WO2018090189A1

WO2018090189A1 - Cell-biomaterial composite stent and preparation method and use thereof

Info

Publication number: WO2018090189A1
Application number: PCT/CN2016/105921
Authority: WO
Inventors: 阮长顺; 翟欣昀; 吕维加; 潘浩波
Original assignee: 深圳先进技术研究院
Priority date: 2016-11-15
Filing date: 2016-11-15
Publication date: 2018-05-24

Abstract

Disclosed is a cell-biomaterial composite stent, comprising a stent matrix and a viscous cell suspension capable of slowly releasing cells. The composite stent has a configuration of ACnB from the bottom up, wherein A is a stent matrix layer, B is a viscous cell suspension layer, C is a mixed layer formed by the viscous cell suspension distributed crosswise in voids of the stent matrix, and n is a positive integer from 3 to 20. The viscous cell suspension comprises cells, a carrier carrying the cells, and water, the carrier being a biocompatible viscous material. Cells on the composite stent have a controllable distribution density and good adherence rate and survival rate, thereby solving the problem that cells on a three-dimensional composite stent grow slowly, have poor proliferation and differentiation, and are not good at promoting tissue regeneration. Further disclosed are a preparation method and the use of the composite stent.

Description

Cell-biomaterial composite scaffold and preparation method and application thereof

Technical field

The invention relates to the field of biomaterial technology, in particular to a cell-biomaterial composite scaffold and a preparation method and application thereof.

Background technique

Tissue engineering is an emerging discipline that applies the principles and techniques of life sciences and engineering to research and develop biological substitutes for repairing, maintaining, and promoting the function and morphology of various tissues or organs after injury. Among them, the selection and construction of biomaterial-based scaffold materials is a key link, and normal human cells are planted into biocompatible scaffold materials. After they are proliferated and differentiated to a certain extent on the scaffold, they can be adhered. The stent of the cell is implanted into the defect or loss site of the tissue to achieve the purpose of tissue repair. In addition to excellent biocompatibility, bioactivity, biodegradability, and good pore structure and mechanical properties, the ideal tissue engineering scaffold should further promote cell adhesion, proliferation and differentiation, and promote tissue regeneration. Ability.

At present, there are many methods for preparing tissue engineering stents. Among them, the mainstream three-dimensional printing technology has realized the fabrication of stents of any shape on demand, but the constructed stent has a large pore size and cannot obtain micropores of microscopic size, which cannot provide benefits. The three-dimensional support environment for cell growth, the adhesion rate of cells on the three-dimensional scaffold is low, and most of the cells fall in the culture dish at the time of inoculation. Therefore, how to enhance the adhesion degree of cells on a three-dimensional scaffold and precisely control the distribution of different kinds of cells in three-dimensional size, and obtain a three-dimensional structure similar to an animal or even a human tissue or an organ by in vitro culture is currently applied by three-dimensional printing technology. A major problem in organizational engineering.

Summary of the invention

In view of the above, the present invention provides a cell-biomaterial composite scaffold comprising a designable three-dimensional scaffold base and a viscous cell capable of slowly releasing cells, which is printed on a three-dimensional scaffold substrate, and a preparation method thereof. The suspension, the composite scaffold can increase the adhesion rate, activity, and the like of the cells thereon. In the preparation method of the composite stent, the cell is printed together with the scaffold matrix for the first time, and the controllability is high, and the adhesion rate and cell survival rate of the cells on the composite scaffold are good.

In a first aspect, the present invention provides a cell-biomaterial composite scaffold comprising a scaffold matrix and a viscous cell suspension capable of slowly releasing cells, the composite scaffold forming an AC _n B arrangement from the bottom Form, wherein A is a scaffold base layer formed by a scaffold matrix, B is a viscous cell suspension layer, C is a mixed layer formed by viscous cell suspension cross-distribution between the scaffold matrix voids, n is a positive integer of 3-20; The viscous cell suspension comprises cells, a cell-loaded carrier, and water, the carrier being a biocompatible viscous material.

The composite stent provided by the first aspect of the present application comprises a stent base, and a viscous cell suspension specifically distributed on the stent base, the composite stent can precisely control the distribution of the cells thereon, and enhance the cells thereon. Distribution density, degree of adhesion, etc., when the composite scaffold is used in a tissue engineering scaffold, the loaded cells can be slowly released and adhered to the surface of the scaffold substrate, growth, differentiation, etc., which can be solved. The cells grow slowly on the three-dimensional composite scaffold, have weak proliferation and differentiation, and have a weak ability to promote tissue regeneration.

Preferably, the n is a positive integer from 3-8.

In the mixed layer, the viscous cell suspension is cross-distributed at the voids of the stent base so as not to affect the stent matrix structure. At this time, since the cells are loaded in the viscous cell suspension and are not adhered to the composite scaffold, after the culture medium is added to the composite scaffold for later culture, the cells are slowly released and adhered to The surface of the composite stent.

The shape of the cell-biomaterial composite scaffold is related to the programming in three-dimensional printing, and the three-dimensional composite scaffolds with different morphologies can be printed according to different requirements.

Preferably, the composite scaffold is a regular geometry (such as a cuboid, a cube, a cylinder, etc., but is not limited thereto) and other irregular three-dimensional porous structures.

Preferably, the composite support is a cube having a bottom side length of 10-30 mm, a height of 3-20 mm, and a line spacing of 0.2-0.5 mm.

In an embodiment of the invention, the composite stent has a size of 15 mm × 15 mm cubes, and the number of layers is 5-10 layers.

Preferably, the viscous cell suspension has a viscosity of from 80 to 200 Pa.s. If the viscosity of the viscous cell suspension is too low, it still has fluidity, and the purpose of gradually releasing the cells cannot be achieved; if the viscosity is too high, the printing pressure used in three-dimensional printing is large, and the damage to the cells is too large. Affects cell survival rate, biological activity, and the like.

Preferably, in the composite scaffold, the distribution density of the cells is (4-10) × 10 ⁵ / scaffold. The distribution density is related to the size of the stent and can be adjusted according to the shape parameters of the stent (such as length, height, width or diameter).

