CN113244460A

CN113244460A - Oriented microchannel bracket for promoting tissue regeneration and preparation method thereof

Info

Publication number: CN113244460A
Application number: CN202110473808.1A
Authority: CN
Inventors: 朱美峰; 孔德领; 李雯
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-08-13

Abstract

The invention belongs to the technical field of preparation of porous natural polymer scaffolds in the field of tissue engineering, and relates to a preparation method of an oriented microchannel scaffold for promoting tissue regeneration, which comprises the following steps: preparing a fiber support framework, filling a natural support material and eluting the fiber support framework. The beneficial effects are that: the natural polymer scaffold with controllable and communicated pore diameter successfully solves the problem that the pore diameter of the natural polymer scaffold material is difficult to control.

Description

Oriented microchannel bracket for promoting tissue regeneration and preparation method thereof

Technical Field

The invention belongs to the technical field of preparation of porous natural polymer scaffolds in the field of tissue engineering, and relates to an oriented microchannel scaffold for promoting tissue regeneration and a preparation method thereof.

Background

The tissue engineering scaffold material needs to simulate the structure and function of extracellular matrix, plays a certain role of supporting and template, provides sites for adhesion, growth, differentiation and proliferation of cells, and further guides the regeneration of damaged tissues so as to repair the damaged tissues and organs. The material properties of the scaffold are thus crucial to determining the response and fate of the cells. The ideal tissue engineering scaffold should have the following conditions: good biocompatibility; a controllable pore structure; high porosity and connectivity; proper mechanical strength; degradability; is favorable for loading biological macromolecules and maintaining the activity of the biological macromolecules. Three-dimensional tissue engineering scaffold materials generally have a highly interconnected porous structure and interconnected networks, thereby promoting the diffusion of nutrients and oxygen and the removal of waste, playing an important role in the adhesion, proliferation and migration of cells, and tissue vascularization and the formation of new tissues, and thus have wide applicability, including drug screening, disease modeling and organotypic modeling, as well as basic biomedical scientific research and in vivo implantation (tissue engineering and regenerative medicine are widely used).

Natural materials such as collagen, fibroin, gelatin and the like have good biocompatibility and biodegradability, so that the natural materials are widely researched and applied in various fields such as clinical application, biomedical engineering, tissue repair and the like. To date, a number of techniques have been used to construct porous scaffolds of natural materials, such as conventional techniques of electrospinning, particle leaching, freeze-drying and phase separation. While these strategies have their own advantages, and the ease of scaffold preparation, these techniques are limited in their ability to fabricate complex tissue structures, and lack precision and controllability. In summary, these techniques produce porous protein scaffolds with random structures, poorly controlled pore sizes, and lack topology-directing structures.

Disclosure of Invention

Aiming at the problem that the structure of a natural scaffold material is difficult to control, the invention discloses a preparation method of the natural scaffold material with a controllable pore structure, which is controllable in structure and simple to prepare. The method is flexible to use, simple and convenient, and can be used for preparing various topological hole structures by using natural materials according to requirements.

The invention discloses a preparation method of an oriented microchannel bracket for promoting tissue regeneration, which comprises the following steps:

step 1, preparing a fiber support framework; preparing a fiber supporting framework by using a biodegradable polymer or a corresponding compound thereof as a raw material and adopting a textile technology;

step 2, filling natural scaffold materials; immersing the fiber support framework prepared in the step 1 into a support material or a solution thereof, and then carrying out curing treatment to obtain a composite support;

step 3, eluting the fiber support skeleton; and (3) placing the composite scaffold prepared in the step (2) in a template eluent to elute the fiber support skeleton, so as to obtain the natural material scaffold with a controllable topological structure.

Further, in the step 1, the spinning technology adopts at least one of electrostatic spinning, wet spinning, melt spinning and 3D printing.

Further, in the step 1, the fiber arrangement included angle of the fiber support skeleton is 0 to 360 °, more preferably 15 to 90 °, and still more preferably 45 °.

Further, in the step 1, the fiber diameter is 400nm-1mm, preferably 120-450 μm, and more preferably 120-280 μm.

Further, in step 1, the biodegradable polymer material is one or more selected from Polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polylactic-co-glycolic acid (PLGA), poly-L-lactide-caprolactone (PLCL), polysebacic acid glyceride (PGS), Polyhydroxyalkanoate (PHA), poly-p-dioxanone (PDS), Polyurethane (PU), polyvinyl alcohol (PVA), and polyethylene glycol (PEO).

