CN111346264A

CN111346264A - Preparation method of multi-branch hollow biomaterial catheter for tissue repair

Info

Publication number: CN111346264A
Application number: CN202010161214.2A
Authority: CN
Inventors: 李贵才; 李沈洁; 韩琦; 张鲁中; 杨宇民; 金春奎
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-03-10
Filing date: 2020-03-10
Publication date: 2020-06-30

Abstract

The invention discloses a preparation method of a multi-branch hollow biomaterial catheter for tissue repair, which can be used for clinically repairing tissue injuries of nerves, trachea, muscles, blood vessels and the like which need branch growth. The method can realize integrated processing of various biological materials and various branched structures, and is prepared by directly utilizing the processes of biological 3D printing, molding, freeze drying and electrostatic spinning. The method comprises the following specific steps: (1) designing an axle center and a sleeve model with different bifurcation structures; (2) preparing a maltose solution; (3) printing and assembling maltose axes and sleeves with different bifurcation structures; (4) pouring the biological material solution into the model; (5) drying and forming; (6) removing the outer sleeve, and dissolving the axis to obtain the hollow biomaterial catheter with the multi-branch structure. The catheter can be used for repairing tissue injuries needing oriented growth, such as peripheral nerve injury trachea, muscle, blood vessels and the like in clinic.

Description

Preparation method of multi-branch hollow biomaterial catheter for tissue repair

Technical Field

The invention relates to a preparation method of a multi-branch hollow biomaterial catheter for tissue repair, belonging to the field of biomedical materials and implantable medical devices.

Background

Because of the frequent occurrence of tissue or organ trauma caused by accident traffic or war conflict, the current gold standard for treating various tissue and organ injuries is still autograft, but because of the limited source of autograft, severe limitation of donor site lesion and size mismatch, the use of artificial graft made of various biological materials is considered as an effective alternative treatment method and has achieved better treatment effect. However, normal tissues and organs such as nerves, trachea, muscles, and blood vessels, etc. are not grown only in a single direction under physiological conditions, but are required to grow divergently at a specific site to form a branched structure for better material transportation or information transfer. Therefore, the construction of the tissue engineering artificial graft with different bifurcation structures has important clinical significance and application value for better realizing the damage repair of tissues and organs. However, to date, there have been few reports of constructing artificial grafts having different branch structures for tissue and organ damage repair.

The continuous progress of tissue engineering research provides a new method for treating tissue and organ injuries, the construction of local damage microenvironment is crucial to tissue regeneration, the biomaterial scaffold provides mechanical and three-dimensional structural support for tissue and organ regeneration, and biological 3D printing, micromolding and electrostatic spinning are 3 different process technologies for preparing the biomaterial scaffold which are established in recent years. The 3D printing technology is also called additive manufacturing or incremental manufacturing, and is a technology for generating a three-dimensional entity by adding materials layer by layer through superposition of continuous physical layers based on a digital model file. The biological 3D printing technology can be used for preparing a microscopic 3D structure body with complex properties and a bracket for creating a unique design, but the problems of difficult catheter forming, limited printing height, insufficient material mechanical property and the like of directly printing a hollow biological material catheter with a bifurcation shape still exist; the micro-molding method can conveniently prepare hollow biomaterial catheters in batches by means of a mold, can form a porous structure, and is more beneficial to tissue and cell growth and migration, but is difficult to prepare the catheter with a multi-branch structure due to the difficulty of demolding; the electrostatic spinning technology can construct a porous fiber scaffold with nanometer and submicron scale, and can realize orientation distribution of fibers so as to be more beneficial to orientation growth of tissues and cells, but the preparation of the multi-branch biomaterial catheter is difficult to realize by the single electrostatic spinning technology.

