泡的聚赖氨酸溶液的质量分数进行调控。
实施例18
本实施例与实施例15的区别在于，聚赖氨酸水溶液质量分数为2％。
实施例19
本实施例与实施例15的区别在于，聚赖氨酸水溶液质量分数为15％。
试验例1生理环境下稳定性测试
将实施例3打印出的物理交联支架和对比例2得到的支架放入PBS溶液中浸泡1、7天，结果见图7。
从图中可以看出，实施例3打印出的物理交联支架能够保持结构稳定，对比例2得到的支架1天时支架表面形貌几乎被破坏，而7天左右就都变成絮状物。
试验例2
将实施例9-19以及对比例2-3得到的支架进行稳定性测试，测试方法如下：将支架浸泡于PBS溶液中，计算1天、7天、14天、21天、28天和56天后支架的质量损失百分比，质量损失％＝(浸泡前支架的质量-浸泡一定时间后支架的质量)/浸泡前支架的质量×100％。
结果如表2所示。
表2
Figure PCTCN2019120103-appb-000001
Figure PCTCN2019120103-appb-000002
从表2的结果可以看出，化学交联后的支架在水中的稳定性明显高于海藻酸钠水凝胶支架。海藻酸钠的羧基与聚赖氨酸的氨基的比例对支架的稳定性具有重要影响，开始随着聚赖氨酸在海藻酸钠墨水中的用量提高，支架的稳定性会提高，羧基与氨基的比例为1:1时，支架会更加稳定，但聚赖氨酸用量过多，会对支架稳定性产生负面影响。
后续浸泡于聚赖氨酸溶液中聚赖氨酸的质量分数也会对支架的稳定性产生影响，随着聚赖氨酸溶液质量分数的提升，支架稳定性上升，但当大于10％后，支架稳定性差别不大。这是因为使用EDC/NHS交联活化支架中海藻酸钠的羧基后，仍有多余的羧基没有反应，继续将支架浸泡于聚赖氨酸溶液中能将 羧基反应完全，形成更致密的交联网络，从而提高稳定性。所以随着浸泡的聚赖氨酸溶液质量分数的提升，支架的稳定性上升，但由于支架中未反应的羧基量是一定的，因此会达到一个平台期。
此外，使用的聚赖氨酸的分子量也会对支架的稳定性产生影响。
试验例3负载生长因子或细胞外基质的活性支架
各自独立地将实施例11得到的化学交联支架的支架、实施例15和实施例17得到的氨基修饰支架、以及对比例2得到的支架进行如下步骤：
将支架浸泡于100ng/mL的血管内皮细胞生长因子(VEGF)溶液或0.5mg/mL，1.0mg/mL，5.0mg/mL硫酸软骨素(CS)溶液中，时间为12小时。测定不同支架对对硫酸软骨素和血管内皮生长因子的吸附和释放，其中浸泡于1.0mg/mL硫酸软骨素溶液中的支架用于测试硫酸软骨素的释放行为。活性支架放置于24孔板中，每孔加入1mL PBS溶液并放置于37℃摇床中，摇速为60转/分。在固定间隔时间后取出100μL溶液用于检测，并加入100μL PBS溶液于原孔中。结果如图8、图9所示。
从图8、图9可以看出，硫酸软骨素是带负电的，血管内皮细胞生长因子带正电，在不同的支架上的吸附量不同，本申请支架能够有效地固定CS以及持续地释放VEGF，以这两者为范例证明了电荷可调控的支架能够有效地搭载细胞外基质和生长因子，提高了海藻酸钠支架的生物活性。
实施例20
制备搭载人骨髓间充质干细胞(hBMSCs)的三维生物打印支架。制备工艺如下:
(1)从培养瓶将hBMSCs消化并重悬浮于5mL培养基中，细胞的浓度为500,000/mL；
(2)将0.731g聚赖氨酸溶解于上述5mL培养基中，继续称取1g海藻酸钠并加入其中搅拌至均匀，得到海藻酸钠的羧基和聚赖氨酸的氨基的比例为1:1且搭载细胞的海藻酸钠/聚赖氨酸生物墨水；
(3)将该生物墨水装进打印料筒中，使用三维生物打印机进行打印。打印压力为100kPa，打印速度为1mm/s。
