WO2022126426A1

WO2022126426A1 - Controllable gradient scaffold for loading drug, active factor and cell, 3d printing method therefor, and dedicated multi-nozzle 3d printer thereto

Info

Publication number: WO2022126426A1
Application number: PCT/CN2020/136803
Authority: WO
Inventors: 阮长顺; 彭刘琪; 陈志刚; 王品品; 吴明明
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2020-12-16
Filing date: 2020-12-16
Publication date: 2022-06-23

Abstract

The present disclosure relates to the technical field of 3D printing for biological tissues and organs, and in particular to a controllable gradient scaffold for loading a drug, an active factor and a cell, a 3D printing method therefor, and a multi-nozzle 3D printer. The controllable gradient scaffold is formed in one step by alternating printing. The integrated gradient scaffold at least comprises two materials, namely a first material and a second material. The first material is loaded with one or more of the load drug, the active factor or the cell, and the second material is loaded with one or more of the load drug, the active factor or the cell. After the first material and the second material are respectively alternatingly printed, the integrated gradient scaffold is constructed. The constructed integrated gradient scaffold for loading the drug, the active factor and the cell is free from the problem that a double-layer gradient interface is loose and prone to falling off. The integrated gradient scaffold for three-dimensional cells is constructed by means of multi-nozzle alternating printing, thereby laying the foundation for constructing complex tissues and organs of organisms.

Description

A controllable gradient scaffold loaded with drugs, active factors and cells, its 3D printing method, and a dedicated multi-jet 3D printer

technical field

The present application relates to the technical field of 3D printing of biological tissues and organs, in particular to a controllable gradient scaffold loaded with drugs, active factors and cells, a 3D printing method thereof, and a multi-jet 3D printer.

Background technique

For a long time, researchers have tried to construct complex tissues and organs in the body through tissue engineering, so as to solve the problem of repairing damaged tissues in the clinic and many problems such as infection, rejection, and shortage of donors faced by organ transplantation. However, the functional microenvironments of different tissues and organs are quite different. Therefore, choosing the appropriate technology to successfully construct tissues and organs with differential functionalities in vitro, realizing the gradient scaffolds that can load cells and can simultaneously regulate the microenvironment to promote repair and reconstruction has a great effect. difficulty.

The currently used gradient tissue and organ construction methods mainly include: 1) traditional injection molding manufacturing method; 2) development of an injectable material system containing living cells to form a multi-phase scaffold after minimally invasive implantation into the defect site; 3) 3D printing Technique for constructing gradient scaffolds.

The first type has a more precise structural design, but the scaffold itself does not contain cells. After implanting the defect site, it is necessary to guide the cells to grow into the scaffold, which may easily lead to the phenomenon of "hollowing" of uneven growth of cells inside the scaffold, or The constructed scaffold has large pores, and it is difficult to obtain micropores below 100 μm, so that it cannot provide a three-dimensional support environment conducive to cell growth, so it is not easy for cells to adhere during the culture process.

In the second type, although exogenous cells are introduced, the formed scaffold still has the problem of poor structural controllability and inability to achieve precise structure.

The third type of current 3D printing technology is a relatively simple printing method, usually a single-jet printing method, which can only print a bracket constructed from a single material. When the local structures of the two scaffolds are required to be combined, the problem that the double-layer gradient interface is weak and easy to fall off easily occurs. Therefore, the construction of complex tissues and organs cannot be realized.

Therefore, in view of the above-mentioned related technologies, the inventors believe that the existing technologies have the defect of being unable to provide a controllable gradient scaffold with high precision and biomimetic complex tissues and organs.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned defects, the present application provides a controllable gradient scaffold loaded with drugs, active factors and cells, a 3D printing method thereof, and a multi-jet 3D printer.

In the first aspect, the present application provides a controllable gradient scaffold loaded with drugs, active factors and cells, using the following technical solutions:

A controllable gradient scaffold loaded with drugs, active factors and cells, including an integrated gradient scaffold, which is alternately printed and formed in one step; the integrated gradient scaffold at least includes two materials: a first material and a second material. two materials; the first material is loaded with one or more of drugs, active factors or cells; the second material is loaded with one or more of drugs, active factors or cells; the first material and the second material are loaded with one or more of drugs, active factors or cells; The two materials were alternately printed to form an integrated gradient scaffold.

