CN109381749B

CN109381749B - Bone tissue repair ink, composition, scaffold, preparation method and kit

Info

Publication number: CN109381749B
Application number: CN201811238197.7A
Authority: CN
Inventors: 徐铭恩; 王玲
Original assignee: Regenovo Biotechnology Co ltd
Current assignee: Regenovo Biotechnology Co ltd
Priority date: 2018-10-23
Filing date: 2018-10-23
Publication date: 2021-11-05
Anticipated expiration: 2038-10-23
Also published as: CN109381749A

Abstract

The invention relates to the field of bone tissue engineering, in particular to bone tissue repair ink, a composition, a stent, a preparation method and a kit. The bone tissue repair ink and the bioactive carrier wrapping the cells are used as raw materials, a biological printer is adopted, and the bone tissue repair support is printed according to a preset three-dimensional structure. Meanwhile, the bone tissue repair ink and the bioactive carrier wrapping the cells are used as raw materials, so that living cell printing is realized. The cells are uniformly distributed on the scaffold model formed by the bone tissue repair ink and are not easy to slide to the bottom of the scaffold, and the problem that certain characteristic proteins of the cells are easy to lose in the prior art is effectively solved. Is favorable for the growth of cells on the bracket. And better simulates the growth environment of human bone cells, promotes the proliferation, directional differentiation and specific protein expression of the cells, is beneficial to the cell extension and migration in the bone tissue scaffold, establishes cell connection and forms an organic construct.

Description

Bone tissue repair ink, composition, scaffold, preparation method and kit

Technical Field

The invention relates to the field of bone tissue engineering, in particular to bone tissue repair ink, a composition, a stent, a preparation method and a kit.

Background

At present, clinically, bone defects caused by infection, tumor and trauma are always difficult to treat clinically due to the limitation of autologous bone sources and the specificity of bone structures, and the traditional bone transplantation technology is difficult to prepare a support material with personalized characteristics according to the structure of a specific damaged part. This is because the scaffold constructed by the conventional methods (including solution casting/ion washing out method, electrostatic spinning hammer method, phase separation/freeze drying method, in-situ forming method, gas/pore-forming fiber weaving method, fiber bonding method, melt molding method, and air pressure pore-forming method, etc.) lacks stable structure, has poor mechanical properties, remains harmful chemical solvent, is difficult to realize the formation of complex heterogeneous material scaffold, accurately controls pore structure, and realizes personalized implant manufacture, etc., and thus cannot meet the preparation requirements of tissue engineering.

But the research and development of bone tissue engineering scaffold materials and autologous cell transplantation open up a new treatment idea for bone defects.

The scaffold is used as a place for cell planting and a template for tissue regeneration, is an important research content in bone tissue engineering, and has become a bottleneck for restricting the clinical transformation and application of the bone tissue engineering. In bone tissue engineering, an ideal scaffold material should have the following properties: good biocompatibility; excellent biomechanical property and easy processing and forming; suitable biodegradability, the degradation rate of which should be matched with the formation rate of new bone; good osteoinductive and osteoconductive properties; moderate price and sufficient source.

In the prior art, in the application of bone tissue engineering, a common three-dimensional scaffold has the problem that the expression of certain characteristic proteins of cells is easy to lose, and the growth of the cells on the scaffold is influenced.

Disclosure of Invention

The first purpose of the invention is to provide bone tissue repair ink and a preparation method thereof.

The second purpose of the invention is to provide a bone tissue repair composition and a preparation method thereof.

The third purpose of the invention is to provide a bone tissue repair scaffold and a preparation method thereof.

The fourth purpose of the invention is to provide a bone tissue repair kit.

In order to achieve the above purpose, the embodiment of the present invention adopts the following technical solutions:

a preparation method of bone tissue repair ink comprises the following steps: mixing at least one biodegradable material and at least one auxiliary forming material to prepare slurry; or at least one biodegradable material is made into a slurry.

A bone tissue repair ink is prepared by adopting the preparation method of the bone tissue repair ink.

A bone tissue repair composition, which comprises the bone tissue repair ink, a bioactive carrier and a cell unit; wherein the bioactive carrier comprises bioactive hydrogel and bioactive factor.

A preparation method of the bone tissue repair composition adopts the preparation method of the bone tissue repair ink to prepare the bone tissue repair ink. Recovering the cells, centrifuging, and adding a culture medium to culture to obtain a cell unit; dissolving the bioactive hydrogel to obtain a bioactive carrier; after mixing the bone tissue repair ink, the cell unit and the bioactive carrier, the pH was adjusted to 7.4.

A method for preparing a bone tissue repair scaffold, comprising: the bone tissue repair ink and the bioactive carrier wrapping the cells are used as raw materials, and a bone tissue repair scaffold is constructed according to a preset three-dimensional structure; the bone tissue repair scaffold is constructed by a bioprinting method. Further, when the bone tissue repair scaffold is constructed by adopting a bioprinting method, a part of bone tissue repair ink is used as a printing material, and the rest of bone tissue repair ink is used as a replenishing material, and the printing is continuously carried out. In the continuous printing process, the preparation proportion of the materials used by the brackets in the same batch can be flexibly adjusted, and the use and proportion adjustment of the printing materials are monitored on line in real time; the proportion of the materials used in the same batch of brackets can be flexibly adjusted, and the preparation of two or more materials can be realized.

A bone tissue repair scaffold is prepared by the preparation method of the bone tissue repair scaffold; the bone tissue repair scaffold comprises a plurality of fiber layers; the multiple fiber layers are arranged in parallel; each fiber layer comprises a bracket structure made of bone tissue repair ink and a bracket structure made of a bioactive carrier wrapping cells; or each two adjacent fiber layers comprise a scaffold structure made of bone tissue repair ink or a scaffold structure made of a bioactive carrier wrapping cells.

A bone tissue repair kit, the bone tissue repair kit comprising: a bioactive carrier, a bone tissue repair ink as described above, a bone tissue repair composition as described above, and a bone tissue repair scaffold as described above.

The invention has the beneficial effects that:

the invention provides a preparation method of bone tissue repair ink, which comprises the following steps: mixing at least one biodegradable material and at least one auxiliary forming material to prepare slurry; or at least one biodegradable material is made into a slurry. The bone tissue repair ink prepared by the method can provide suitable mechanical properties.

The preparation method of the bone tissue repair composition provided by the invention comprises the steps of recovering cells, centrifuging, adding a culture medium, and culturing to obtain a cell unit; dissolving the bioactive hydrogel to obtain a bioactive carrier; after mixing the bone tissue repair ink, the cell unit and the bioactive carrier, the pH was adjusted to 7.4. The bone tissue repairing composition prepared by the method better simulates the growth environment of human bone cells, promotes the proliferation and the directional differentiation of the cells and the expression of specific proteins.

The invention provides a preparation method of a bone tissue repair scaffold, which takes bone tissue repair ink with biocompatibility and a bioactive carrier wrapping cells as raw materials and constructs the bone tissue repair scaffold according to a preset three-dimensional structure. The bone tissue repair scaffold is constructed by a bioprinting method. The method simultaneously adopts bone tissue repair ink and a bioactive carrier wrapping cells as raw materials, and realizes living cell printing. The cells are uniformly distributed on the scaffold model formed by the bone tissue repair ink and are not easy to slide to the bottom of the scaffold, and the problem that certain characteristic proteins of the cells are easy to lose in the prior art is effectively solved. Is favorable for the growth of cells on the bracket. And better simulates the growth environment of human bone cells, promotes the proliferation, directional differentiation and specific protein expression of the cells, is beneficial to the cell extension and migration in the bone tissue scaffold, establishes cell connection and forms an organic construct.