The type of the cells is determined according to the application site of the composite stent. When the composite scaffold is used as a bone tissue engineering scaffold, the cells include bone marrow stem cells, osteoblasts, chondrocytes, vascular endothelial cells, and osteosarcoma cells. One or more of them, but is not limited thereto.

Preferably, in the viscous cell suspension, the cells are loaded in the carrier without contacting the scaffold matrix.

Preferably, the carrier comprises one or more of hyaluronic acid, gelatin, collagen, chitosan. The carrier is a material with good biocompatibility, has a certain viscosity, and can be dissolved in water, thereby realizing the purpose of slowly releasing the cells on the stent during the cell culture process.

In an embodiment of the invention, the carrier capable of slowly releasing cells is hyaluronic acid.

Preferably, in the viscous cell suspension, the mass fraction of the carrier is 10-50%.

Preferably, in the viscous cell suspension, the density of the cells is (0.5-10) × 10 ⁶ /mL. Further preferred is (2-8) × 10 ⁶ /mL. More preferably, it is 4 × 10 ⁶ /mL.

In an embodiment of the present invention, the material of the stent base comprises one or more of a natural polymer, a synthetic polymer, and a bioceramic, wherein the natural polymer comprises fibrin, gelatin, collagen, and shell. At least one of sugar, hyaluronic acid, sodium hyaluronate, and alginate; the synthetic polymer includes polylactic acid (PLA), polyamino acid, polyglycolic acid (PGA), polyvinyl alcohol (PVA), lactic acid At least one of a glycolic acid copolymer (PLGA) and a polycaprolactone; the bioceramic comprising at least hydroxyapatite, octacalcium phosphate, calcium phosphate, calcium metaphosphate, tricalcium phosphate, and bioactive glass One. The bioactive glass contains elements such as Sr, B, Cu, P, and Mg.

Further preferably, the raw material of the stent base is a mixture of the natural polymer and/or the synthetic polymer and the bioceramic. That is, a mixture of one or both of the natural polymer and the synthetic polymer and the bioceramic.

In another embodiment of the invention, the stent base is a gel stent. Preferably, the three-dimensional gel scaffold is formed by ultraviolet light polymerization of inorganic clay, a crosslinking agent (a biocompatible macromolecule containing a carbon-carbon double bond), and an ultraviolet photoinitiator.

Preferably, the material of the stent base is a clay-based hydrogel matrix comprising the following mass percentages of the raw material components:

Crosslinking agent: 10-50%;

Inorganic clay: 3-20%;

Ultraviolet light initiator: 0.05-0.1%;

Water: 30-86%;

The total mass percentage of each of the above raw material components is 100%; wherein the crosslinking agent is a carbon-carbon double bond

Biocompatible macromolecules, which are polyethylene glycol, polyvinyl alcohol, chitosan, gelatin, and transparent

One or more of the acids.

The clay-based hydrogel matrix obtained by the synergistic action of various raw materials of the above specific mass ratio of the present invention (i.e., clay-based hydrogel printing paste) has a suitable printing viscosity and strength, and has a certain pre-shape before UV crosslinking. The clay-based hydrogel matrix can be printed as a stable gel scaffold precursor with good mechanical strength and tensile properties at room temperature, which facilitates uniform UV curing of the gel composite scaffold precursor at a later stage to obtain three-dimensional condensation. Glue composite bracket. The clay-based hydrogel matrix is suitable for batch printing to obtain a three-dimensional printing gel scaffold, which greatly improves the three-dimensional printing efficiency, and is very suitable for industrial production.

Under the action of ultraviolet light, in the clay-based hydrogel matrix of the present application, the cross-linking of the carbon-carbon double bonds of the cross-linking agent forms a long-chain polymer intercalated in an ordered piece of inorganic clay. In the layer structure, a hydrogel of a three-dimensional network structure is formed, so that the gel shape is maintained, and the inorganic clay also functions as a physical crosslink, and a hydrogen bond formed between the biocompatible macromolecule with a carbon-carbon double bond. The effect, van der Waals force, etc., also further enhance the strength of the printed three-dimensional gel scaffold, and fully exert the synergistic effect of physical cross-linking and chemical cross-linking. Improve the stability of the gel scaffold.

The inorganic clay may be selected from the group consisting of kaolin, bentonite, montmorillonite, laponite (lithosite), hectorite, beidellite, saponite, stevensite, magnesium aluminum silicate, Other aluminum silicates and various other natural and/or synthetic clays, and combinations thereof.

Preferably, the inorganic clay has a particle size of not more than 500 nm. More preferably, it is 50-200 nm.

In a preferred embodiment of the invention, the inorganic clay is a laponite clay. The laponite clay can be quickly peeled off and dispersed into a single layer in water to form a colorless transparent colloidal dispersion with good stability. The clay can be purchased from Rockwood's Laponite XLG. Laponite XLG is a synthetic layered clay similar to natural montmorillonite.

In the present application, the addition of inorganic clay can effectively increase the strength of the hydrogel stent, and the inorganic clay acts as a physical crosslinking agent. In addition, controlling the content of the inorganic clay can control the viscosity of the clay-based hydrogel matrix, so that the clay-based hydrogel matrix has a certain pre-shape, and can be post-printed and post-cured.

Preferably, the clay-based hydrogel matrix has a viscosity of from 30 to 350 Pa.s. The clay-based hydrogel matrix to be printed should have a certain viscosity, the viscosity is too high, the fluidity is poor, the pressure required for printing is too large, and the material is not easily mixed; the solution viscosity is too low, and the fluidity is too high. It is difficult to shape when printing, and the bracket will collapse and cannot maintain the pre-shape. In the present application, the content of the inorganic clay is controlled to be between 3% and 20%, and the viscosity of the clay-based hydrogel matrix can be controlled to be 30 to 350 Pa·s.

Further preferably, the inorganic clay has a mass percentage of 5-15%. More preferably, it is 8-15%. When the mass percentage of the inorganic clay is increased to 8% or more, the clay-based hydrogel matrix reaches a higher viscosity, and the gel stent is stretched after the gel stent precursor is cured. Can reach about 5000%, the mechanical strength is obvious improve.