By adopting the above technical solution, this solution uses an integrated gradient scaffold with controllable gradients constructed by alternately printing multiple nozzles. It provides the basis for the construction of tissues and organs with high precision and complex structure. The first material and the second material in this scheme can be loaded with any one or any two or three of drugs, active factors or cells, and then be constructed together with other printable materials, so that the function of the biological material can be affected. Optimized to provide a basis for the construction of integrated gradient scaffolds with complex functions. In summary, this solution can provide a controllable gradient scaffold with high precision and biomimetic complex tissues and organs.

The number of layers of the controllable gradient stent constructed in this scheme can be several layers or multi-layer stents, and the gradient can be a single gradient or a complex gradient, which is determined according to the actual clinical needs.

The integrated gradient scaffold loaded with drugs, active factors, and cells in this solution is an integrated scaffold, which is alternately printed in one step, and there is no problem that the double-layer gradient interface of the related technology is not firm and easy to fall off.

The integrated gradient scaffold of the scheme is alternately printed and formed in one step in one overall step, with high production efficiency and good structural stability of the obtained complex tissues and organs of the organism.

Preferably, the first material is a thermoplastic material mixed with active factors or drugs; the second material is a hydrogel material mixed with cells or active factors or drugs; the first material and the second material Alternately print out one structural layer respectively.

By adopting the above technical solution, in this solution, the thermoplastic polymer in the first material has supporting ability and can be loaded with drugs or active factors; the second material is responsible for constructing a microbial environment with good biocompatibility and is responsible for the function of loading cells. The two materials are alternately printed in combination to obtain a structural layer.

Preferably, the active factor is one of β-tricalcium phosphate and biological glass;

The thermoplastic material is one or more of polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, and poly-L-lactide-caprolactone;

The cells are one or more of chondrocytes, bone marrow mesenchymal stem cells, endothelial cells, and nerve cells;

The medicine is one or both of anti-inflammatory and analgesic.

By adopting the above technical scheme, the bioactive factors in this scheme can provide the necessary biological microenvironment for the loaded cells and induce the specific differentiation of the cells, such as the cartilage layer loaded cells differentiate into cartilage, and the subchondral bone layer towards osteogenic differentiation, Or other functions, etc., so it can be replaced by other factors that induce biological activity. The hydrogel material in this scheme can also be replaced with other materials with good biocompatibility, and its porous structure can provide a place for cell adhesion and proliferation. The thermoplastic material in this scheme can also be replaced with other materials with the function of printing pre-shapes. The morphology of the integrated gradient scaffold in this scheme is a human or animal tissue or organ, such as bone, ear, liver and other tissues and organs. In this scheme, the cells can be a single type of cells, and can also be multi-cells of various types mixed in proportion, which can realize the construction of complex tissues and organs of organisms. The first material of this scheme can also be loaded with drugs with different functions.

Preferably, the structural layer alternately printed with the first material and the second material is further provided with a top layer, and the top layer is coated with a third material; the third material is a hydrogel material mixed with active factors or drugs.

By adopting the above technical solution, the third material added in this solution is used for compounding other functions, so as to satisfy the construction of an integrated gradient scaffold with complex and precise bionic structure. The second loading site of the drug is mainly provided here, and the drug loaded by the third material can be used to slowly release the drug to assist the adjuvant therapy after the human or animal body is loaded into the integrated gradient stent.

Preferably, a bottom layer printed from a fourth material is also connected to the bottom of the structural layer alternately printed with the first material and the second material, and the fourth material is a thermoplastic material mixed with active factors or drugs.

By adopting the above technical solutions, the bottom layer and the fourth material provide the possibility for the construction of complex tissues and organs. In specific use, the fourth material is similar to the first material, and the difference lies in the different active factors.