从活死染色的结果如图10所示，可以看出死细胞较少，说明该生物墨水满足生物相容性的需求。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本申请。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本申请的精神或范围的情况下，在其它实施例中实现。因此，本申请将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种用于3D打印的浆料，其特征在于，所述浆料包括海藻酸盐、聚赖氨酸和水，其中海藻酸盐的质量是水质量的20-40％，海藻酸盐的羧基和聚赖氨酸的氨基的摩尔比为1:0.1-2。
  2. 根据权利要求1所述的用于3D打印的浆料，其特征在于，海藻酸盐的羧基和聚赖氨酸的氨基的摩尔比为1:0.5-1，优选为1:1。
  3. 根据权利要求1所述的用于3D打印的浆料，其特征在于，聚赖氨酸的重均分子量为750-5000，优选为1000-4200；
    和/或，聚赖氨酸的熔点为170-175℃，优选为172-175℃。
  4. 根据权利要求1-3任一项所述的用于3D打印的浆料，其特征在于，所述浆料还包括有效生物成分，有效生物成分包括营养物质、细胞外基质、生长因子、细胞或药物活性成分中的一种或几种。
  5. 一种权利要求1-4任一项所述的用于3D打印的浆料的制备方法，其特征在于，包括以下步骤：
    将海藻酸盐、聚赖氨酸和水混合均匀，得到用于3D打印的浆料；
    优选地，将海藻酸盐加入聚赖氨酸和水的溶液中，混合均匀得到浆料；
    优选地，聚赖氨酸和水的溶液中还包括有效生物成分。
  6. 一种3D结构体，其特征在于，采用权利要求1-4任一项所述的用于3D打印的浆料或权利要求5所述的用于3D打印的浆料的制备方法制得的浆料制得；
    所述3D结构体包括组织修复支架、活组织或类器官体，优选为组织修复支架。
  7. 一种权利要求6所述的3D结构体的制备方法，其特征在于，包括以下 步骤：
    使用所述用于3D打印的浆料进行3D打印，得到3D结构体；
    优选地，3D打印的打印压力为50-600kPa，打印速度为5-10mm/s。
  8. 根据权利要求7所述的3D结构体的制备方法，其特征在于，所述方法还包括将得到的3D结构体置于缩合剂溶液或缩合剂和稳定剂的混合溶液中，得到化学交联3D结构体的步骤；
    缩合剂和稳定剂的摩尔比为1-2:1；
    缩合剂包括水溶性或油溶性缩合剂，优选为水溶性缩合剂，进一步优选为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；
    稳定剂包括酰亚胺或苯并三唑类稳定剂，优选为N-羟基丁二酰亚胺或1-羟基苯并三唑。
  9. 根据权利要求8所述的3D结构体的制备方法，其特征在于，所述方法还包括将得到的化学交联3D结构体置于聚赖氨酸溶液中，得到氨基修饰的3D结构体的步骤；
    聚赖氨酸溶液的质量分数为2-10％，优选为4-6％；
    所述方法还包括将得到的氨基修饰的3D结构体置于有效生物成分溶液中，得到负载有效生物成分的3D结构体。
  10. 一种权利要求6所述的3D结构体或权利要求7-9任一项所述的3D结构体的制备方法制得的3D结构体在组织工程中的应用。
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CN114099770A (zh) * 2021-11-02 2022-03-01 常州大学 一种自固化3d打印生物墨水及其制备方法和应用
WO2023231762A1 (zh) * 2022-06-01 2023-12-07 苏州大学 一种粘弹膏体材料高精度3d打印设备及打印方法

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