How to construct a hollow biomaterial catheter with different branch structures in vitro actually solves the problems of the stent forming process and the requirements of the stent on the aspects of materials, mechanics, structural performance and the like. The biological 3D printing technology can well realize the forming processing of a material fine structure with excellent mechanical property, the micro-molding technology is convenient for mass production preparation of the biomaterial conduit, and the electrostatic spinning technology can realize the further regulation and control of the conduit space structure. Therefore, the combination of biological 3D printing, micromolding and electrospinning is expected to overcome the problems in the preparation of multi-branched hollow biomaterial catheters.

In view of the thermal stability and water solubility of maltose, it is contemplated that maltose is processed into a sacrificial template and used as the axis of the biomaterial conduit, and the hollow biomaterial conduit is prepared after the axis is dissolved away by water.

In summary, in the present invention, we disclose a method of preparing a mold with a multi-branched maltose axle as a sacrificial template by means of biological 3D printing technology, and then preparing a catheter of hollow biomaterial with multi-branched structure by combining micro-molding, freeze-drying and electrospinning technologies, which provides a beneficial reference for the development of tissue engineering artificial graft for repairing damaged tissues or organs with multi-branched structure.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems existing in the prior art of preparing the hollow biomaterial catheter with a multi-branch structure, the invention provides a preparation method of a multi-branch hollow biomaterial catheter for tissue repair. The method takes maltose with a multi-branch structure prepared by biological 3D printing as the axis of a catheter mould, takes various natural or synthetic biomaterials as raw materials, adopts micro-molding, freeze-drying, electrostatic spinning or extraction methods to prepare the hollow biomaterial catheter with the multi-branch structure, has better mechanical property and stability, can be used for clinically repairing tissues needing oriented growth, such as peripheral nerve injury trachea, muscle, blood vessels and the like, and has important clinical application significance and value.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

a multi-branch hollow biological material conduit for tissue repair is prepared from natural or synthetic biological materials, a multi-branch axial component is composed of maltose, a multi-branch structure is prepared by adopting a 3D biological printing technology, and the multi-branch hollow biological material conduit is prepared by adopting a micro-molding combined freeze drying method, an electrostatic spinning method or an extraction method.

Further: the specific operation steps of the multi-branch hollow biomaterial conduit adopting the micro-molding and freeze-drying method are as follows:

step 1) designing and drawing an axis and sleeve model with different bifurcation structures by adopting computer professional software, exporting the model to a file capable of being printed in a 3D mode, and importing the file to a computer connected with a biological 3D printer for later use;

step 2), preparing a maltose solution, printing the axis and the sleeve for preparing the multi-branched hollow biomaterial catheter mould by adopting a biological 3D printing technology, and assembling;

step 3), preparing a biological material solution, pouring the biological material solution into the assembled multi-branch type conduit mould for drying treatment;

step 4) post-treatment of the bracket: removing the outer sleeve of the mould, and then soaking the maltose axle coated with the biomaterial scaffold in water to obtain the multi-branch hollow biomaterial catheter.

Further: the multi-branch hollow biomaterial conduit adopts the following operation steps in an electrostatic spinning method: providing an electrostatic spinning device, wherein the electrostatic spinning device comprises a collector, a multi-branch type maltose axle center is arranged at an electrostatic spinning receiver end, and a biomaterial bracket is prepared by spinning on the multi-branch type maltose axle center by adopting an electrostatic spinning technology;

and (3) post-treatment of the bracket: and soaking the multi-branch type maltose axle coated with the biomaterial scaffold in water to obtain the multi-branch type hollow biomaterial catheter.

Further: the operation steps of the multi-branch type hollow biomaterial conduit adopting the leaching method are as follows: directly soaking a multi-branched maltose axle in a biological material solution for a certain time by adopting an extraction method, extracting, and then drying;

and (3) post-treatment of the bracket: and soaking the dried multi-branch maltose axle coated with the biomaterial scaffold in water to obtain the multi-branch hollow biomaterial catheter.