In the second aspect, the present application provides a 3D printing method for a controllable gradient scaffold loaded with drugs, active factors and cells, using the following technical solutions:

A 3D printing method for a controllable gradient scaffold loaded with drugs, active factors and cells, the steps of which are as follows:

S1. Preparation of printing materials: mixing thermoplastic materials with active factors or drugs as the first printing ink; using hydrogels mixed with cells or active factors or drugs as the second printing ink;

S2. Load each printing ink in the channel of each nozzle of the 3D bioprinter;

S3. One-step alternate printing: the first printing ink is ejected through the nozzle with a temperature control channel of the 3D bioprinter, the second printing ink is ejected through another nozzle of the 3D bioprinter, and the first material and the second material are alternately printed , to obtain the semi-finished product of the integrated gradient stent;

S4. Solid type: After the semi-finished product of the integrated gradient bracket is printed, it is placed in a UV cross-linking apparatus for photo-solidification, so that the semi-finished product of the integrated gradient bracket is plasticized and formed;

S5. Culture and proliferation: Take out the plasticized integrated gradient scaffold, add medium, and place it in an incubator for culture, so that the cells can adhere and proliferate in the pores of the hydrogel, and finally the finished product of the integrated gradient scaffold is obtained.

By adopting the above technical solution, at least two printing inks can be prepared in this solution, and the inks can be adjusted according to actual clinical needs. The two inks use the alternate printing method of multiple nozzles to construct an integrated gradient scaffold with controllable gradient, which can realize the personalized customization of the structure. This solution can finally provide a controllable gradient with high precision and biomimetic complex tissues and organs. bracket.

Preferably, before the step S4, it also includes the step of coating: coating the third material on the top layer of the semi-finished product of the integrated gradient stent.

By adopting the above technical solution, the third material is loaded with drugs, which can be used to slowly release the drugs to help the human or animal body in the adjuvant therapy after the integrated gradient stent is installed.

In a third aspect, the present application provides a multi-nozzle 3D printer, which adopts the following technical solutions:

A multi-nozzle 3D printer includes a multi-nozzle 3D printer body, the multi-nozzle 3D printer body includes at least two nozzles, a first nozzle and a second nozzle; the first nozzle has a temperature control channel, and the temperature control channel A first printing ink formed by mixing thermoplastic materials with active factors or drugs is loaded therein; the channels of the second nozzle are loaded with a second printing ink containing hydrogel materials mixed with cells, active factors or drugs.

By adopting the above technical solution, a dedicated multi-nozzle 3D printer loaded with multiple inks and used for printing an integrated gradient bracket is provided. This multi-nozzle 3D printer has multiple nozzles, which can be alternately printed, and are used to construct complex and controllable gradient scaffolds with precise biomimetic structures.

Preferably, the printing method of the multi-nozzle 3D printer is as follows:

Step 1: Correct the position of each channel used in printing, and make the bottoms of all nozzles connected to the channel are on the same horizontal line;

Step 2: The first nozzle and the second nozzle move relatively up, down, left and right to print: the printing substrate is stationary during the printing process, and each time the first or second material is printed, the positions of the first nozzle and the second nozzle are both up move; print according to the shape of the pre-designed printing substrate. When printing the next material, the first nozzle or the second nozzle descends to print; in this cycle, the first nozzle and the second nozzle print alternately until the printing is completed.

By adopting the above technical solution, a printing method for a multi-nozzle 3D printer is provided, the printing process is simple and flexible, and can be applied to the printing of complex and controllable gradient stents with precise bionic structures.