Preferably, the clay-based hydrogel matrix comprises a mass percent of the raw material component as follows:

Crosslinking agent: 10-50%;

Inorganic clay: 5-15%;

Ultraviolet light initiator: 0.05-0.1%;

Water: 35-84%;

The total mass percentage of each of the above raw material components was 100%.

Further preferably, the clay-based hydrogel matrix has a viscosity of 50 to 250 Pa·s. More preferably, it is 100-200 Pa.s.

In the present application, the biocompatible macromolecule containing carbon-carbon double bond has a good gel-forming effect, and in the dispersion formed by the inorganic clay and water, no additional chemical crosslinking agent is needed, and ultraviolet polymerization is possible. A higher strength gel structure is obtained. The formed three-dimensional gel scaffold matrix has low toxicity and good cell compatibility. While providing a three-dimensional environment required for cell growth, the cells are promoted to promote adhesion, growth and proliferation.

In an embodiment of the invention, at least one end of the molecular chain of the crosslinking agent has a carbon-carbon double bond. The crosslinking agent may be methacrylic acid, acrylic acid or polyethylene glycol diacrylate (PEGDA) modified gelatin, hyaluronic acid, polyvinyl alcohol, polyvinyl alcohol or the like. For example, it may be methacrylic acid modified gelatin, acrylic acid modified hyaluronic acid, methacrylic acid modified polyvinyl alcohol, low molecular weight (molecular weight less than 1000 Dalton) PEGDA modified chitosan, and the like.

In another embodiment of the present invention, at least one end of the molecular chain of the crosslinking agent has a carbon-carbon double bond, and the molecular chain of the crosslinking agent is a main chain structure of polyethylene glycol.

Further preferably, the crosslinking agent is polyethylene glycol diacrylate.

In addition to promoting gel formation and improving the mechanical strength of the gel, the cross-linking agent can also control the adhesion, growth, proliferation and even differentiation of the cells on the prepared scaffold by controlling the molecular weight and solid content thereof. For example, when the molecular weight of polyethylene glycol is 4000 and the solid content is 20%, the cells can adhere and stretch well on the stent; but when the molecular weight of polyethylene glycol is 10000 or more, the cells are spherical on the stent, which cannot be very Well developed, cell proliferation behavior is relatively weak.

Preferably, among the crosslinking agents, the main chain structure of the polyethylene glycol has a molecular weight of from 1,000 to 10,000. For example, it may be 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000 or 9000. More preferably, it is 2000-8000.

In the present application, the crosslinking agent has a mass percentage of 10-50%. For example, it may be 15%, 20%, 25%, 30%, 35%, 40% or 45%. More preferably, it is 20-40%.

Crosslinking agent: 20-40%;

Inorganic clay: 3-20%;

Ultraviolet light initiator: 0.05-0.1%;

Water: 40-76%;

Further preferably, the clay-based hydrogel matrix comprises a mass percent of the raw material component as follows:

Crosslinking agent: 20-40%;

Inorganic clay: 5-15%;

Ultraviolet light initiator: 0.05-0.1%;

Water: 45-74%;

The UV photoinitiator selected in the present application is a biocompatible initiator which is used in a very small amount and hardly affects the cell experiments of the late composite scaffold.

In an embodiment of the invention, the ultraviolet photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2 -methylpropiophenone. The ultraviolet photoinitiator may also be 1-[4-(2-hydroxyethoxy)-phenylene]-2-hydroxy-2',2'-dimethylethyl ketone (Irgacure 2959), 1-hydroxycyclohexyl One of phenyl ketone, α,α'-dimethoxy-α-phenylacetophenone and 2-methyl-1-(4-methylthiophenyl)-2-morpholine-1-propanone Kind or more. It is not limited to the ones listed in this application.

In a second aspect, the present invention provides a method for preparing a cell-biomaterial composite scaffold comprising the following steps:

(1) preparing a material of the stent base into a stent matrix precursor slurry; providing a viscous cell suspension comprising cells, a carrier capable of slowly releasing the cells, and water, the carrier being biocompatible material;

(2) printing by multi-channel three-dimensional printing method, wherein one channel is used for controlling printing of the viscous cell suspension, and at least one of the remaining channels is used for controlling printing of the precursor substrate slurry of the stent;

First printing the scaffold matrix precursor slurry to form a bottom layer of the composite scaffold precursor, starting from the second layer, cross-printing the scaffold matrix precursor slurry and the viscous cell suspension to cross the viscous cell suspension Arranged between the voids of the precursor of the stent substrate to form a mixed layer, repeating printing of the mixed layer n times; finally printing the viscous cell suspension to form a top layer of the composite stent precursor to obtain cells - a biomaterial composite scaffold precursor, wherein the composite scaffold precursor forms an A'C' _n B arrangement from the bottom, wherein A' is a scaffold matrix precursor layer, B is a viscous cell suspension layer, C' a mixed layer formed by interstitial distribution of the viscous cell suspension between the interstices of the scaffold matrix precursor, n being a positive integer of 3-20;

(3) curing the printed composite stent precursor to obtain a cell-biomaterial composite stent, wherein the composite stent forms an AC _n B arrangement from the bottom, wherein A is a stent base layer, B For the viscous cell suspension layer, C is a mixed layer formed by the viscous cell suspension cross-distributing between the voids of the stent matrix, n being a positive integer of 3-20.

Preferably, in the step (3), the curing mode is ultraviolet light curing, heat curing, ion crosslinking or freeze drying. The curing method is determined according to the raw material of the stent base. The curing method can be selected according to whether the raw material of the stent substrate is sensitive to light, heat, ions, or the like. For example, for the raw material system of the gel scaffold, ultraviolet curing or thermal curing can be used; for the raw material system of the alginate-containing scaffold substrate, curing is carried out by divalent ion crosslinking; for bioceramics It can be cured by freeze drying.

Further preferably, the curing mode is ultraviolet light curing. The curing method is mild and the damage to the cells is relatively small.