Further: in the step 1), the computer professional software is Solidworks software, the different branched structures comprise Y-shaped and more than 3 branched structures, the file capable of being printed in a 3D mode is in an STL format, and the biological 3D printer is provided with double nozzles;

the axis component is maltose, and the sleeve component is silica gel; the branched structure can be a Y-shaped structure and can also be a multi-branched structure which is symmetrically arranged; the multi-branched maltose axle has at least 3 and more branches.

Further: in the step 2), the 3D printing technology is completed by adopting biological 3D printers of various models, the axis of the mold is composed of maltose, the printing of the axis is completed in a low-temperature environment, and an outer sleeve of the mold is prepared from silica gel and can be spliced and combined;

the axis diameter of the 3D printed maltose is 1-5mm, the inner diameter of the silica gel outer sleeve is 3-8mm, and the wall thickness of the outer sleeve is 1-2 mm; the concentration of the maltose solution is 20-40%, and the maltose solution is in a semi-fluid state;

the printing temperature of the multi-branch maltose axle center and the silica gel sleeve is 0-4 ℃, the printing line width is 50-100 mu m, and the printing length is 0.5-3 cm;

the assembly process of the printed maltose axle center and the silica gel sleeve is as follows: one end of the axis of the maltose is vertically fixed, and then the silica gel sleeve is assembled outside the axis of the maltose to form the maltose-axis-silica gel sleeve structure.

Further: in the step 3), the drying treatment method of the biological material solution in the mold adopts natural drying or freeze drying technology, wherein the natural drying is drying for 48 hours at room temperature, and the freeze drying technology adopts a freeze dryer to freeze and dry for 48 hours.

Further: the electrostatic spinning device comprises a high-voltage power supply, a micro-propulsion system and a receiving device, wherein the high voltage is 10-25Kv, the micro-propulsion speed is 0.01-2ml/min, the spinning receiving distance is adjustable within 5-50cm, and the rotating speed of a rotor at a receiving end is adjustable within 0-1000 rpm;

the multi-branch maltose shaft center is vertically or horizontally suspended and fixed on a receiving end rotor, and the spinning time is at least more than 6 h.

Further: the biological material leached by the multi-branch maltose axle has high viscosity and can be stably attached to the maltose axle, and the leaching time is 5-10 s.

Further: the biomaterial solution is all degradable and non-degradable natural or synthetic polymer material solutions, including extracellular matrix protein, chitosan, silk fibroin, collagen, hyaluronic acid, PLA, PLGA, PCL and PU;

the stent post-treatment specifically operates as follows: the maltose axle coated with the biomaterial scaffold is soaked in water for at least 4 hours at the water temperature of 25-40 ℃.

Has the advantages that: the invention designs the maltose axle center and the silica gel sleeve mold with the multi-branch structure, and prepares the mold with the multi-branch structure by a biological 3D printing technology, the maltose axle center is convenient to degrade to form a hollow structure of the conduit, and the hollow biomaterial conduit with different branch structures is further prepared and obtained by means of a micro-molding technology, a freeze-drying technology and an electrostatic spinning technology, the conduit is made of biodegradable high polymer materials, thereby providing a guiding function for the growth of tissues and cells, accelerating the repair of the damaged tissues or organs, the used materials have certain mechanical properties, are favorable for the proliferation and growth of the cells, and have no toxicity and good tissue compatibility. The method has the advantages of simple process, good controllability, high efficiency and the like. Is expected to be used for repairing and treating clinically relevant tissues or organs after being damaged, and has important guiding significance and reference value for the design and preparation of tissue engineering artificial grafts with different branch structures.

Drawings

FIG. 1 is a flow diagram of a multi-pronged hollow biomaterial catheter for tissue repair; wherein, (1) Y-type bifurcation structure, (2) interdigital bifurcation structure, (3) contralateral arrangement type bifurcation structure;

FIG. 2 is a schematic view of a Y-catheter model designed using software;

fig. 3 is a physical diagram of a multi-branched (Y-shaped) hollow biomaterial catheter.