To sum up, the present application includes at least one of the following beneficial technical effects:

1. The integrated gradient scaffold loaded with drugs, active factors, and cells in this application is an integrated scaffold, which is alternately printed in one step, and there is no problem that the double-layer gradient interface is not firm and easy to fall off;

2. The integrated gradient scaffold of the present application can provide a better growth space for cells, and effectively solve the problems of low adhesion rate of cells on the scaffold and slow growth into the scaffold;

3. The integrated gradient scaffold of the present application has certain components and structural design, which can accurately biomimulate the complexity and heterogeneity of in vivo tissues and organs, and further promote the feasibility of 3D bioprinting to construct tissues and organs in vitro;

4. In the actual construction process of the integrated gradient stent of the present application, a specific shape and internal structure can be designed according to clinical needs, so as to realize the personalized customization of the structure;

5. The printing method of the integrated gradient scaffold of this application adopts the method of temperature-controlled printing and multi-nozzle alternate printing. This printing method can construct scaffolds loaded with different gradients of drugs, active factors, and cells, and even construct other more complex scaffolds. tissues and organs;

6. The solidification step in the integrated gradient scaffold printing method of the present application adopts the ultraviolet light curing method, which is mild and causes less damage to cells;

7. The integrated gradient scaffold printing method of the present application realizes the possibility of accurately constructing human or animal tissues or organs in vitro;

8. The shape of the integrated gradient support printed by the multi-nozzle 3D printer of the present application is related to the program design, and the program can be designed according to different requirements to print the integrated gradient support with different shapes.

Description of drawings

FIG. 1 is a process diagram of the construction of a cell-containing biomimetic osteochondral integrated scaffold with sustained-release anti-inflammatory drugs with multiple nozzles in Example 1 of the present application.

FIG. 2 is the topography of the subchondral bone layer scaffold printed in Example 1 of the present application.

FIG. 3 is the surface morphology of the subchondral bone layer scaffold observed by scanning electron microscope in Example 1 of the present application.

FIG. 4 is the topography of the cartilage layer scaffold printed in Example 1 of the present application.

FIG. 5 is the surface morphology of the cartilage layer scaffold observed by scanning electron microscope in Example 1 of the present application.

6 is a picture of the osteochondral integrated scaffold after culture and proliferation in Example 1 of the present application; in the figure, the display surface A is the front side, and the display surface B is the front side after incision.

FIG. 7 is the topography of the large-sized bone tissue scaffold in Example 2 of the present application.

In the figure, 1. Printing ink 1; 2. Printing ink 2; 3. Printing ink 3; 4. Cell-containing osteochondral integrated scaffold with slow-release anti-inflammatory drugs; 5. Coating.

Detailed ways

The present application will be further described in detail below in conjunction with accompanying drawings 1-7.

The embodiment of the present application discloses a controllable gradient scaffold loaded with drugs, active factors, and cells, which is a kind of drug, active factors, or cells loaded with any one or any two or three of the three, and then combined with other An integrated gradient scaffold constructed with printed materials. Wherein, the loaded drug can be a single drug or drugs mixed in proportion; the loaded active factor can be a single or multiple active factor mixed in proportion; the cells used can be a single species Cells, or multicellular mixtures of various types in proportions. The number of layers of the constructed integrated gradient stent can be several layers or a multi-layer stent, and the gradient can be a single gradient or a complex gradient, which is determined according to the actual clinical needs. The integrated gradient scaffold of the present application can be used for complex tissues or organs of the human or animal body.

The integrated gradient scaffold of the present application is alternately printed in one step, and it includes at least two materials, a first material, a second material and a third material. The first material is a thermoplastic material mixed with active factors or drugs. The second material is a hydrogel material mixed with cells or active factors or drugs. The first material and the second material are alternately printed to build a structural layer. The structural layer can also be provided with a top layer, and the top layer is coated with a third material, and the third material is a hydrogel material mixed with active factors or drugs. A bottom layer printed from a fourth material is also connected to the bottom of the structural layer, and the fourth material is a thermoplastic material mixed with active factors or drugs. The fourth material of the bottom layer is similar to the first material, and the difference lies in the different active factors in the application example; the third material and the second material of the top layer are also similar.

The following description takes the osteochondral integrated scaffold and the large-scale bone tissue scaffold as examples. The structure of the osteochondral integrated scaffold constructed below can also be designed into other complex structures, such as bone, ear, liver and other tissues and organs. For example, these complex tissues and organs have other structural layers, such as the second structural layer. The material of the second structural layer can refer to the two materials of the above-mentioned structural layer, and the two materials are alternately printed, or the second structural layer can also be made of only Thermoplastic material mixed with active factors.