Preferably, after the step (3), the method further comprises:

(4) taking the composite scaffold, adding the cell culture medium, immersing it in the cell culture medium, culturing at 25-37 ° C, slowly releasing the cells loaded in the vector, and expanding Adhering to the composite scaffold to obtain a cell-biomaterial composite scaffold for adhering cells. When immersed in a culture medium, the resulting cell-biomaterial composite scaffold of adherent cells is free of sticky cell suspension, and the viscous cell suspension has gradually dissolved in the medium, leaving only the scaffold and adhering to it. The cells on the stent.

Preferably, in the step (4), the cell culture medium is α-MEM medium. Additional nutrients may be added depending on the type of the cells.

Preferably, the culture is carried out for a period of from 1 to 30 days. It is further preferably 5-10 days or 18-25 days. In general, the cells will be substantially full of scaffolds in seven days yesterday; for observing the differentiation of cells on the scaffolds, it usually takes about 21 days to culture.

Preferably, each channel of the three-dimensional printer is provided with a material replenishing cavity. Used to replenish the printing materials in each channel.

Preferably, before the start of the three-dimensional printing, the position of each channel used in the printing is corrected, and the first head of the stent base precursor slurry is used as a reference to connect the channels used. The bottoms of all the tips are on the same horizontal line. This facilitates cross-printing of the late stent substrate precursor slurry with the cell suspension.

Preferably, the gun heads connected to the respective channels are movable relative to the top, bottom, left and right, and are not mutually restricted.

Preferably, the driving medium in the three-dimensional printing process is pneumatic or voltage driven. This can be used for the base of the bracket Both the slurry and the cells in the viscous cell suspension can exert a force without causing damage to them.

Preferably, in the three-dimensional printing process, the holes of the adjacent layers are staggered and communicate with each other.

Preferably, the three-dimensional printing is performed in a biosafety cabinet. This will ensure that the overall operating environment is sterile.

Preferably, in the three-dimensional printing process, each time a layer of the composite stent precursor is printed, all the nozzle positions connected to the respective channels are moved up, and the gun head is moved upwardly from the gun connected to each channel. Head diameter is related to material properties.

Further preferably, the tip of the gun is moved upward by (0.6-1) times the diameter of the tip.

For example, when printing with a nozzle with a diameter of 150 μm, the thickness of the printed material is about 150 μm. For each layer printed, the tip should be moved up to 150 μm and the next layer printed to prevent the distance from moving too small or too much. If the viscosity of the printed material is low (for example, for a slurry of a gel matrix, if the viscosity of the slurry is less than 30 Pa·s), the stent line is likely to collapse after being printed. The height will be lower than the diameter of the tip. At this time, after the printing of one layer, the upward movement distance of the tip should be smaller than the diameter of the tip, so that the contact between the lines of the bracket is good, and no line detachment occurs.

In the present application, the at least one channel is used for controlling the printing of the precursor matrix slurry of the stent, and may be a channel for printing a matrix precursor slurry of a certain fixed composition, or may be composed of multiple channels (2). More than one) to print the matrix precursor slurry of the same composition and different ratios, or a matrix precursor slurry composed of different materials by a plurality of channels to form a three-dimensional composite stent of cell-biomaterials of different substrates.

In the present application, in the step (2), the lowermost layer (bottom layer) of the composite stent precursor is a separate stent matrix precursor, starting from the bottom second layer, the stent matrix precursor slurry and The viscous cell suspensions are cross-distributed (staggered side by side), and the scaffold matrix precursor slurry can be printed into a porous structure, and then the viscous cell suspension is printed, and the viscous cell suspension is staggered in the scaffold matrix to form a mixed layer. This completes the printing of the second layer, repeating the printing mode of the second layer n times to form the intermediate layer of the precursor of the composite stent; when printing the uppermost layer (top layer) of the precursor of the composite stent, separately printing the sticky cells Suspension. This can increase the seeding density of the composite scaffold for cells.

Preferably, the n is a positive integer from 3-8.

Preferably, the viscous cell suspension has a viscosity of from 80 to 200 Pa.s.

Preferably, the carrier comprises one or more of hyaluronic acid, gelatin, collagen, chitosan.

Preferably, in the viscous cell suspension, the density of the cells is (0.5-10) × 10 ⁶ /mL.

Preferably, the scaffold matrix precursor slurry is a clay-based hydrogel matrix comprising the following mass percentages of the raw material components:

Crosslinking agent: 10-50%;

Inorganic clay: 3-20%;

Ultraviolet light initiator: 0.05-0.1%;

Water: 30-86%;

One or more of the acids.

The clay-based hydrogel matrix composed of the above specific ratio of inorganic clay, crosslinking agent, ultraviolet light initiator, water, the clay-based hydrogel matrix has a suitable viscosity and a certain pre-shape, and is suitable for continuous , batch printing, solidified to get a three-dimensional gel stent, greatly improving the efficiency of three-dimensional printing. The preparation method of the three-dimensional gel scaffold has the advantages of simple process, strong controllability, low energy consumption, no need to change the pressure in the printing process, low manufacturing cost and strong practicability.

Preferably, when the stent matrix precursor slurry is a clay-based hydrogel matrix, the curing mode is ultraviolet light curing.

Preferably, the crosslinking cures for a time of 20-50 min.

Preferably, the ultraviolet light used has a wavelength of from 230 to 400 nm. More preferably, it is 250-350 nm. More preferably, it is 254 nm.

The preparation method of the cell-biomaterial composite scaffold provided by the second aspect of the invention has the advantages of simple steps and mild preparation conditions. In the preparation of the composite scaffold, the cell suspension is printed together with the raw material of the scaffold base for the first time, and various types can be constructed. The composite scaffold system of different cells and different scaffold bases precisely controls the distribution of cells on the composite scaffold, and enhances the adhesion rate and cell survival rate of the cells on the composite scaffold.