The bone tissue repair scaffold provided by the invention is prepared by adopting the preparation method of the bone tissue repair scaffold. The bone tissue repair scaffold comprises a plurality of fiber layers; the multiple fiber layers are arranged in parallel; each fiber layer comprises a bracket structure made of bone tissue repair ink and a bracket structure made of a bioactive carrier wrapping cells; or each two adjacent fiber layers comprise a scaffold structure made of bone tissue repair ink or a scaffold structure made of a bioactive carrier wrapping cells. The bone bioremediation ink can provide adaptive mechanical properties, the bioactive carrier simulates extracellular matrix components, and the support is favorable for promoting the growth of bone cells and the formation of a cartilage integrated structure.

The invention provides a bone tissue repair kit, which comprises: a bioactive carrier, a bone tissue repair ink as described above, a bone tissue repair composition as described above, and a bone tissue repair scaffold as described above. The bone tissue repair kit can be used for bone tissue repair.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic structural diagram of a bone tissue repair scaffold prepared by a bioprinting method according to an embodiment of the present invention;

FIG. 2 is a schematic flow chart of a process for preparing a bone tissue repair scaffold by a bioprinting method according to an embodiment of the present invention;

FIG. 3 is a schematic view of a continuous printing method for preparing a bone tissue repair scaffold by using a bio-printing method according to an embodiment of the present invention;

FIG. 4 is a schematic structural diagram of a bone tissue repair scaffold provided in an embodiment of the present invention;

FIG. 5 is a flow chart of a continuous printing method for preparing a bone tissue repair scaffold by a bio-printing method according to an embodiment of the present invention;

fig. 6 is a flowchart of another continuous printing method for preparing a bone tissue repair scaffold by using a bioprinting method according to an embodiment of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In the description of the present invention, it should be noted that the terms "first", "second", and the like are used only for distinguishing the description, and are not intended to indicate or imply relative importance.

The bone tissue repair ink, the composition, the scaffold, the preparation method and the kit according to the embodiments of the present invention are described in detail below.

Some embodiments of the present invention provide a bone tissue repair ink comprising a biodegradable material and other auxiliary molding materials.

The bone tissue repair ink comprises at least one biodegradable material and can provide a good substrate for bone defect repair, such as no cytotoxicity, no immunogenicity or low immunogenicity; can be biodegraded and replaced by new bone in a relatively short time; has a certain structure, and can play a role of a bracket of a bone defect area (bone conduction function); has the effect of inducing osteogenesis.

The biodegradable material comprises degradable inorganic substances, natural polymer materials, degradable synthetic polymer materials, composite materials of different types or the same type and the like.

Further, the biodegradable material has osteoconductivity or osteoinductivity. Can be integrated into host bone in different degrees, and can provide suitable environment for seed cells to promote tissue regeneration and provide conditions for adhesion, differentiation, proliferation or migration of the seed cells.

Furthermore, the biodegradable material has certain mechanical strength and can realize three-dimensional stacking; has good mechanical property and can match the supporting performance required by the corresponding bone defect part.

Further, degradation of the biodegradable material can provide a microenvironment that maintains or promotes bone cell vital activities, e.g., the degradation products are small molecule compounds including organic acids, monosaccharides (e.g., glucose), oligosaccharides, amino acids, lipids, and the like. Such degradation products may be involved in metabolic activities of the cells (e.g. for the synthesis of extracellular matrix), or used for the synthesis of extracellular matrix or converted into energy required for the activity.

Further, inorganic substances such as: tricalcium phosphate, hydroxyapatite, calcium phosphate, calcium silicate, magnesium oxide, magnesium hydroxide, magnesium chloride, magnesium carbonate, magnesium phosphate, magnesium silicate, iron oxide, ferroferric oxide, iron hydroxide, iron chloride, strontium carbonate and the like, have good biocompatibility and osteoconductivity similar to the components of natural bone matrixes, and can form a bioactive cell-free layer on the surface after being implanted into a body, wherein the bioactive cell-free layer is rich in calcium phosphate, mucopolysaccharide and glycoprotein, provides a suitable environment for the deposition of collagen and bone mineral, and provides support for the realization of cell biological functions. Thus, in certain preferred embodiments, the biodegradable material used to prepare the bone tissue repair ink is or contains a degradable mineral.

Furthermore, the natural polymer material refers to bone extracellular matrix or its analogues, such as: type I collagen fibers and an amorphous matrix, wherein the amorphous matrix is a gel comprising a plurality of non-collagenous proteins, glycoproteins, proteoglycans, polypeptides, carbohydrates, lipids, and the like; hydroxyapatite and amorphous calcium phosphates, and some CO₃ ^2-、C1^-、F^-、Na⁺、K⁺、Mg²⁺MiscellaneousThe micro-environment of bone tissue in human body can be simulated by using the micro-environment of proton ions and microelements such as Sr and Zn, which is more beneficial to promoting the adhesion, extension, growth, proliferation and differentiation of cells and the establishment of cell information interaction.

Still further, degradable synthetic polymeric materials include Polycaprolactone (PCL), polydioxanone (PPDO), polyacrylic acid and its derivatives (e.g., polymethacrylic acid, copolymers of acrylic acid and methacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid copolymer (PLGA), Polyorthoesters (POE), Polycaprolactone (PCL), Polyhydroxybutyrate (PHB), polyaminoacids (e.g., polylysine), degradable polyurethanes, and any combination thereof.

Further, different types or the same type of composite materials include Hyaluronic Acid (HA)/Hydroxyapatite (HA), extracellular matrix deprived (dmemc)/Hydroxyapatite (HA) composite, Collagen (COL)/Polycaprolactone (PCL) composite, Collagen (COL)/tricalcium phosphate (TCP), tricalcium phosphate (TCP)/polylactic acid-glycolic acid copolymer (PLGA) composite, and the like, and any combination thereof.

Assisted molding material, refers to a biomaterial that can assist the molding of the construct and is compatible with cells, and includes, but is not limited to, one or more combinations of bio-dispersants, bio-binders, bio-lubricants, bio-anticoagulants, cross-linking agents, solvents, and the like.

Further, "biodispersant" refers to a degradable material, such as polyacrylamide, that is compatible with cells and organisms for dispersion.

Further, "bio-lubricant" refers to degradable materials that act to lubricate, smooth the flow of bone tissue repair ink, and are compatible with cells and organisms, such as glycerol;

further, "biological adhesive" refers to degradable polymers (including but not limited to hyaluronic acid, chitosan, collagen or sodium alginate, etc.) which have an adhesive effect and can realize crosslinking or solidification in other forms, such as alginate crosslinked by calcium chloride, collagen crosslinked by genipin, methacrylate gelatin crosslinked by light, etc., and the biological adhesive not only can adjust the viscosity of bioactive ceramic slurry, but also has a crosslinkable property of avoiding a scaffold sintering step, simplifying a scaffold preparation process, and simultaneously realizing synchronous printing with cells, solving the problem of uneven cell distribution caused by scaffold inoculation, thereby being more beneficial to the growth, proliferation and differentiation of cells.