Example 1 Osteochondral integrated scaffold

The osteochondral integrated scaffold of the present embodiment 1 includes, from bottom to top, a subchondral bone layer scaffold, a cartilage layer scaffold and a hydrogel layer containing anti-inflammatory drugs, and the three-layer structure is firmly connected.

Among them, the subchondral bone layer scaffold is printed by printing ink-1 containing polycaprolactone and β-tricalcium phosphate, the molecular weight of the used polycaprolactone is 10,000-100,000, and the content of β-tricalcium phosphate is 5 %～40%;

The cartilage layer scaffold is alternately printed with two ink materials, namely the printing ink 2 containing polycaprolactone and the small molecular organic compound Kartogenin (KNG), the printing ink 2 containing double bond hyaluronic acid, human mesenchymal stem cells hBMSCs, UV photoinitiator and water printing ink 3 3; wherein, the molecular weight of polycaprolactone used in printing ink 2 2 is 10,000-100,000, and the mass percentage of KNG is 1 wt %; printing ink 3 3 contains double bond hyaluronate The mass fraction of acid is 5% to 30%, the density of human mesenchymal stem cells hBMSCs is 1×10 ⁶ to 5×10 ⁶ CFU/mL, the content of ultraviolet light initiator is 0.05% to 0.1%, and the rest of the components for water;

The hydrogel layer is composed of a mixture of double-bonded hyaluronic acid mixed with a protease-sensitive polypeptide and diclofenac sodium; the mass fraction of double-bonded hyaluronic acid used is 5% to 30%, and the content of the polypeptide is 4 to 30%. 16 mg/mL, and the content of diclofenac sodium is 20-80 mg/mL.

The main function of the printing ink 1 and the printing ink 2 2 is to provide support for the osteochondral integrated scaffold. The main function of printing ink 33 is to achieve the precise delivery of cells on the osteochondral integrated scaffold, so as to print an osteochondral integrated scaffold with functions similar to real tissues and organs.

1, the specific method for obtaining the osteochondral integrated scaffold of the present embodiment 1 is as follows:

S1. Printing material preparation: Dissolve and mix polycaprolactone (Mw=14000, molecular weight 100000) and β-tricalcium phosphate (content 20%) at a weight ratio of 4:1 at 60°C to obtain a printing ink 1; Mix polycaprolactone (Mw=14000, molecular weight 100000) and KGN (1wt%) in a weight ratio of 1:1 to obtain printing ink 2; Mix double bond hyaluronic acid (mass fraction of 5%) ) and human mesenchymal stem cells hBMSCs (2×10 ⁶ CFU/mL), ultraviolet photoinitiator (content 0.01%), and the rest of the components are water, and the four were mixed to obtain printing ink three 3; the double bond was transparent Ronic acid was mixed with polypeptide (10 mg/mL) and diclofenac sodium (50 mg/mL) in a volume ratio of 1:0.5:0.5 to obtain a coating containing anti-inflammatory drugs.

S2. Load each printing ink in the channel of each nozzle of the 3D bioprinter, the temperature control channel of the first nozzle is loaded with printing ink 1; the temperature control channel of the second nozzle is loaded with printing ink 2; the temperature control channel of the third nozzle is loaded with printing ink 2; The channel is loaded with printing ink three 3.

The temperature control channel of the first nozzle of the S3.3D bioprinter first prints the printing ink 1 to build the subchondral bone layer scaffold, and the subchondral bone layer scaffold is printed in 4 layers with a total of 1mm. The morphology of the subchondral bone layer scaffold is shown in Figure 2 and Figure 3.