In the prior art, the scaffold matrix material is first mixed with the cell suspension to form a mixed slurry and then printed, which can satisfy the carrier matrix material which is mixed with the cells and then printed, and the cells are printed in the bracket after mixing. The activity of the stent; the mechanical strength of the stent after the mixed slurry is usually formed is low. The preparation method provided by the application has more application scopes, is applicable to various matrix materials, and is easy to maintain the activity of cells on the stent, and the resulting cell-biomaterial composite stent has high mechanical strength and can well satisfy the composite. The requirements of tissue engineering for the mechanical strength of materials.

In a third aspect, the present invention provides a cell-biomaterial composite scaffold according to the first aspect of the present invention or an adherent cell-biomaterial composite scaffold according to the third aspect of the present invention, in the preparation of a tissue repair material Applications. It is preferably used in bone tissue engineering scaffold materials.

Preferably, the application comprises the following steps:

Taking the cell-biomaterial composite scaffold, adding the cell culture medium, immersing in the cell culture medium, culturing at 25-37 ° C, slowly releasing the cells loaded in the vector, and expanding The cell-biomaterial composite scaffold for adhering cells is obtained by adhering to the composite scaffold. The cell-biomaterial composite scaffold for adhering cells can be implanted into a tissue defect or loss site of a mammal.

Further preferably, the cell culture medium is α-MEM medium. Additional nutrients may be added depending on the type of the cells.

Further preferably, the culture is carried out for a period of from 1 to 30 days. It is further preferably 5-10 days or 18-25 days. In general, the cells will be substantially full of scaffolds in seven days yesterday; for observing the differentiation of cells on the scaffolds, it usually takes about 21 days to culture.

In a fourth aspect, the present invention also provides a cell-biomaterial composite scaffold for adhering cells. The resulting cell-biomaterial composite scaffold of adherent cells is free of sticky cell suspensions, and the viscous cell suspension has gradually dissolved in the medium, leaving only the scaffold matrix and the cells adhering to the scaffold matrix, but The scaffold base structure in the original cell-biomaterial composite scaffold was retained.

Preferably, the cell-biomaterial composite scaffold for adhering cells comprises a scaffold matrix and cells (proliferating and/or differentiated cells) adhering to the surface of the scaffold substrate.

The cell-biomaterial composite scaffold for adhering cells is a cube having a bottom side length of 10-30 mm, a height of 3-20 mm, and a line spacing of 0.2-0.5 mm.

The advantages of the embodiments of the present invention will be set forth in part in the description which follows.

DRAWINGS

1 is a nuclear magnetic spectrum of polyethylene glycol (B, D) having a molecular weight of 4000 and 10,000, respectively, and polyethylene glycol (A, C) having a molecular weight of 4000 and 10,000 after double bond modification;

2 is a schematic structural view of a precursor of a cell-biomaterial composite scaffold;

Figure 3 is a distribution diagram of osteoblasts in a cell-biomaterial composite scaffold after solidification;

Figure 4 is a distribution diagram of osteoblasts in a cell-biomaterial composite scaffold after culture.

detailed description

The following are the preferred embodiments of the embodiments of the present invention, and it should be noted that those skilled in the art can make some improvements and refinements without departing from the principles of the embodiments of the present invention. And retouching is also considered to be the scope of protection of the embodiments of the present invention.

The embodiments of the present invention are further described below in various embodiments. The embodiments of the present invention are not limited to the following specific embodiments. Changes can be implemented as appropriate within the scope of the invariable primary rights.

In one embodiment of the present invention, a polyethylene glycol modified with a double bond is used as a crosslinking agent, and the preparation method is as follows: 1.2 ml of acryloyl chloride is dissolved in 10 ml of dichloromethane to prepare an acryloyl chloride solution. Subsequently, 10 g of polyethylene glycol (Mn = 4000) was placed in a 50 ml three-necked flask, dissolved in 20 ml of dichloromethane, and stirred at room temperature. Under ice water bath conditions, 2.1 ml of triethylamine solution was slowly added. Subsequently, an acryloyl chloride solution prepared in advance was added dropwise under ice water bath conditions. The temperature of the control system was zero degrees until all the acryloyl chloride was added dropwise, and the reaction was carried out at room temperature for 24 hours under nitrogen protection. After the reaction was completed, the triethylamine hydrochloride formed in the system was filtered off, and the synthesized double bond-modified polyethylene glycol (Mn=4000) was precipitated with anhydrous diethyl ether, and the obtained crude product was dried by vacuum for 1 d, dialysis. 7d, filtered, lyophilized for 2d to obtain a higher purity double bond modified polyethylene glycol macromolecular crosslinker, which is polyethylene glycol diacrylate, the molecular formula is CH ₂ =CHCO-(OCH ₂ CH ₂ _n COCOCH=CH ₂ (when the molecular weight of the polyethylene glycol is 4000, n=88, when the molecular weight is 10000, n=224).

Figure 1 shows polyethylene glycol (B, D) having a molecular weight of 4000 and 10,000, respectively, and polyethylene glycol (A, C) having a molecular weight of 4000 and 10,000 after modification with a double bond (i.e., polyethylene glycol diacrylate). Nuclear magnetic spectrum. That is, (A) is PEGDA4000, (B) is PEG 4000, (C) is PEGDA 10000, and (D) PEG 10000. The structural formula of the polyethylene glycol diacrylate is as shown in the following formula (I):

As can be seen from Figure 1, c, d, and e are characteristic peaks of hydrogen on the double bond modified on polyethylene glycol diacrylate, indicating that the double bond was successfully modified onto polyethylene glycol.

Example 1:

A method for preparing a cell-biomaterial composite scaffold comprises the following steps:

1. Preparation of stent matrix precursor slurry:

The inorganic nano-clay-based hydrogel is used as a precursor slurry of the support matrix of the cell-biomaterial composite three-dimensional scaffold. The preparation of the scaffold matrix precursor slurry is as follows:

Taking the above double bond modified polyethylene glycol macromolecular crosslinking agent, Laponite XLG hectorite clay, ultraviolet photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone and water mixed at room temperature The stent substrate precursor slurry was obtained, and the viscosity thereof was 160 Pa·s, wherein each of the above raw materials was mixed in the following mass percentage:

Polyethylene glycol diacrylate (polyethylene molecular weight Mn = 4000) 20%

Laponite XLG clay: 5%

UV photoinitiator: 0.05%

Water: 74.95%.