Some embodiments of the present invention provide a method for preparing bone tissue repair ink, comprising the steps of:

(1) a mixture of one or more biodegradable materials is provided.

(2) A mixture of one or more secondary molding materials is provided.

(3) Selecting a material formula 1 from one or more biodegradable materials in the step (1), selecting a formula 2 from one or more auxiliary forming materials in the step (2), and slurrying the

formulas

1 and 2 to form the bone tissue repair ink.

Further, the biodegradable material in the bone tissue repair ink is hydroxyapatite or tricalcium phosphate, and the mass ratio of the hydroxyapatite to the tricalcium phosphate in the bone tissue repair ink is about 0:10-10:0, such as 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and 10: 0. The mass percentage of the mixture of the hydroxyapatite and the tricalcium phosphate in the bone tissue repair ink is 50-80%. In certain preferred embodiments, the binder is sodium alginate, the cross-linking agent is calcium chloride, the lubricant is glycerol, and the dispersant is polyacrylamide, wherein the bone tissue repair ink auxiliary molding material accounts for 20-50% by mass.

Further, the biodegradable material in the bone tissue repair ink is hydroxyapatite or magnesium phosphate, and the mass ratio of the hydroxyapatite to the magnesium phosphate in the bone tissue repair ink is about 0:10-10:0, such as 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and 10: 0. The mass percentage of the mixture of hydroxyapatite and magnesium phosphate in the bone tissue repair ink is 50-90%. In some preferred embodiments, the binder is collagen, the cross-linking agent is genipin, the lubricant is glycerol, and the dispersant is polyacrylamide, wherein the bone tissue repair ink auxiliary forming material accounts for 10-50% by mass.

Further, the biodegradable material in the bone tissue repair ink is hydroxyapatite or polylactic acid-glycolic acid copolymer, and the mass percentage of the hydroxyapatite in the bone tissue repair ink is about 0% -100%, such as 0%, 1%, 2%, 3%, and 4%. The mixture of hydroxyapatite and polylactic acid-glycolic acid copolymer accounts for 50-90% by mass of the bone tissue repair ink, such as 50%, 51%, 52%. the. 77%, 78%, 79%, 80%, 90%. In some preferred embodiments, the binder is methylcellulose, and the solvent is 1, 4-dioxane, wherein the bone tissue repair ink auxiliary forming material accounts for 10-50% by mass.

Further, the biodegradable material in the bone tissue repair ink is tricalcium phosphate or polycaprolactone polymer, for example, tricalcium phosphate accounts for about 0-80% by mass of the bone tissue repair ink, for example, 0%, 1%, 2%, 3%, 4%. 78%, 79%, 80%. The polycaprolactone accounts for 20-100% of the bone tissue repair ink, such as 20%, 21%, 22%, 97%, 98%, 99%, 100%. In certain preferred embodiments, the bone tissue repair ink does not comprise a secondary modeling material.

Further, the bone tissue repair ink includes, but is not limited to, the following combinations:

some embodiments of the invention provide a bone tissue repair composition comprising a bone tissue repair ink, a bioactive carrier, and a cell unit, wherein the bioactive carrier comprises a bioactive hydrogel and a bioactive factor.

The bioactive hydrogel comprises: natural bioactive hydrogel, artificially synthesized biological hydrogel, modified biological hydrogel, or biological hydrogel compounded by materials of the same type or different types, or any combination of the above biological hydrogels.

Further, natural bioactive hydrogels, including collagen, fibrin, silk fibroin, chitosan, alginate, starch, hyaluronic acid, laminin, agarose, gelatin, dextran, and any combination thereof;

further, artificially synthesized biological hydrogel comprises polyacrylic acid and derivatives thereof, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene oxide and derived copolymers thereof, polyphosphazene and the like, and any combination form thereof;

further, modified biological hydrogels, including modified gelatin, modified hyaluronic acid, modified chondroitin, such as methacrylate gelatin (GelMA), methacrylate hyaluronic acid (GelHA), methacrylate chondroitin, and the like, and any combination thereof;

furthermore, biological hydrogel compounded by materials of the same type or different types comprises methacrylic acid gelatin composite hyaluronic acid, sodium polyacrylate composite collagen, methacrylic acid hyaluronic acid composite collagen and the like, which are compounded in any form;

the bioactive factor refers to a class of active ingredients which can promote bone growth, induce bone formation or/and prevent bone loss in the bone formation process and have beneficial effects, and the bioactive factor comprises bone morphogenetic factors, osteogenic factors, bone induction factors, bone resorption resisting agents, growth promoters, antibacterial agents and the like.

Further, osteoinductive factors, such as: certain Bone Morphogenetic Proteins (BMPs), growth and differentiation factors (GDPs), the intracellular factor LIM mineralization protein-1 (LIM)), osteoprotegerin, RUNX2, transforming growth factor beta, fibroblast growth factor, Bone Morphogenetic Proteins (BMPs), and any combination thereof;

further, anti-bone resorption agents, are bisphosphonates such as: zoledronic acid, pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, risedronic acid, and the like, as well as any combinations thereof.

Further, antibacterial agents such as silver, copper, fosfomycin, antibiotics.

Cell units, including but not limited to cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, multicellular bodies, subcellular structures (e.g., organelles and cell membranes).

Some embodiments of the present invention provide a method for preparing a bone tissue repair composition, comprising the steps of:

(1) preparing bone tissue repair ink;

a) a mixture of one or more biodegradable materials is provided.

b) A mixture of one or more secondary molding materials is provided.

c) Selecting a material formula 1 from one or more biodegradable materials in the step (a), selecting a formula 2 from one or more auxiliary molding materials in the step (b), and slurrying the

formulas

1 and 2 to form the bone tissue repair ink.

(2) Preparation of cell units

a) Putting the frozen cells into a shaking water bath at 30-37 deg.C for 50-100s for resuscitation

b) The cells were transferred to a centrifuge tube containing the culture medium and centrifuged.

c) And (4) absorbing and removing the centrifuged supernatant culture solution, and adding a proper amount of culture medium to enable the final concentration of the cells to be suitable for the experimental requirements for later use.

(3) Preparing a bioactive carrier;

dissolving the bioactive hydrogel with corresponding solvent to proper concentration, mixing the bioactive hydrogel with culture medium, bioactive factor and prepared cell in certain proportion, and regulating pH to 7.4 with buffer solution and acid-base solution.

The prepared bone tissue repair ink, the cell unit and the bioactive carrier jointly form the bone tissue repair composition.

Referring to fig. 1-2, some embodiments of the present invention provide a method for preparing a bone tissue repair scaffold, including the following steps:

s1, preparing the biodegradable bone tissue repair ink.

Specifically, in the present embodiment, the bone tissue repair ink prepared in the foregoing embodiment is used.

S2, preparing the bioactive carrier for wrapping the cell.

Specifically, the bioactive carrier for wrapping the cells is prepared by mixing bioactive hydrogel and bioactive factors according to the volume ratio of 1: 1.