S4. One-step alternate printing of the cartilage layer scaffold: the temperature-controlled channel of the second nozzle of the 3D bioprinter prints the printing ink two 2 on the subchondral bone layer scaffold, and the channel of the third nozzle of the 3D bioprinter prints the ink two 2 The printing ink three 3 is printed on the constructed substrate; the temperature control channel of the second nozzle and the channel of the third nozzle are then alternately printed to ensure the distribution of human mesenchymal stem cells in the cartilage layer scaffold to construct a cartilage layer scaffold . The morphology of the cartilage layer scaffold is shown in Figure 4 and Figure 5. The second nozzle and the third nozzle complete a group of alternately printed cartilage layer scaffolds as one layer, and the cartilage layer scaffolds are printed in 6 layers with a total of 1.5mm.

S5. Coating: Coating the anti-inflammatory drug-containing coating 5 on the top layer of the cartilage layer scaffold to slowly release the anti-inflammatory drug, and finally form an osteochondral integrated scaffold. The thickness of the hydrogel layer is preferably controlled at 0.5mm.

S6. Solid type: After the osteochondral integrated scaffold is printed, it is placed in a UV cross-linking apparatus for light-solidification for 300s, so that the osteochondral integrated scaffold is plasticized and formed.

S7. Culture and proliferation: Take out the plasticized osteochondral integrated scaffold, add α-MEM medium, and place it in a 37°C incubator for culture, so that human bone marrow mesenchymal stem cells adhere to the pores of double-bonded hyaluronic acid and proliferating to obtain the final cell-containing osteochondral integrated scaffold 4 with sustained-release anti-inflammatory drugs, the morphology of which is shown in FIG. 6 .

The layer height ratios of the subchondral bone layer scaffold and the cartilage layer scaffold in Example 1 are designed and printed according to clinical needs.

Example 2 Large-sized bone tissue scaffolds

Large-scale bone tissue scaffolds are alternately printed in one step from two materials in a multi-jet 3D bioprinter.

Wherein, the first material is a printing ink 1 composed of polycaprolactone and β-tricalcium phosphate, the molecular weight of the used polycaprolactone is 10,000-100,000, and the content of β-tricalcium phosphate is 5%-40%;

The second material is a printing ink mixed with double bond-grafted gelatin, double-bond-grafted sodium alginate, ultraviolet photoinitiator, water, human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells; The mass fraction of the grafted gelatin is 5% to 20%, the mass fraction of the double bond modified sodium alginate is 1% to 10%, the ultraviolet photoinitiator is 0.05% to 0.1%, and the remaining components are water; The densities of stem cells and human umbilical vein endothelial cells were controlled at 1×10 ⁶ to 3×10 ⁶ CFU/mL.

The specific obtaining method of the large-size bone tissue scaffold of the present embodiment 2 is as follows:

S1. Printing material preparation: Dissolve and mix polycaprolactone (Mw=14000) and β-tricalcium phosphate at a weight ratio of 4:1 at 60°C to obtain printing ink one; the double bond-grafted gelatin (8 %), double bond-grafted sodium alginate (2%), ultraviolet photoinitiator (0.01%) and water, the hydrogel obtained by mixing the four, and then encapsulating the hydrogel with human bone marrow mesenchymal stem cells (1.5%) ×10 ⁶ CFU/mL) and human umbilical vein endothelial cells (1.5×10 ⁶ CFU/mL), the volume ratio of the two cells was set to 1:1, and finally the printing ink four was obtained;

S2. Load each original printing ink in the channel of each nozzle of the 3D bioprinter, load the printing ink in the temperature control channel of the first nozzle of the 3D bioprinter, and load the printing ink in the channel of the second nozzle of the 3D bioprinter Four.

S3. One-step alternate printing of large-sized bone tissue: the temperature-controlled channel of the first nozzle of the 3D bioprinter prints the printing ink one; the channel of the second nozzle of the 3D bioprinter prints the printing ink four on the support scaffold layer; the first The temperature-controlled channels of the first nozzle and the channels of the second nozzle are then alternately printed to construct large-scale bone tissue scaffolds. The large-size bone tissue scaffold obtained by alternately printing a set of two nozzles is one layer. A total of 134 layers of large-sized bone tissue were printed with a height of about 4 cm, an inner diameter of 8 mm and an outer diameter of 20 mm.