2. Prepare a viscous cell suspension:

The hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution, and the human osteoblasts are resuspended in a hyaluronic acid solution to obtain a viscous cell suspension having a viscosity of 150 Pa·s. The quality of hyaluronic acid in the viscous cell suspension is obtained. The score was 20% and the density of the cells was 4 × 10 ⁶ /mL.

3. Print the composite bracket precursor:

Printing by two-channel three-dimensional printing method, wherein one channel is used for controlling the printing of the above viscous cell suspension, and the distribution of cells in the three-dimensional scaffold is controlled; and another channel is used for controlling the printing of the precursor matrix slurry of the scaffold;

First printing the stent substrate precursor slurry to form a bottom layer of the composite stent precursor, and from the layer upward, that is, starting from the second layer, cross-printing the stent substrate precursor slurry and the viscous cell suspension, so that The viscous cell suspension is arranged alternately between the voids of the precursor of the stent substrate to form a mixed layer, and the printing of the mixed layer is repeated n times to form an intermediate layer of the precursor of the composite stent; The viscous cell suspension forms a top layer of the composite scaffold precursor to obtain a cell-biomaterial composite scaffold precursor, and the composite scaffold precursor forms an A'C' _n B arrangement from the bottom, wherein A 'For the precursor matrix of the scaffold, B is the viscous cell suspension layer, C' is the mixed layer of the viscous cell suspension cross-distributed between the precursors of the scaffold matrix, n is 3, and the cell density is controlled at 6×10 ⁵ / scaffold. The size of the stent is a cube having a length and width of 15 mm × 15 mm, and the number of layers is five.

4. Curing of the composite stent precursor:

The printed composite stent precursor is placed in an ultraviolet cross-linking device for photocuring for 2000 s to plasticize the gel to obtain a cell-biomaterial composite scaffold, and the composite scaffold is formed from the bottom to form an AC _n B arrangement. Form, wherein A is a scaffold base layer, B is a viscous cell suspension layer, C is a mixed layer formed by a viscous cell suspension cross-distributed between the scaffold matrix voids, n is 3; the scaffold matrix is polyethylene glycol diacrylate A gel scaffold matrix formed by solidification of an inorganic clay and an ultraviolet photoinitiator.

5. The three-dimensional composite scaffold was taken out and immersed in the α-MEM medium, and placed in a 37 ° C incubator to slowly release the osteoblasts loaded in the hyaluronic acid and adhered to the gel scaffold base. , a cell-biomaterial three-dimensional composite scaffold obtained by adhering cells.

2 is a schematic view showing the structure of a precursor of a cell-biomaterial composite scaffold synthesized in the present invention. In FIG. 2, the gray scale is a precursor of the scaffold matrix, and the gray scale is a viscous cell suspension. The bottom layer is the precursor of the scaffold matrix, and each layer in the middle is a mixed layer in which the viscous cell suspension is staggered between the precursors of the scaffold matrix, and the uppermost layer is a viscous cell suspension layer.

Fig. 3 is a distribution diagram of osteoblasts in a cell-biomaterial composite scaffold after solidification (the scale is 200 μm in Fig. 3). In Fig. 3, the lighter columnar portion is a viscous cell suspension; the darker columnar portion is the scaffold matrix. As can be seen from Fig. 3, the viscous cell suspension is staggered with the scaffold matrix, and the osteoblasts are still Wrapped inside the cell suspension layer (white dots in the lighter columnar portion of Figure 3 are cells) have not been released.

Figure 4 is a graph showing the growth of cell-biomaterial composite scaffolds (i.e., cell-biomaterial three-dimensional composite scaffolds adhering to cells) after osteoblasts were cultured for 7 days in the medium; the scale in Fig. 4 was 200 μm. As can be seen from Figure 4, there is no longer a staggered cell suspension in the space of the stent base, and the osteoblasts are released and adhere to the surface of the stent base.

Example 2

1. Preparation of stent matrix precursor slurry:

An inorganic nanoclay-based hydrogel is used as a precursor slurry of a scaffold matrix of a cell-biomaterial composite three-dimensional scaffold having a viscosity of 150 Pa·s, including the following mass percentage of raw material components:

Crosslinking agent (methacrylic acid modified gelatin): 20%;

Laponite XLG clay: 7%;

Ultraviolet light initiator: 0.05%;

Water: 72.95%;

Wherein the ultraviolet photoinitiator is 1-[4-(2-hydroxyethoxy)-phenylene]-2-hydroxy-2',2'-dimethylethylketone (Irgacure 2959).

2. Prepare a viscous cell suspension:

The hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution, and the human osteoblasts are resuspended in a hyaluronic acid solution to obtain a viscous cell suspension having a viscosity of 80 Pa·s. The quality of hyaluronic acid in the viscous cell suspension is obtained. The score was 10% and the density of the cells was 8 × 10 ⁶ /mL.

3. Print the composite bracket precursor:

First printing the stent substrate precursor slurry to form a bottom layer of the composite stent precursor, and from the layer upward, that is, starting from the second layer, cross-printing the stent substrate precursor slurry and the viscous cell suspension, so that The viscous cell suspension is arranged alternately between the voids of the precursor of the stent substrate to form a mixed layer, and the printing of the mixed layer is repeated n times to form an intermediate layer of the precursor of the composite stent; The viscous cell suspension forms a top layer of the composite scaffold precursor to obtain a cell-biomaterial composite scaffold precursor, and the composite scaffold precursor forms an A'C' _n B arrangement from the bottom, wherein A 'For the precursor matrix of the scaffold, B is the viscous cell suspension layer, C' is the mixed layer of the viscous cell suspension cross-distributed between the precursors of the scaffold matrix, n is 5, and the cell density is controlled at 4×10 ⁵ / scaffold. The size of the stent is a cube having a length and width of 20 mm × 25 mm, and the number of layers is 7 layers.