Detailed Description

In order to make the present invention more fully understood by those skilled in the art, the following technical solutions of the present invention are described with reference to examples and drawings, but the present invention is not limited thereto in any way.

Fig. 1 is a flow chart of a multi-branched hollow biomaterial duct for tissue repair, which includes (a) a mold design, (B) a mold for 3D printing of a multi-branched structure, (C) a multi-branched hollow biomaterial duct manufactured by micro-molding, (D) a multi-branched hollow biomaterial duct manufactured by electrospinning, and (E) a real figure of a multi-branched hollow biomaterial duct, wherein (1) a Y-branched structure, (2) an interdigitated branched structure, and (3) an opposite side arrangement branched structure.

Specific example 1: preparation of multi-branched hollow silk fibroin/PCL catheter

The method comprises the following specific steps:

1) preparation of silk fibroin solution:

silk fibroin has excellent biocompatibility and has been approved by FDA as a clinical biomaterial. Putting 150g of silkworm raw silk into 5000mL of 0.06% sodium carbonate solution, treating for 3 times at the temperature of 98-100 ℃ for 45 minutes each time, cleaning, drawing, drying in an oven (55 ℃) to obtain refined silk for later use, and adding CaCl₂、CH₃CH₂OH、H₂Preparing solution by using O according to the molar ratio of 1:2:8, dissolving the refined silk for 1h at the temperature of 72 ℃ to obtain mixed solution, filling the mixed solution into a dialysis bag, changing water every two hours, dialyzing for 72h to obtain pure silk fibroin solution, and then putting the pure silk fibroin solution into a freeze dryer for freeze drying for 48 h.

2) Preparing a mixed solution of silk fibroin and PCL:

polycaprolactone (PCL) is biodegradable polyester with shape memory property, has low melting point (about 60 ℃) and several biocompatibility, has the glass transition temperature of-60 ℃, and is in a rubber state at room temperature. The fibroin has better biocompatibility, can support the adhesion, proliferation and differentiation of cells on the surface of a material and the secretion of extracellular matrix, and the PCL has good toughening effect.

Putting the prepared silk fibroin into a cosolvent HFIP (high frequency plasma injection) according to the mass ratio of 6%, putting PCL into the HFIP according to the mass ratio of 3%, dissolving to obtain a silk fibroin/PCL mixed solution, and placing the mixed solution at the temperature of 4 ℃ for later use.

3) Preparation of maltose solution:

adding 5g of edible maltose into 100g of deionized water, heating to 80 ℃, fully stirring and dissolving to prepare a maltose solution, naturally cooling, and then placing into one of syringes of a 3D printer for storage and standby.

4) Construction of a multi-branch catheter mold:

placing an injector filled with silica gel on one nozzle of a 3D printer, placing an injector filled with maltose on the other nozzle of the 3D printer, setting the temperature of a temperature controller of a heating device to 40 ℃, heating the maltose, respectively extruding the maltose (axis) and the silica gel (outer sleeve) by controlling air pressure by software, controlling the temperature of a printing platform to be 0-4 ℃, and simultaneously enabling a receiving platform to regularly move according to a constructed forked conduit model, and quickly curing to form the mold with the forked structure. The detachable type catheter is printed by using a 3D printing technology to manufacture a mold. The diameter of the printed axis is 1-5mm, the inner diameter of the silica gel outer sleeve is 3-8mm, and the wall thickness of the outer sleeve is 1-2 mm. And after printing, taking down the mold with the maltose as the axis and the silica gel as the outer sleeve for later use.

5) Preparing a multi-branched hollow silk fibroin/PCL catheter:

pouring the silk fibroin/PCL mixed solution into the printed mould, respectively carrying out natural drying and freeze drying, then peeling off the silica gel outer sleeve, soaking the maltose axes coated with the silk fibroin/PCL into pure water at 35 ℃ for 6h, fully dissolving maltose, obtaining the multi-branched hollow silk fibroin/PCL catheter, and finishing the whole preparation process of the catheter.