S4. Solid type: After the constructed large-size bone tissue scaffold is printed, it is placed in a UV cross-linking apparatus for light-solid type for 60-100 s, so that the large-size bone tissue scaffold is plasticized and formed.

S5. Culture and proliferation: Take out the plasticized large-sized bone tissue scaffold, add α-MEM medium, and place it in a 37°C incubator for culture, so that human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells are in the hydrogel Adhesion and proliferation in the pores of the layer to obtain a real large-scale bone tissue scaffold, the morphology of which is shown in Figure 7.

In above-mentioned embodiment 1 or embodiment 2:

(1) Polycaprolactone can be replaced with other thermoplastic and pre-printed materials, such as one or more of polylactic acid, polylactic acid-glycolic acid copolymer, poly L-lactide-caprolactone, etc.;

(2) β-tricalcium phosphate with osteogenic effect can be replaced with other active materials with the function of regulating the microenvironment, such as bioglass, etc.;

(3) Kartogenin (KGN), a small-molecule organic compound with cartilage-forming effect, can be replaced with other active components that promote cartilage differentiation;

(4) The cells used can be one or more cells such as chondrocytes, bone marrow mesenchymal stem cells, endothelial cells, nerve cells, etc., according to actual clinical needs;

(5) Double bond-modified hyaluronic acid acts as a cell carrier, providing a place for cell adhesion and proliferation, so double-bond-modified hyaluronic acid can also be replaced with modified gelatin, collagen, chitosan and other biocompatible good biological material;

(6) The double-base modification method of hyaluronic acid is not limited to double-bond grafting, and other methods, such as the double-bond-modified hyaluronic acid obtained by the method of enzymatic cross-linking of grafted tyramine groups, are equally feasible;

(7) Solid type method In addition to the ultraviolet curing method, other polymerization methods can also be used, such as enzymatic cross-linking, ion cross-linking and other methods.

The embodiment of the present application also discloses a special multi-jet 3D printer for a 3D printing method of a controllable gradient scaffold loaded with drugs, active factors and cells. The multi-nozzle 3D printer body includes a plurality of nozzles, but at least two nozzles, a first nozzle and a second nozzle; the first nozzle has at least a temperature control channel, and the temperature control channel is loaded with thermoplastic materials and active factors or The printing ink 1 formed by mixing the medicines; the second nozzle is a common channel, and the printing ink 2 of the hydrogel material mixed with cells, active factors or medicines is loaded in the channel. Temperature-controlled printing and alternate printing are performed by at least the two types of nozzles.

The first and second nozzles of the multi-jet 3D printer are both driven by air pressure or voltage to eject ink, and the air pressure or voltage drive produces a force on the printing ink 1 with active factors and the printing ink 2 with cells. Cause damage to the printing material, or affect the activity of the printing material.

The printing method of the special-purpose multi-nozzle 3D printer of the embodiment of the present application is as follows:

Step 1: Correct the position of each channel used in printing, and make the bottom of all nozzles connected to the channel on the same horizontal line. Adjust the position of the nozzle on the printing platform based on the nozzle that discharges the material first.

Step 2: The first nozzle and the second nozzle move relatively up, down, left and right to print: the printing substrate is stationary during the printing process, and the positions of the first nozzle and the second nozzle move up each time the printing of the first material or the second material is completed; According to the shape of the pre-designed printing substrate, when printing the next material, the first nozzle or the second nozzle descends to print; in this cycle, the first nozzle and the second nozzle print alternately until the printing is completed.

The above printing process is all completed in a biological safety cabinet to ensure that the overall operating environment is sterile. The shape of the printing substrate is related to the program design of the multi-nozzle 3D printer. The program can be designed according to different requirements to print integrated gradient scaffolds with different shapes, sizes or gradients.

The above are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore: all equivalent changes made according to the structure, shape and principle of the present application should be covered within the scope of the present application. Inside.