4. Curing of the composite stent precursor:

The printed composite stent precursor was placed in an ultraviolet cross-linking instrument for photo-curing for 45 min to plasticize the gel to obtain a cell-biomaterial composite scaffold, and the composite scaffold was formed from the bottom to form an AC _n B arrangement. Form, wherein A is a scaffold base layer, B is a viscous cell suspension layer, C is a mixed layer formed by a viscous cell suspension cross-distributed between the scaffold matrix voids, n is 5; the scaffold matrix is polyethylene glycol diacrylic acid A gel scaffold matrix formed by curing an ester, an inorganic clay, or an ultraviolet photoinitiator.

Example 3:

1. Preparation of stent matrix precursor slurry:

A sodium alginate-based hydrogel is used as a precursor slurry of a stent matrix of a cell-biomaterial composite three-dimensional scaffold. The precursor slurry has a viscosity of 130 Pa·s, and includes the following mass percentage of raw material components:

Crosslinking agent (methacrylic acid modified modified polyvinyl alcohol): 20%;

Sodium alginate: 10%;

Water: 70%.

2. Prepare a viscous cell suspension:

The hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution, and the human bone marrow stem cells are resuspended in a hyaluronic acid solution to obtain a viscous cell suspension having a viscosity of 200 Pa·s, and the mass fraction of hyaluronic acid in the viscous cell suspension. At 30%, the density of the cells was 2 x 10 ⁶ /mL.

3. Print the composite bracket precursor:

First printing the stent substrate precursor slurry to form a bottom layer of the composite stent precursor, and from the layer upward, that is, starting from the second layer, cross-printing the stent substrate precursor slurry and the viscous cell suspension, so that The viscous cell suspension is arranged alternately between the voids of the precursor of the stent substrate to form a mixed layer, and the printing of the mixed layer is repeated n times to form an intermediate layer of the precursor of the composite stent; The viscous cell suspension forms a top layer of the composite scaffold precursor to obtain a cell-biomaterial composite scaffold precursor, and the composite scaffold precursor forms an A'C' _n B arrangement from the bottom, wherein A 'For the precursor matrix of the scaffold matrix, B is the viscous cell suspension layer, C' is the mixed layer formed by the viscous cell suspension cross-distribution between the voids of the scaffold matrix precursor, n is 8, and the cell density is controlled at 4×10 ⁵ / bracket, the bracket size is a cube with a length and width of 15mm × 20mm, the number of layers is 10 layers.

4. Curing of the composite stent precursor:

The printed composite stent precursor was placed in a calcium chloride solution having a concentration of 0.3 mol/ml for 15 min, and solidified to obtain a cell-biomaterial composite scaffold. The composite scaffold was formed from the bottom from the bottom to form AC _n B. Arrangement form, wherein A is the base layer of the stent, B is the viscous cell suspension layer, C is a mixed layer formed by the viscous cell suspension cross-distribution between the gaps of the stent matrix, n is 8; the stent matrix is polyethylene glycol II A gel scaffold matrix formed by curing acrylate, inorganic clay, and ultraviolet photoinitiator.

5. The three-dimensional composite scaffold is taken out and immersed in the α-MEM culture medium, and placed in an incubator at 37 ° C to slowly release the bone marrow stem cells loaded in hyaluronic acid and adhere to the gel scaffold base. A three-dimensional composite scaffold of cell-biomaterial with adherent cells is obtained.

Example 4:

1. Preparation of stent matrix precursor slurry:

Taking the above double bond modified polyethylene glycol macromolecular crosslinking agent, Laponite XLG hectorite clay, ultraviolet photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and The water was mixed at room temperature to obtain a scaffold matrix precursor slurry having a viscosity of 170 Pa·s, wherein each of the above raw materials was mixed in the following mass percentages:

Polyethylene glycol diacrylate (polyethylene molecular weight Mn = 4000) 30%

Laponite XLG clay: 8%

UV photoinitiator: 0.05%

Water: 61.95%.

Step 2-5 is the same as Embodiment 1.

Example 5:

Step 1. Prepare the stent matrix precursor slurry:

Taking the above double bond modified polyethylene glycol macromolecular crosslinking agent, Laponite XLG hectorite clay, ultraviolet photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and The water was mixed at room temperature to obtain a scaffold matrix precursor slurry having a viscosity of 150 Pa·s, wherein each of the above raw materials was mixed in the following mass percentages:

Polyethylene glycol diacrylate (polyethylene molecular weight Mn = 10000) 20%;

Laponite XLG clay: 7%;

Ultraviolet light initiator: 0.05%;

Water: 72.95%.

The remaining steps 2-5 are the same as in the first embodiment.

Example 6

Step 1. Prepare the stent matrix precursor slurry:

The double-bond modified polyethylene glycol macromolecule is used as a crosslinking agent, bentonite is used as inorganic clay, and Irgacure 2959 is used as an ultraviolet light initiator to mix with water at room temperature to obtain a precursor matrix slurry of the stent, and the viscosity is 200 Pa·s. Wherein each of the above raw materials is mixed in the following mass percentages:

Polyethylene glycol diacrylate (polyethylene molecular weight Mn = 4000) 25%;

Inorganic clay: 10%;

Ultraviolet light initiator: 0.1%;

Water: 64.9%;

The remaining steps 2-5 are the same as in the first embodiment.

Example 7

Step 1. Prepare the stent matrix precursor slurry:

The inorganic nanoclay-based hydrogel is used as a precursor slurry of a scaffold matrix of a cell-biomaterial composite three-dimensional scaffold, and the scaffold matrix precursor slurry comprises the following mass percentage of raw material components:

Crosslinking agent: 40%;

Inorganic clay: 15%;

Ultraviolet light initiator: 0.05%;

Water: 44.95%;

Wherein the crosslinking agent is acrylic acid modified hyaluronic acid; the inorganic clay is kaolin, and the ultraviolet photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylbenzene. acetone.

The remaining steps 2-5 are the same as in the first embodiment.

The above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and those skilled in the art can understand all or part of the process of implementing the above embodiments, and according to the claims of the present invention. The equivalent change is still within the scope of the invention.