Further, the bioactive hydrogel is selected from any one of a collagen solution, a mixed solution of gelatin and sodium alginate, or a chitosan solution.

Further, the bioactive factor is selected from any one of osteoblast or mesenchymal stem cell containing bone marrow.

Alternatively, the bioactive hydrogel used in combination with the bone tissue repair composition may be a natural biological material (e.g., collagen, fibrin, silk fibroin, chitosan, alginate, starch, hyaluronic acid, laminin, agarose, gelatin, dextran, and any combination thereof), a synthetic biological hydrogel (e.g., polyacrylic acid and its derivatives, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene oxide and its derivative copolymers, polyphosphazene, and the like, and any combination thereof), a modified biological hydrogel, a biological hydrogel compounded from the same or different types of materials, or any combination thereof.

Alternatively, the biodegradable hydrogel used in combination with the bone tissue repair composition is a naturally occurring degradable polymer. Preferably, the degradable polymer is selected from the group consisting of collagen, fibrin, chitosan, alginate (e.g. sodium alginate), starch, hyaluronic acid, laminin, agarose, gelatin, silk fibroin, dextran, and any combination thereof.

Alternatively, the biodegradable hydrogel for use in combination with the bone tissue repair composition is a modified degradable polymer, such as modified gelatin, modified hyaluronic acid, modified chondroitin, such as methacrylate gelatin (GelMA), methacrylate hyaluronic acid (GelHA), methacrylate chondroitin, and the like, and any combination thereof.

Alternatively, the biodegradable hydrogel used in combination with the bone tissue repair composition is a synthetic degradable polymer such as polyacrylic acid and its derivatives, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene oxide and its derived copolymers, polyphosphazene, and the like, and any combination thereof.

Alternatively, the biodegradable hydrogel for use in combination with the bone tissue repair composition is a degradable polymer compounded with different types of materials, such as methacrylic acid gelatin complex hyaluronic acid, sodium polyacrylate complex collagen, methacrylic acid hyaluronic acid complex collagen, and the like, in any form thereof.

Alternatively, the biologically active factors of the biologically active carrier (e.g., bioactive agents that promote bone growth) and their degradation products are non-toxic to the cell and/or non-immunogenic to the host and secrete specific proteins in the form of peptides that promote adherent growth of seed cells, induce directed differentiation thereof. In certain preferred embodiments, the bioactive factor may be an osteoinductive factor (e.g., certain Bone Morphogenetic Proteins (BMPs) and certain growth and differentiation factors (GDPs), as well as certain intracellular factors LIM mineralization protein-1 (LIM)), an anti-bone resorption agent (e.g., an anti-phosphonate), a growth promoter, an antimicrobial agent (e.g., silver, copper, fosfomycin, antibiotics), other bioactive agents, and the like, as well as any combination thereof.

Alternatively, the bioactive factor in the bioactive carrier is an osteoinductive factor, such as certain Bone Morphogenetic Proteins (BMPs) and some growth and differentiation factors (GDPs), some intracellular factors LIM mineralization protein-1 (LIM), and any combination thereof.

Alternatively, the biologically active factor in the biologically active carrier is an osteogenic factor, such as osteoprotegerin, RUNX2, transforming growth factor beta, fibroblast growth factor, bone morphogenic protein, and any combination thereof.

Alternatively, the biologically active carrier encapsulating the cells is an anti-resorptive agent, which in certain preferred embodiments is a bisphosphonate. Bisphosphonates are preferably used as specific inhibitors of osteoclasts, inhibiting bone loss. A bisphosphonate selected from the group consisting of zoledronic acid, pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, risedronic acid, and the like, and any combination thereof.

Alternatively, degradation of the bioactive carrier degradable material can provide a microenvironment, such as nutrients, that maintains or promotes bone cell vital activities. In certain preferred embodiments, the degradation products are small molecule compounds, such as organic acids, monosaccharides (e.g., glucose), oligosaccharides, amino acids, lipids, and the like. Such degradation products may be involved in metabolic activities of cells (e.g. for the synthesis of extracellular matrix), for the synthesis of extracellular matrix or for conversion to energy required for the activity.

The microenvironment refers to the environment in which cells grow, and comprises elements including physical factors such as spatial structure, mechanical strength, temperature, humidity, osmotic pressure and the like; chemical factors such as ph, ion concentration, etc.; biological factors including cells, cytokines, etc. These elements together constitute the environment in which cells live and dynamically regulate the proliferation, differentiation, migration, secretion and metabolism of cells growing in this environment.

And S3, constructing the bone tissue repair scaffold according to the preset three-dimensional structure.

Further, the bone tissue repair scaffold is constructed by any one of bioprinting, coating, pouring or filling.

In the present embodiment, a bioprinting method is selected for construction.

The method comprises the following specific steps:

1. a biological printer is adopted, bone tissue repair ink preparation slurry and biological activity carrier ink (capable of mixing cells for synchronous printing) are respectively added into the charging barrel 1 and the charging barrel 2.

2. The corresponding functions (such as, but not limited to, cooling or/and heating and/or curing) are initiated and the material is processed to the corresponding requirements according to the characteristics of the bone tissue repair ink slurry and the bioactive carrier ink.

3. And importing a corresponding CAD model according to the model requirement, and properly adjusting the printing parameters of the biological printer to generate a corresponding reasonable path. And starting a printing program, and printing the three-dimensional construct of the corresponding bone tissue repair ink and the bioactive carrier combination on a clean slide.

4. And soaking the printed bone tissue repair combined scaffold in a cross-linking agent solution for a period of time to ensure that the scaffold is completely cross-linked, thereby improving the stability of the scaffold.

The CAD model is a bone defect repair module which is used for reconstructing a three-dimensional image of a bone defect of a patient by using medical image processing software according to full bone segment image data obtained by CBCT examination of a damaged part of the patient, establishing a bone model, determining the size, the structure and the area of the bone defect and individually designing the bone defect repair module matched with the appearance of the defect area.

The proper adjustment of the printing parameters means that the influence of the printing parameters (such as printing layer thickness, filling angle, filling interval, spray head temperature and platform temperature) on the structural precision and physicochemical characteristics of the bracket is revealed by adjusting the printing parameters; designing a bioactive ceramic combined bracket which is identical with the internal structure and performance of the natural bone.

Furthermore, according to the formula characteristics of different bone tissue repair ink slurries and bioactive carrier inks, different forms of stent post-treatment modes can be selected. Specific stent post-treatment means include, but are not limited to, crosslinking, photocuring, freeze-drying, coating, and any combination thereof.

Further optionally, bioprinting is used to construct a three-dimensional construct that is required to meet the 4F criteria design requirements. Among them, the 4F guidelines, including shape complaints (Form), are that the shape of the scaffold material must completely fill the three-dimensional defect and can induce regeneration of the corresponding tissue; performance requirements (functions), namely mechanical strength, performance and the like of the stent, can temporarily play a role in replacing the missing tissue before the missing tissue; functional appeal (format), material needs corresponding biological activity, can provide suitable environment for seed cells to promote tissue regeneration, and provide conditions of adhesion, differentiation, proliferation or migration for seed cells. Implantable (i.e., scaffolding) materials can be implanted into the body by surgical procedures, requiring that the scaffolding material be capable of being secured to a bone defect and provide a suitable surface to meet the Fixation requirements and to perform its intended function.