Claims

A controllable gradient scaffold loaded with drugs, active factors and cells, including an integrated gradient scaffold, characterized in that: the integrated gradient scaffold is alternately printed and formed in one step; the integrated gradient scaffold comprises at least two materials: a first a material and a second material; the first material is loaded with one or more of drugs, active factors or cells; the second material is loaded with one or more of drugs, active factors or cells; the first material is loaded with one or more of drugs, active factors or cells The first material and the second material are alternately printed to form an integrated gradient scaffold.
The controllable gradient stent loaded with drugs, active factors and cells according to claim 1, wherein the first material is a thermoplastic material mixed with active factors or drugs; the second material is mixed with A hydrogel material of cells or active factors or drugs, the first material and the second material alternately print a structural layer respectively.
A controllable gradient scaffold loaded with drugs, active factors and cells according to claim 2, characterized in that:

The active factor is one of β-tricalcium phosphate and biological glass;

The thermoplastic material is one or more of polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, and poly-L-lactide-caprolactone; the cells are chondrocytes, bone marrow mesenchymal One or more of stem cells, endothelial cells, and nerve cells;

The medicine is one or both of anti-inflammatory and analgesic.
The controllable gradient scaffold loaded with drugs, active factors and cells according to any one of claims 1 to 3, wherein the structural layer alternately printed with the first material and the second material is further provided with The top layer is coated with a third material; the third material is a hydrogel material mixed with active factors or drugs.
The controllable gradient scaffold loaded with drugs, active factors and cells according to claim 4, wherein the bottom of the structural layer alternately printed with the first material and the second material is also connected with a fourth material. For the printed bottom layer, the fourth material is a thermoplastic material mixed with active factors or drugs.
A 3D printing method for a controllable gradient scaffold loaded with drugs, active factors and cells, characterized in that the steps are as follows:

S1. Preparation of printing materials: mixing thermoplastic materials with active factors or drugs as the first printing ink; using hydrogels mixed with cells or active factors or drugs as the second printing ink;

S2. Load each printing ink in the channel of each nozzle of the 3D bioprinter;

S3. One-step alternate printing: the first printing ink is ejected through the nozzle with a temperature control channel of the 3D bioprinter, the second printing ink is ejected through another nozzle of the 3D bioprinter, and the first material and the second material are alternately printed , to obtain the semi-finished product of the integrated gradient stent;

S4. Solid type: After the semi-finished product of the integrated gradient bracket is printed, it is placed in a UV cross-linking apparatus for photo-solidification, so that the semi-finished product of the integrated gradient bracket is plasticized and formed;

S5. Culture and proliferation: Take out the plasticized integrated gradient scaffold, add medium, and place it in an incubator for culture, so that the cells can adhere and proliferate in the pores of the hydrogel, and finally the finished product of the integrated gradient scaffold is obtained.
A 3D printing method for a controllable gradient stent loaded with drugs, active factors, and cells according to claim 6, characterized in that, before the step S4, the method further comprises the step of coating: on the top layer of the semi-finished product of the integrated gradient stent A third material is applied.
A dedicated multi-nozzle 3D printer, comprising a multi-nozzle 3D printer body, characterized in that: the multi-nozzle 3D printer body at least includes two nozzles, a first nozzle and a second nozzle; the first nozzle has a warm A control channel, the temperature control channel is loaded with a first printing ink formed by mixing a thermoplastic material with an active factor or a drug; the channel of the second nozzle is loaded with a second printing ink mixed with a hydrogel material of cells, an active factor or a drug printing ink.
A kind of special-purpose multi-nozzle 3D printer according to claim 8, is characterized in that: the multi-nozzle 3D printer printing method is as follows:

Step 1: Correct the position of each channel used in printing, and make the bottoms of all nozzles connected to the channel are on the same horizontal line;

Step 2: The first nozzle and the second nozzle move relatively up, down, left and right to print: the printing substrate is stationary during the printing process, and each time the first or second material is printed, the positions of the first nozzle and the second nozzle are both up move; print according to the shape of the pre-designed printing substrate. When printing the next material, the first nozzle or the second nozzle descends to print; in this cycle, the first nozzle and the second nozzle print alternately until the printing is completed.