Claims

A cell-biomaterial composite scaffold, characterized in that the composite scaffold comprises a scaffold substrate and a viscous cell suspension capable of slowly releasing cells, wherein the composite scaffold forms an AC n B arrangement from the bottom, wherein A is a scaffold base layer formed by a scaffold matrix, B is a viscous cell suspension layer, C is a mixed layer formed by viscous cell suspension cross-distribution between the scaffold matrix voids, n is a positive integer of 3-20; Suspensions include cells, cell-loaded carriers, and water, which are biocompatible viscous materials.
The composite scaffold according to claim 1, wherein said composite scaffold has a distribution density of (4-10) × 10 5 / scaffold; said cells include bone marrow stem cells, osteoblasts, One or more of chondrocytes, vascular endothelial cells, and osteosarcoma cells.
The composite stent according to claim 1, wherein the viscous cell suspension has a viscosity of 80 to 200 Pa·s.
The composite scaffold according to claim 1, wherein the viscous cell suspension has a mass fraction of 10-50%; and the carrier comprises hyaluronic acid, gelatin, collagen, and chitosan. Or a variety.
The composite scaffold according to claim 1, wherein the density of the cells in the viscous cell suspension is (0.5 - 10) × 10 6 /mL.
The composite stent of claim 1 wherein the material of the stent substrate is a clay-based hydrogel matrix comprising a mass percent of a raw material component as follows:

Crosslinking agent: 10-50%;

Inorganic clay: 3-20%;

Ultraviolet light initiator: 0.05-0.1%;

Water: 30-86%;

The total mass percentage of each of the raw material components is 100%; wherein the crosslinking agent is a biocompatible macromolecule containing a carbon-carbon double bond, and the biocompatible macromolecule is polyethylene glycol or polyethylene. One or more of alcohol, chitosan, gelatin and hyaluronic acid.
The composite stent according to claim 6, wherein at least one end of the molecular chain of the crosslinking agent has a carbon-carbon double bond, and the molecular chain of the crosslinking agent is a main chain structure of polyethylene glycol. The molecular weight of the main chain structure of the polyethylene glycol is from 1000 to 10,000.
The composite stent of claim 7 wherein said crosslinking agent is polyethylene glycol diacrylate.
The composite stent according to claim 6, wherein the inorganic clay has a mass percentage of 5-15%.
The composite stent according to claim 6, wherein the crosslinking agent has a mass percentage of 20-40%.
The composite stent of claim 6 wherein said clay-based hydrogel matrix comprises a mass percent of a raw material component as follows:

Crosslinking agent: 20-40%;

Inorganic clay: 5-15%;

Ultraviolet light initiator: 0.05-0.1%;

Water: 45-74%;

The total mass percentage of each of the above raw material components was 100%.
The composite stent of claim 6 wherein said clay-based hydrogel matrix has a viscosity of from 30 to 350 Pa.s.
A method for preparing a cell-biomaterial composite scaffold, comprising the steps of:

(1) preparing a material of the stent base into a stent matrix precursor slurry; providing a viscous cell suspension comprising cells, a carrier capable of slowly releasing the cells, and water, the carrier being biocompatible material;

(2) printing by multi-channel three-dimensional printing method, wherein one channel is used for controlling printing of the viscous cell suspension, and the remaining at least one channel is used for controlling printing of the carrier substrate slurry;

First printing the scaffold matrix precursor slurry to form a bottom layer of the composite scaffold precursor, starting from the second layer, cross-printing the scaffold matrix precursor slurry and the viscous cell suspension to cross the viscous cell suspension Arranged between the voids of the precursor of the stent substrate to form a mixed layer, repeating printing of the mixed layer n times; finally printing the viscous cell suspension to form a top layer of the composite stent precursor to obtain cells - a biomaterial composite scaffold precursor, wherein the composite scaffold precursor forms an A'C' n B arrangement from the bottom, wherein A' is a scaffold matrix precursor layer, B is a viscous cell suspension layer, C' a mixed layer formed by interstitial distribution of the viscous cell suspension between the interstices of the scaffold matrix precursor, n being a positive integer of 3-20;

(3) curing the printed composite stent precursor to obtain a cell-biomaterial composite stent, wherein the composite stent forms an AC n B arrangement from the bottom, wherein A is a stent base layer, B For the viscous cell suspension layer, C is a mixed layer formed by the viscous cell suspension cross-distributing between the scaffold matrix voids, n being a positive integer of 3-20.
The preparation method according to claim 13, wherein the curing method is ultraviolet light curing, heat curing, ion crosslinking or freeze drying.
The preparation method according to claim 13, wherein before the start of the three-dimensional printing, the position of each channel used in the printing is corrected, and the tip of the stent substrate precursor slurry which is first discharged is Benchmark, use and use The bottoms of all the tips attached to each channel are on the same horizontal line.
The preparation method according to claim 13, wherein the driving medium in the three-dimensional printing process is pneumatic or voltage driven.
The preparation method according to claim 13, wherein in the three-dimensional printing process, the holes of the adjacent layers are staggered and communicated with each other.
The method according to claim 13, wherein the material of the stent base is a clay-based hydrogel matrix, and the clay-based hydrogel matrix comprises the following mass percentage of the raw material component:

Crosslinking agent: 10-50%;

Inorganic clay: 3-20%;

Ultraviolet light initiator: 0.05-0.1%;

Water: 30-86%;

The total mass percentage of each of the raw material components is 100%; wherein the crosslinking agent is a biocompatible macromolecule containing a carbon-carbon double bond, and the biocompatible macromolecule is polyethylene glycol or polyethylene. One or more of alcohol, chitosan, gelatin and hyaluronic acid.
The cell-biomaterial composite scaffold according to any one of claims 1 to 12 or the cell-biomaterial composite scaffold according to any one of claims 13 to 18 for use in preparing a tissue repair material.
The application of claim 19 comprising the steps of:

Taking the cell-biomaterial composite scaffold, adding the cell culture medium, immersing in the cell culture medium, culturing at 25-37 ° C, slowly releasing the cells loaded in the vector, and expanding The cell-biomaterial composite scaffold for adhering cells is obtained by adhering to the composite scaffold.