Further optionally, the method of bioprinting a build scaffold enables continuous printing (fig. 3), comprising the steps of: the ink used during printing is divided into printing material and supply material, wherein the printing material divides the material into residual material a1 and printing material b1 during printing.

Printing material b1 will complete the printing of object 1; the residual material a1 will be divided into residual material a2 and printed material b2 when the stent is continuously printed.

The printing material b2, if its composition is insufficient, may be supplemented completely by the supply material c1, and then the object 2 is printed.

The residual material a2 is to be re-divided into residual material a3 and printing material b 3; when the printed material b3 is insufficient in some components, the object 3 can be printed after the replenishment material c2 is completed.

In the process of printing on the bracket, when the situation that the printing material is insufficient is monitored for the first time, the materials are divided into a supply material c1 and a residual material d1, wherein the supply material c1 supplies the printing material b2 to finish printing; when the printing material shortage is monitored for the second time, the residual material d1 is divided into a supply material c2 and a residual material d2, wherein the supply material c2 supplies the printing material b3 to finish printing; in the subsequent printing process, the supply materials are sequentially and circularly distributed according to the number of the printed objects.

The preparation method can realize the continuous printing of the bracket, can flexibly adjust the preparation proportion of the materials used by the bracket in the same batch in the continuous printing process, and can realize the real-time online monitoring of the use and proportion adjustment of the printing materials; wherein, the flexible adjustment of the proportion of the materials used in the same batch of the stent is understood to realize the preparation of two or more materials.

Some embodiments of the present invention further provide a bone tissue repair scaffold, which is prepared by the above method for preparing a bone tissue repair scaffold.

Specifically, the bone tissue repair scaffold comprises a plurality of fibrous layers; the multiple fiber layers are arranged in parallel. Each fiber layer comprises a bracket structure made of bone tissue repair ink and a bracket structure made of a bioactive carrier wrapping cells; or each two adjacent fiber layers comprise a scaffold structure made of bone tissue repair ink or a scaffold structure made of a bioactive carrier wrapping cells.

Further optionally, the bracket comprises: by supreme multilayer fibrous layer together of fixed connection in proper order down, multilayer fibrous layer parallel arrangement, every layer fibrous layer includes a plurality of parallel arrangement's cellosilk, and the cellosilk of adjacent two-layer fibrous layer is crisscross to be set up.

Further optionally, the stent has the fiber filaments in two adjacent fiber layers staggered by an angle of 0 DEG theta-180 deg. The cross-sectional shape of the holes formed by the plurality of fiber filaments of the at least two fiber layers in a staggered manner is a polygon or a circle.

Further optionally, for the same material, in the same fiber layer, the distance between two adjacent fiber filaments is the pore diameter; in the same fiber unit, the fiber filaments in the same layer of fiber layer have the same pore size of the formed pores.

In this embodiment, specifically, referring to fig. 4, the scaffold is formed such that the fiber yarn formed by the bone tissue repair ink (white fiber yarn) and the fiber yarn formed by the bioactive carrier (black fiber yarn) constitute one fiber unit. As shown in fig. 4, the bone tissue repair ink fiber (white fiber) and the bioactive carrier fiber (black fiber) are located in the same fiber layer or different fiber layers, and the bioactive carrier fiber (black fiber) is located between the adjacent fibers of the bone tissue repair ink interval. Different fiber layers, the bioactive carrier fiber (black fiber) and the bone tissue repair ink fiber (white fiber) are positioned in two adjacent layers in parallel or perpendicular. For the same material (such as bone tissue repair ink or bioactive carrier), the fiber filaments in the fiber layer are parallel and not coplanar or arranged in parallel and coplanar with the fiber filaments in other fiber layers. Further, a plurality of fiber units form the three-dimensional scaffold of the bone tissue repair ink composition.

Further, the shape of the scaffold is polygonal column, cylinder, cartilage model or hard bone model.

Some embodiments of the invention provide a bone tissue repair kit, which comprises one or more of a bone tissue repair ink, a bioactive carrier, a bone repair composition, a three-dimensional construct scaffold, or other components.

The kit comprises at least one bioremediation ink.

The three-dimensional construct scaffold in the kit is based on the natural structure and cell arrangement pattern of the construct to be printed.

Other components of the kit include containers, media, buffers, other reagents required for bioprinting, instructions, and the like.

The kit may be placed in any suitable packaging including, but not limited to, bottles, jars, and flexible packaging (e.g., mylar or plastic bags).

The kit can have various uses, such as the bone tissue repair ink and the composition thereof can be used for culturing cells (for example, the three-dimensional culture of osteoblasts); or for cell growth, proliferation, differentiation, secretion, or migration; or for bioprinting (e.g., 3D bioprinting); or for constructing a three-dimensional construct scaffold (e.g., a three-dimensional construct, a tissue precursor, a tissue, or an organ); or other applications, such as analyzing changes in cells in response to stimuli or agents (e.g., morphological changes or functional changes), for drug screening or drug discovery, for treating a subject in need thereof, for studying stem cell differentiation, for assessing the effect of factors (e.g., chemical agents, compounds; physical stimuli, such as radiation or heat) on cells in tissue or tissues, for three-dimensional tissue culture, for in vivo or in vitro assays, for implantation into a host, for tissue engineering, or for tissue regeneration.

The features and properties of the present invention are further described in detail below with reference to examples:

example 1

The embodiment provides a bone tissue repair scaffold, which is prepared by the following steps:

(1) the slurry with the solid content of 38 vol% is prepared by taking beta-tricalcium phosphate (beta-TCP) and Hydroxyapatite (HAP) mixed powder with the mass ratio of 7:3 as a raw material, taking a mixture of deionized water and glycerol (with the volume ratio of 6:4) as a solvent and sodium polyacrylate as a dispersing agent, and performing gradient centrifugal stirring for 1h (the maximum rotating speed is set to 3000rpm) by using a vacuum defoaming machine. Adding collagen as biological binder into the slurry, wherein the addition amount is 1 wt% of the mixed powder, mixing for 2h by using a vacuum defoaming machine, and obtaining the bioactive ceramic slurry by ultrasonic oscillation (frequency 100Hz, time 30min and temperature 30 ℃) and low-temperature defoaming (time 12h and temperature 4 ℃) modes, wherein the viscosity of the slurry is lower than 100 Pa.S, and the solidification time in the air is less than 1 min.

(2) Dissolving the I-type collagen solution with 100mL/L and V/V acetic acid, fixing the volume to 50mg/L, then uniformly mixing the I-type collagen solution with 10-DMEM (containing osteoblasts) in a ratio of 1:1 to obtain a final concentration of 4.0 × 105cells/mL, and preferably adjusting the pH of the solution to 7.4 by using 1mol/L NaOH to obtain the cell-coated bioactive carrier.