Further, in the step 1, the prepared fiber support skeleton controls the bonding between the fibers by heating.

Further, in the step 2, the scaffold material is made of a natural material.

Preferably, the natural material is one or more of silk fibroin, collagen, gelatin, chitosan, sodium alginate, hyaluronic acid, proteoglycan, elastin, agarose and tissue extracellular matrix material.

Further, in the step 2, the solidification treatment adopts freeze drying, and preferably the temperature is-20 to-196 ℃, and more preferably-80 to-196 ℃.

The invention discloses application of an oriented microchannel bracket for promoting tissue regeneration, which is used for tissue repair or research of experimental animals, such as hemostasis research, and repair of blood vessels, nerves, tendons, muscles, esophagus and heart.

The invention also discloses a surgical navigation marking reagent composition which comprises the oriented microchannel bracket for promoting the tissue regeneration.

Compared with the existing prepared natural stent material, the invention has the beneficial effects that:

1. the invention uses the polymer bracket as a template to prepare the natural polymer bracket with controllable and communicated aperture, successfully solving the problem that the aperture of the natural polymer bracket material is difficult to control;

2. the pore size and the connectivity of the natural polymer scaffold channel can be regulated and controlled by controlling the fiber diameter, the fiber cross arrangement angle and the bonding degree of the polymer template scaffold;

3. the matrix pore structure of the scaffold can be adjusted by controlling the freezing temperature;

4. the natural scaffold material can accurately simulate the structure and components of damaged tissues, so that the natural scaffold material can be widely used in the aspects of tissue defect repair, hemostatic materials, in-vitro cell planting, stem cell treatment, drug delivery and the like.

Drawings

FIG. 1 is a representation of microchannel scaffold prepared from fibrous support scaffolds at different angles;

FIG. 2 is a graph of Young's modulus and maximum compressive stress of microchannel scaffolds prepared from fiber support scaffolds at different angles;

FIG. 3 is a representation of a microchannel scaffold prepared by treating a fibrous support scaffold at different temperatures;

FIG. 4 is a graph of Young's modulus and maximum compressive stress of microchannel scaffolds prepared by treating a fiber support scaffold at different temperatures;

FIG. 5 is a representation of a microchannel scaffold prepared from fibrous support scaffolds of different diameters;

FIG. 6 is a graph of Young's modulus and maximum compressive stress for microchannel scaffolds made from fiber-supported scaffolds of different diameters.

Detailed Description

The following examples are given to illustrate the technical examples of the present invention more clearly and should not be construed as limiting the scope of the present invention.

Example 1

Nano-scale porous fibroin scaffold prepared by electrostatic spinning with PCL as template

Preparing a PCL nanofiber scaffold: 2.5 g of PCL with the molecular weight of 80000 is weighed, added into 10ml of dichloromethane, stirred and dissolved at room temperature until the mixture is clear, and a PCL solution with the concentration fraction of 25% (m/v) is prepared. Electrospinning was carried out at room temperature with a room relative humidity of 60%. The aluminum foil paper was covered on the electrospinning roller receiving device and grounded, 25% (m/v) PCL spinning solution was charged into a disposable syringe having a diameter of about 14.95mm, and a high voltage dc power source was connected to the syringe needle. The syringe needle was adjusted to point at the center of the cylindrical receiver, the distance between the needle and the receiver was set to 12cm, the solution flow rate was 2ml/h, the dc voltage was 16 kv, and the spinning time was 40 min. After the preparation is finished, the obtained membrane bracket material is dried in vacuum at room temperature for 48 hours so that the solvent is completely volatilized.

Preparation of the fibroin solution: taking 0.2M Na for natural silk₂CO₃Decocting the solution for 3 times, each for 20min, washing with distilled water, and air drying; and (3) mixing silk fibroin: after lithium bromide (10 g: 40g) was dissolved in a water bath at 60 ℃ for 4 hours, it was dialyzed for three days using a dialysis bag having a molecular weight of 12000, and then dialyzed with 15% polyethylene glycol for 1 day to obtain a fibroin solution having a certain concentration.

Preparing a porous fibroin scaffold: immersing the PCL of the prepared oriented structure into a fibroin solution, fully immersing the solution into a bracket, freezing at-20 ℃ and-80 ℃ or liquid nitrogen, and freeze-drying in a freeze dryer after complete freezing. And finally, soaking the scaffold in a dichloromethane solution to remove PCL, and then airing to obtain the porous fibroin scaffold.