Specific example 2: preparation of multi-branch hollow chitosan catheter

The method comprises the following specific steps:

this example 2 is essentially the same as example 1, except that: the biological material adopted in the step 1) is chitosan; step 5) soaking the catheter in a 4% NaOH solution; the specific steps 1) and 5) are as follows:

1) preparation of chitosan solution

The chitosan has excellent biocompatibility, mechanical property, degradability and antibacterial property, and has important application value in the field of biomedicine. Weighing 1-5g of chitosan powder and dissolving the chitosan powder into 100mL of 2% acetic acid to prepare 1% -5% chitosan solution.

5) Preparing a multi-branched hollow chitosan catheter:

pouring the chitosan solution into the mold printed in the step 4) of the example 1, respectively carrying out natural drying and freeze drying, then peeling off the silica gel outer sleeve, soaking the maltose axle coated with the dried chitosan into 4% NaOH for 6 hours together, fully dissolving the maltose to obtain the multi-branched chitosan catheter, and finishing the whole preparation process of the catheter.

Specific example 3: preparation of multi-branch silk fibroin catheter

The method comprises the following specific steps:

1) preparation of silk fibroin solution:

putting 150g of silkworm raw silk into 5000mL of 0.06% sodium carbonate solution, treating for 3 times at the temperature of 98-100 ℃ for 45 minutes each time, cleaning, drawing, drying in an oven (55 ℃) to obtain refined silk for later use, and adding CaCl₂、CH₃CH₂OH、H₂Preparing solution by using O according to the molar ratio of 1:2:8, dissolving the refined silk for 1h at the temperature of 72 ℃ to obtain mixed solution, filling the mixed solution into a dialysis bag, changing water every two hours, dialyzing for 72h to obtain pure silk fibroin solution, and then putting the pure silk fibroin solution into a freeze dryer for freeze drying for 48 h. And (3) putting the prepared silk fibroin into a cosolvent HFIP according to the mass ratio of 6%, dissolving, and then placing in an environment at 4 ℃ for later use.

2) Preparation of maltose solution:

3) Construction of a multi-branch catheter mold:

the injector filled with maltose is placed on a nozzle of a 3D printer, the temperature of a temperature controller of a heating device is set to be 40 ℃, the maltose is heated, the maltose (axis) is extruded out respectively by controlling air pressure through software, the temperature of a printing platform is controlled to be 0-4 ℃, and meanwhile, the receiving platform moves regularly according to a constructed bifurcate axis model and is rapidly solidified into a mold axis with a bifurcate structure. The diameter of the printed axis is 1-5mm, the inner diameter of the silica gel outer sleeve is 3-8mm, and the wall thickness of the outer sleeve is 1-2 mm. And taking down the maltose as the axis for standby after printing.

4) Preparing a multi-branched hollow silk fibroin catheter:

adding 5mL of silk fibroin solution into an injector and fixing the silk fibroin solution on an electrostatic spinning instrument, fixing a maltose axle center on an electrostatic spinning receiving end rotor, setting the voltage to be 20Kv, the receiving distance to be 15cm, the rotating speed of the receiving end rotor to be 800rpm, spinning time to be 6h, soaking the maltose axle center coated with silk fibroin in pure water at 35 ℃ for 6h after spinning is finished, fully dissolving maltose, obtaining a multi-branched hollow silk fibroin catheter, and finishing the whole preparation process of the catheter.

Specific example 4: preparation of a Multi-bifurcation PCL catheter

The method comprises the following specific steps:

1) preparation of PCL solution:

10g of PCL particles are dissolved in 100ml of HFIP for 4 hours to obtain a 10% PCL solution, and then the solution is placed in an environment at 4 ℃ for standby.