(3) A biological printer is adopted, and bone tissue repair ink (beta-tricalcium phosphate-hydroxyapatite) and bioactive carrier ink (capable of mixing cells and synchronously printing) are respectively added into the charging barrel 1 and the charging barrel 2. The normal temperature control function is started according to the characteristics of the beta-tricalcium phosphate-hydroxyapatite, and the refrigeration function is started according to the characteristics of the collagen by the charging barrel 2. A CAD model of 10X 3mm3 was introduced, and printing parameters of bioprinter air pressure (0.25MPa), speed (8mm/s), layer thickness (0.32mm), filling pitch (1.0mm), head temperature (25 ℃), and platen temperature (controlled at 15 ℃) were adjusted to generate a corresponding rational path. And starting a printing program, and printing the three-dimensional construct of the corresponding bone tissue repair ink and the bioactive carrier combination on a clean slide. And soaking the printed combined three-dimensional construct in a calcium chloride cross-linking agent solution for a period of time to ensure that the scaffold is completely cross-linked, thereby improving the stability of the scaffold.

Example 2

(1) the slurry with the solid content of 38 vol% is prepared by taking beta-tricalcium phosphate (beta-TCP) and Hydroxyapatite (HAP) mixed powder with the mass ratio of 7:3 as a raw material, taking a mixture of deionized water and glycerol (with the volume ratio of 6:4) as a solvent and sodium polyacrylate as a dispersing agent, and performing gradient centrifugal stirring for 1h (the maximum rotating speed is set to 3000rpm) by using a vacuum defoaming machine. Adding sodium alginate as biological adhesive in 0.8 wt% of the mixed powder into the slurry, mixing for 2 hr in a vacuum defoaming machine, ultrasonic vibrating at 100Hz for 30min at 30 deg.c, and defoaming at low temperature for 12 hr at 4 deg.c to obtain bioactive ceramic slurry with viscosity lower than 100 Pa.S and setting time in air lower than 1 min.

(2) Respectively dissolving 0.8g of gelatin and 0.5g of sodium alginate in 10ml of deionized water to obtain a gelatin solution with the concentration of 8 wt% and a sodium alginate solution with the concentration of 5 wt%, uniformly mixing the gelatin solution and the sodium alginate solution at 37 ℃ according to the volume ratio of 1:2 of the gelatin to the sodium alginate, then uniformly mixing the gelatin solution and 10 × DMEM (containing mesenchymal stem cells) according to the ratio of 1:1, preferably using 1mol/L of NaHCO3 to adjust the pH of the solution to 7.4, and keeping the solution at 4 ℃ for later use.

(3) A biological printer is adopted, and bone tissue repair ink (beta-tricalcium phosphate-hydroxyapatite) and bioactive carrier ink (capable of mixing cells and synchronously printing) are respectively added into the charging barrel 1 and the charging barrel 2. The normal temperature control function is started according to the characteristics of the beta-tricalcium phosphate-hydroxyapatite, and the refrigeration function is started according to the characteristics of the gelatin and the sodium alginate in the charging barrel 2. A CAD model of 10X 3mm3 was introduced, and printing parameters of bioprinter air pressure (0.25MPa), speed (8mm/s), layer thickness (0.32mm), filling pitch (1.0mm), head temperature (25 ℃), and platen temperature (controlled at 15 ℃) were adjusted to generate a corresponding rational path. And starting a printing program, and printing the three-dimensional construct of the corresponding bone tissue repair ink and the bioactive carrier combination on a clean slide. And soaking the printed combined three-dimensional construct in a calcium chloride cross-linking agent solution for a period of time to ensure that the scaffold is completely cross-linked, thereby improving the stability of the scaffold.

Example 3

(1) the slurry with the solid content of 38 vol% is prepared by taking beta-tricalcium phosphate (beta-TCP) and Hydroxyapatite (HAP) mixed powder with the mass ratio of 7:3 as a raw material, taking a mixture of deionized water and glycerol (with the volume ratio of 6:4) as a solvent and sodium polyacrylate as a dispersing agent, and performing gradient centrifugal stirring for 1h (the maximum rotating speed is set to 3000rpm) by using a vacuum defoaming machine. Adding methacrylic gelatin as a biological binder into the slurry, wherein the adding amount is 1.2 wt% of the mixed powder, mixing for 2h by using a vacuum defoaming machine, and obtaining the bioactive ceramic slurry by ultrasonic oscillation (frequency 100Hz, time 30min, temperature 30 ℃) and low-temperature defoaming (time 12h, temperature 4 ℃) modes, wherein the viscosity of the slurry is lower than 100 Pa.S, and the solidification time in the air is less than 1 min.

(2) Dissolving chitosan with deacetylation degree of 85-95% in 0.1mol/L diluted hydrochloric acid, fully stirring for 2h to obtain clear chitosan solution, dissolving beta-sodium glycerophosphate in 0.5mL deionized water to obtain beta-sodium glycerophosphate solution, dropwise adding the beta-sodium glycerophosphate solution into the chitosan solution until the pH of the solution is neutral, uniformly mixing the solution with 10 × DMEM (containing mesenchymal stem cells) in a ratio of 1:1, and standing at 4 ℃ for later use.

(3) A biological printer is adopted, and bone tissue repair ink (beta-tricalcium phosphate-hydroxyapatite) and bioactive carrier ink (capable of mixing cells and synchronously printing) are respectively added into the charging barrel 1 and the charging barrel 2. The normal temperature control function is started according to the characteristics of the beta-tricalcium phosphate-hydroxyapatite, and the refrigeration function is started according to the characteristics of the chitosan by the charging barrel 2. A CAD model of 10X 3mm3 was introduced, and printing parameters of bioprinter air pressure (0.25MPa), speed (8mm/s), layer thickness (0.32mm), filling pitch (1.0mm), head temperature (25 ℃), and platen temperature (controlled at 15 ℃) were adjusted to generate a corresponding rational path. And starting a printing program, and printing the three-dimensional construct of the corresponding bone tissue repair ink and the bioactive carrier combination on a clean slide. And soaking the printed combined three-dimensional construct in a calcium chloride cross-linking agent solution for a period of time to ensure that the scaffold is completely cross-linked, thereby improving the stability of the scaffold.

Example 4

(1) weighing polycaprolactone with the molecular weight of 15w, and mixing the weighed polycaprolactone with the molecular weight of 1: adding hydroxyapatite according to the mass ratio of 1, heating the mixture of the two to 80 ℃, stirring at a high temperature for 30min until the two are uniformly mixed, and cooling to room temperature to obtain the polycaprolactone-hydroxyapatite composite biological ink.

(3) A biological printer is adopted, and bone tissue repair ink (polycaprolactone-hydroxyapatite) and bioactive carrier ink (capable of being synchronously printed by mixed cells) are respectively added into a charging barrel 1 and a charging barrel 2. The normal temperature control function is started according to the characteristics of polycaprolactone-hydroxyapatite, and the refrigeration function is started according to the characteristics of chitosan by the charging barrel 2. A CAD model of 10X 3mm3 was introduced, and printing parameters of bioprinter air pressure (0.25MPa), speed (8mm/s), layer thickness (0.32mm), filling pitch (1.0mm), head temperature (25 ℃), and platen temperature (controlled at 15 ℃) were adjusted to generate a corresponding rational path. And starting a printing program, and printing the three-dimensional construct of the corresponding bone tissue repair ink and the bioactive carrier combination on a clean slide.

Example 5

(3) A biological printer is adopted, and bone tissue repair ink (polycaprolactone-hydroxyapatite) and bioactive carrier ink (capable of being synchronously printed by mixed cells) are respectively added into a charging barrel 1 and a charging barrel 2. The normal temperature control function is started according to the characteristics of polycaprolactone-hydroxyapatite, and the refrigeration function is started according to the characteristics of gelatin and sodium alginate in the charging barrel 2. A CAD model of 10X 3mm3 was introduced, and printing parameters of bioprinter air pressure (0.25MPa), speed (8mm/s), layer thickness (0.32mm), filling pitch (1.0mm), head temperature (25 ℃), and platen temperature (controlled at 15 ℃) were adjusted to generate a corresponding rational path. And starting a printing program, and printing the three-dimensional construct of the corresponding bone tissue repair ink and the bioactive carrier combination on a clean slide.

Example 6

(3) A biological printer is adopted, and bone tissue repair ink (polycaprolactone-hydroxyapatite) and bioactive carrier ink (capable of being mixed with cells for synchronous printing) are respectively added into the charging barrel 1 and the charging barrel 2. The normal temperature control function is started according to the characteristics of polycaprolactone-hydroxyapatite, and the refrigeration function is started according to the characteristics of collagen in the charging barrel 2. A CAD model of 10X 3mm3 was introduced, and printing parameters of bioprinter air pressure (0.25MPa), speed (8mm/s), layer thickness (0.32mm), filling pitch (1.0mm), head temperature (25 ℃), and platen temperature (controlled at 15 ℃) were adjusted to generate a corresponding rational path. And starting a printing program, and printing the three-dimensional construct of the corresponding bone tissue repair ink and the bioactive carrier combination on a clean slide.

The bone tissue repair scaffolds provided in examples 1-6 and the scaffold provided in comparative example 1 were placed in a well plate for culture, 1mL of H-DMEM medium was added to the well plate, and the plate was left to stand in an incubator (37 ℃, 5% CO2) for culture, after which the medium was changed every 3d of the total amount. The modulation of cell behavior (including cell viability, cell adhesion, cell proliferation) by the scaffold was determined throughout the 7d culture period.

Comparative example

A bioactive ceramic scaffold is provided. The preparation method comprises the following steps:

(1) beta-tricalcium phosphate and hydroxyapatite bone tissue repair ink prepared according to the protocol of example 1 above, a slurry prepared from the bone tissue repair ink was added to a low temperature cartridge using a bioprinter.

(2) A CAD model of 10X 3mm3 was introduced, and printing parameters of bioprinter air pressure (0.25MPa), speed (8mm/s), layer thickness (0.32mm), filling pitch (1.0mm), head temperature (25 ℃), and platen temperature (controlled at 37 ℃) were adjusted to generate a corresponding rational path. And starting a printing program, and printing the three-dimensional construct of the corresponding bone tissue repair ink and the bioactive carrier combination on a clean slide.

(3) The obtained three-dimensional construction body is subjected to gradient sintering in a sintering furnace to improve the stability of the scaffold, and the scaffold obtained after sintering is ready for use.

Inoculating the bioactive ceramic scaffold:

(1) cryopreserved mBMSCs (rat bone marrow mesenchymal stem cells) were placed in a 37 ℃ shaking water bath for approximately 80s to facilitate resuscitation.

(2) The mBMSCs were transferred into a centrifuge tube containing H-DMEM medium and centrifuged at 100g for 5 min.

(3) The supernatant culture solution after centrifugation was aspirated by a pipette gun, and an appropriate amount of H-DMEM medium was added thereto to give a final concentration of 4.0X 105 cells/mL.

(4) 50ul of cell suspension (1.0X 105mBMSCs) were seeded on BCP scaffolds and placed in an incubator for 1 h.

(5) 1mL of H-DMEM medium was added to each well, incubated in an incubator (37 ℃, 5% CO2), and then the medium was changed every 3d of the total volume.

(6) Modulation of cell behavior (including cell viability, cell adhesion, cell proliferation) was measured throughout the 7d culture period.

Experimental example:

the bone tissue repair scaffolds provided in examples 1-6 were cultured in a well plate, 1mL of H-DMEM medium was added to the well plate, and the plate was left to stand in an incubator (37 ℃, 5% CO2), after which the medium was replaced every 3d of the total volume. The modulation of cell behavior (including cell viability, cell adhesion, cell proliferation) by the scaffold was determined throughout the 7d culture period.

The experimental results are as follows:

the cell activity, cell adhesion and cell proliferation of the scaffolds provided by the comparative examples and experimental examples were compared for 1,3,5 and 7 days.

The experimental results show that the cell activities of the bone tissue repair scaffolds provided in examples 1-6 all reach 98% compared to the comparative example (cell activity is 90%); the scaffold of comparative example 1 had a significant cell leakage at the bottom and poor cell adhesion relative to the experimental group. The cells of the bone tissue repair scaffold provided in examples 1-6 are all coated in a bioactive carrier, and can be uniformly dispersed in the scaffold, which is more beneficial to the growth of the cells. The cell proliferation was observed to be slightly different between the experimental example and the comparative example on day 3, but on days 5 and 7, the cell proliferation on the bone tissue repair scaffolds provided in examples 1-6 was significantly greater than that provided in the comparative example due to the presence of the bioactive carrier substance.

Therefore, experimental results show that the bone tissue repair scaffolds provided in the embodiments 1 to 6 have good cell biocompatibility and have a great application prospect in the field of bone tissue defect repair.

Example 7

This example provides a method of continuous printing of two materials (FIG. 5)

Step 1, dividing a printing material into polycaprolactone powder (100 parts) and bioceramic powder (100 parts); the supplementary material includes supplementary polycaprolactone powder and supplementary bioceramic powder, but is not limited.

And 2, extracting 90 parts of polycaprolactone powder of the printing material and 10 parts of biological ceramic powder to form a mixed material 1, wherein the ratio of the polycaprolactone to the biological ceramic material of the printing support 1 is 90:10, and at the moment, 10 parts of residual polycaprolactone material powder and 90 parts of residual biological ceramic powder in the printing material.

And 3, 10 parts of the residual polycaprolactone powder of the printing material, namely complementing 90 parts of the supply material polycaprolactone powder to 100 parts, extracting 50 parts of the printing material polycaprolactone powder in the state, forming a mixed material 2 by 50 parts of the biological ceramic powder, and printing the support 2, wherein the ratio of the polycaprolactone to the biological ceramic material of the support is 50:50, and at the moment, the residual polycaprolactone powder of the printing material is 50 parts and the residual biological ceramic powder is 40 parts.

And 4, 40 parts of residual biological ceramic powder, namely supplementing 60 parts of supplementary material biological ceramic powder to 100 parts of supplementary material biological ceramic powder, then extracting 30 parts of printing material polycaprolactone powder in the state, wherein 70 parts of biological ceramic powder form a mixed material 3, the printing support 3 is provided, the ratio of polycaprolactone to biological ceramic material of the support is 30:70, and at the moment, the residual polycaprolactone powder of the printing material is 20 parts and the residual biological ceramic powder is 30 parts.

And 5, if the support is continuously printed, repeating the process.

Example 8

This example provides a method of continuous printing of three materials (FIG. 6)

Step 1, dividing a printing material into a cell suspension (100 parts), gelatin (100 parts) and sodium alginate (100 parts); the supplementary materials include, but are not limited to, cell suspension supplement, gelatin supplement, and sodium alginate supplement.