2) Preparation of maltose solution:

3) Construction of a multi-branch catheter mold:

4) Preparing a multi-branched hollow silk fibroin catheter:

the multi-branched maltose solid structure is soaked in PCL solution for 5 seconds and then taken out immediately, so that the PCL solution is uniformly coated on the surface of maltose, and air-dried. And (3) soaking the air-dried PCL maltose composite structure in pure water at 35 ℃ for 6h to fully dissolve maltose to obtain the multi-branched hollow silk fibroin catheter, and finishing the whole preparation process of the catheter.

5) The multi-branched maltose solid structure is soaked in PCL solution for 5 seconds and then taken out immediately, so that the PCL solution is uniformly coated on the surface of maltose, and air-dried. And (3) soaking the air-dried PCL-maltose composite structure in pure water at 35 ℃ for 6h to fully dissolve maltose to obtain the multi-branched PCL molecular polymer hollow catheter, and completing the whole preparation of the catheter.

Claims

1. A preparation method of a multi-branch hollow biomaterial catheter for tissue repair is characterized by comprising the following steps: the multi-branch hollow biomaterial conduit is made of natural or synthetic biomaterial, the multi-branch axial center component is composed of maltose, the multi-branch structure is prepared by adopting a 3D biological printing technology, and the multi-branch hollow biomaterial conduit is prepared by adopting a micro-molding combined freeze drying method, an electrostatic spinning method or an extraction method.

2. The method of preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claim 1, wherein: the specific operation steps of the multi-branch hollow biomaterial conduit adopting the micro-molding and freeze-drying method are as follows:

3. The method of preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claim 1, wherein: the multi-branch hollow biomaterial conduit adopts the following operation steps in an electrostatic spinning method: providing an electrostatic spinning device, wherein the electrostatic spinning device comprises a collector, a multi-branch type maltose axle center is arranged at an electrostatic spinning receiver end, and a biomaterial bracket is prepared by spinning on the multi-branch type maltose axle center by adopting an electrostatic spinning technology;

4. The method of preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claim 1, wherein: the operation steps of the multi-branch type hollow biomaterial conduit adopting the leaching method are as follows: directly soaking a multi-branched maltose axle in a biological material solution for a certain time by adopting an extraction method, extracting, and then drying;

5. The method of preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claim 2, wherein: in the step 1), the computer professional software is Solidworks software, the different branched structures comprise Y-shaped and more than 3 branched structures, the file capable of being printed in a 3D mode is in an STL format, and the biological 3D printer is provided with double nozzles;

6. The method of preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claim 2, wherein: in the step 2), the 3D printing technology is completed by adopting biological 3D printers of various models, the axis of the mold is composed of maltose, the printing of the axis is completed in a low-temperature environment, and an outer sleeve of the mold is prepared from silica gel and can be spliced and combined;

7. The method of preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claim 2, wherein: in the step 3), the drying treatment method of the biological material solution in the mold adopts natural drying or freeze drying technology, wherein the natural drying is drying for 48 hours at room temperature, and the freeze drying technology adopts a freeze dryer to freeze and dry for 48 hours.

8. The method of preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claim 3, wherein: the electrostatic spinning device comprises a high-voltage power supply, a micro-propulsion system and a receiving device, wherein the high voltage is 10-25Kv, the micro-propulsion speed is 0.01-2ml/min, the spinning receiving distance is adjustable within 5-50cm, and the rotating speed of a rotor at a receiving end is adjustable within 0-1000 rpm;

9. The method of preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claim 4, wherein: the biological material leached by the multi-branch maltose axle has high viscosity and can be stably attached to the maltose axle, and the leaching time is 5-10 s.

10. The method for preparing a multi-branched hollow biomaterial catheter for tissue repair as claimed in claims 2 to 4, wherein: the biomaterial solution is all degradable and non-degradable natural or synthetic polymer material solutions, including extracellular matrix protein, chitosan, silk fibroin, collagen, hyaluronic acid, PLA, PLGA, PCL and PU;