And 2, extracting 10 parts of cell suspension of the printing material, 60 parts of gelatin and 40 parts of sodium alginate to form a mixed material 1, and printing the support 1, wherein the ratio of the cell suspension of the support to the gelatin to the sodium alginate is 10:60:40, and at the moment, 90 parts of the cell suspension of the printing material, 60 parts of the gelatin and 60 parts of the sodium alginate are remained.

And 3, complementing 40 parts of the rest gelatin of the printing material to 100 parts of supplemented gelatin, then extracting 10 parts of the printing material cell suspension in the state, 50 parts of gelatin and 50 parts of sodium alginate to form the mixed material 2, and printing the support 2, wherein the ratio of the cell suspension of the support, the gelatin and the sodium alginate is 10:50:50, and at the moment, 80 parts of the rest cell suspension of the printing material, 50 parts of the rest gelatin and 10 parts of the rest sodium alginate are obtained.

And 4, complementing 90 parts of supplemented sodium alginate to 100 parts of the rest 10 parts of the printing material, then extracting 10 parts of the printing material cell suspension in the state, 40 parts of gelatin and 60 parts of sodium alginate to form a mixed material 3, and printing the support 3, wherein the ratio of the cell suspension of the support, the gelatin and the sodium alginate is 10:40:60, and at the moment, 70 parts of the printing material rest cell suspension, 10 parts of rest gelatin and 40 parts of rest sodium alginate.

And 5, if the support is continuously printed, repeating the process.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of a bone tissue repair scaffold is characterized by comprising the following steps:

mixing at least one biodegradable material and at least one auxiliary forming material to prepare slurry; or

Preparing slurry from at least one biodegradable material to obtain bone tissue repair ink;

wherein the biodegradable material is selected from at least one of degradable inorganic substances, degradable synthetic high molecular materials and composite materials of different types or the same type;

the auxiliary molding material is selected from at least one of a biological dispersant, a biological binder, a biological lubricant, a biological anticoagulant, a cross-linking agent or a solvent;

the bone tissue repair composition is prepared by the steps of:

recovering the cells, centrifuging, and adding a culture medium to culture to obtain a cell unit;

mixing the bioactive hydrogel or bisphosphonate with a culture medium, bioactive factors and the cell units, and adjusting the pH of the mixed solution to be neutral by using a buffer solution and an acid-base solution to prepare a bioactive carrier;

the bone tissue repair ink, the cell unit and the bioactive carrier jointly form a bone tissue repair composition;

the biological active hydrogel is at least one of natural biological active hydrogel, artificially synthesized biological hydrogel, modified biological hydrogel and biological hydrogel formed by compounding materials of the same type or different types;

the bisphosphonate comprises at least one of zoledronic acid, pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid and risedronic acid;

the bioactive factor is at least one of bone morphogenetic factor, osteogenic factor, osteoinductive factor, anti-bone resorption agent, growth promoter and antibacterial agent;

constructing by a biological 3D printing method using the bone tissue repair composition;

during the construction process, a part of the bone tissue repair composition is used as a printing material, the rest of the bone tissue repair composition is used as a replenishing material, and the bone tissue repair scaffold is continuously printed and formed; when in printing, the fiber silk formed by the bone tissue repair ink and the fiber silk formed by the bioactive carrier form a fiber unit; the bone tissue repair ink fiber yarn and the bioactive carrier fiber yarn are positioned on the same fiber layer or different fiber layers, and the bioactive carrier fiber yarn is positioned between the adjacent fiber yarns spaced by the bone tissue repair ink on the same fiber layer; different fiber layers, the fiber yarn of the bioactive carrier and the fiber yarn of the bone tissue repair ink are positioned in two adjacent layers in parallel or vertical; for the same material, the fiber filaments in the fiber layer are parallel to and not coplanar with the fiber filaments in other fiber layers or are arranged in parallel and coplanar with each other; a plurality of the fiber units form a three-dimensional scaffold of the bone tissue repair ink composition;

after the bone tissue repair scaffold is printed, performing crosslinking, freeze drying or coating treatment on the bone tissue repair scaffold;

the continuous printing includes the steps of:

dividing the ink used in the printing process into printing material and supply material, wherein the printing material divides the material into residual material a1 and printing material b1 in the printing process;

printing material b1 will complete the printing of object 1; the residual material a1 is divided into a residual material a2 and a printing material b2 when continuously printing the bracket;

the printing material b2, if some components are insufficient, can be completely supplemented by the supply material c1, and then the object 2 is printed;

the residual material a2 is to be re-divided into residual material a3 and printing material b 3; when the printing material b3 has insufficient components, the replenishment material c2 can be supplemented completely, and then the object 3 is printed;

2. The method for preparing a bone tissue repair scaffold according to claim 1,

the biodegradable material is selected from the degradable inorganic substances;

the degradable inorganic matter is at least one selected from tricalcium phosphate, hydroxyapatite, calcium silicate, magnesium oxide, magnesium hydroxide, magnesium chloride, magnesium carbonate, magnesium phosphate, magnesium silicate, ferric oxide, ferroferric oxide, ferric hydroxide, ferric chloride or strontium carbonate;

the auxiliary molding material comprises sodium alginate, calcium chloride, glycerol and polyacrylamide;

wherein, the degradable inorganic substance accounts for 50 to 80 percent by mass; 20% -50% of the auxiliary forming material.

3. The method for preparing a bone tissue repair scaffold according to claim 1,

the degradable inorganic substance is at least one of hydroxyapatite or magnesium phosphate;

the auxiliary molding material comprises collagen, genipin, glycerol and polyacrylamide;

wherein, the degradable inorganic substance accounts for 50 to 90 percent by mass percent; 10% -50% of the auxiliary molding material.

4. The method for preparing a bone tissue repair scaffold according to claim 1,

the biodegradable material comprises degradable inorganic substances and degradable synthetic high polymer materials;

the degradable inorganic substance is selected from hydroxyapatite; the degradable synthetic high molecular material comprises at least one of polycaprolactone, polydioxanone, polyacrylic acid and derivatives thereof, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polyorthoester, polyhydroxybutyrate, polyamino acid and degradable polyurethane; the auxiliary molding material comprises methyl cellulose and 1, 4-dioxane;

wherein, the biodegradable material accounts for 50 to 90 percent by mass percent; 10% -50% of the auxiliary molding material.

5. A bone tissue repair scaffold is characterized in that,

the bone tissue repair scaffold is prepared by the preparation method of the bone tissue repair scaffold according to claim 1;

the bone tissue repair scaffold comprises a plurality of fibrous layers;

the fiber layers are arranged in parallel;

each fiber layer comprises a bracket structure made of bone tissue repair ink and a bracket structure made of a bioactive carrier wrapping cells; or

Every two adjacent fiber layers comprise a scaffold structure made of bone tissue repair ink or a scaffold structure made of a bioactive carrier wrapping cells.

6. A bone tissue repair kit, comprising:

the bone tissue repair scaffold prepared by the method for preparing a bone tissue repair scaffold according to claim 1, at least one of the bone tissue repair scaffolds according to claim 5;

a container, a culture medium, a buffer, and instructions.