Example 2

Micron-sized porous chitosan scaffold prepared by using PLGA as template and adopting melt spinning

Preparing a PLA coarse fiber scaffold: the scaffold was prepared using melt spinning at room temperature. Add 20 grams of PLA to the barrel. Heating the charging barrel to 100 ℃, keeping the temperature for 5 hours, adjusting the distance between the needle head and the receiving rod of the charging barrel, setting the propelling speed and the flow speed of the charging barrel to be 2ml/h, the moving speed of the x axis and the y axis of the receiving plate to be 1mm/s, the moving distance to be 15cm and the spinning time to be 60 min.

Preparation of chitosan solution: 10g of chitosan was weighed and dissolved in 50ml of acetic acid solution to prepare a 2% chitosan solution.

Preparation of porous chitosan scaffold: immersing a PLA fiber template bracket into a chitosan solution, freezing at-20 ℃ and-80 ℃ or liquid nitrogen respectively after the solution is fully immersed into the bracket, and freeze-drying a sample in a freeze dryer after the solution is completely frozen. And finally, soaking the scaffold in a dichloromethane solution to remove PLA, and then airing to obtain the porous chitosan scaffold.

Example 3

Micron-sized porous sodium alginate scaffold prepared by taking PLCL as template and adopting wet spinning

Preparation of a PLCL micrometer fiber scaffold: the scaffold was prepared in a room temperature fume hood using wet spinning. 1.5g of PLCL was weighed out and dissolved in 10ml of tetrahydrofuran, and the solution was stirred at room temperature overnight to prepare a PLCL solution having a concentration fraction of 15% (mass/volume). A cylindrical receiving rod with a diameter of 2cm was connected to a rotating motor. The PLCL spinning solution was drawn into a syringe, the distance between the syringe needle and the receiving rod immersed in the ethanol coagulation bath was set at 1cm, the spinning speed was 2ml/h, the receiving rod rotation speed was 500rpm, and the spinning time was 30 min. After the preparation is finished, the micron fiber support is dried in vacuum for standby.

Preparing a sodium alginate solution: 10g of chitosan was weighed and dissolved in 50ml of distilled water to prepare a 2% sodium alginate solution.

Preparing a porous sodium alginate scaffold: immersing the PLCL fiber template support into a sodium alginate solution, freezing at-20 ℃ and-80 ℃ or liquid nitrogen respectively after the solution is fully immersed into the support, and freeze-drying the sample in a freeze dryer after the solution is completely frozen. And finally, soaking the scaffold in a dichloromethane solution to remove PLCL, and then airing to obtain the porous sodium alginate solution scaffold.

Example 4

Micron-sized vascular-like fibroin scaffold prepared by melt spinning with PLA as template

Preparing a PLA vascular stent: the scaffold was prepared using melt spinning at room temperature. Add 20 grams of PLA to the barrel. Heating the charging barrel to 100 ℃ and keeping the temperature for 5 hours, adjusting the distance between the needle head of the charging barrel and the receiving rod, setting the propelling speed and the flow rate of the charging barrel to be 2ml/h, the moving speed of the receiving plate to be 1mm/s, the rotating speed to be 200rpm/min, the moving distance to be 15cm and the spinning time to be 20 min. Preparing a PLA vascular stent with a circumferentially oriented structure.

Immersing the PLA vascular stent into the fibroin solution, freezing at-20 ℃ and-80 ℃ or liquid nitrogen respectively after the solution is fully immersed into the stent, and freeze-drying the sample in a freeze dryer after the solution is completely frozen. And finally, soaking the stent in a dichloromethane solution to remove PLA, and then airing to prepare the porous vascular stent with the circumferentially oriented structure.

Example 5

And (3) preparing the oriented micron-sized tendon-like scaffold by using PLA as a template and adopting 3D printing.

Preparation of PLA tendon scaffolds: the method comprises the steps of designing and storing an STL format file by using a 3D printing technology and CAD software, converting the STL file into a printable G code by using 3D Simply software, and printing an oriented micron fiber support required by an experiment by using a matched PLA material. Rolling a PLA membranous scaffold into a cylinder according to the sizes of natural tendons and nerves, completely immersing the PLA membranous scaffold into a fibroin solution, fully immersing the solution into the scaffold, respectively freezing the scaffold at-20 ℃ and-80 ℃ or liquid nitrogen, and freeze-drying a sample in a freeze dryer after the scaffold is completely frozen. Finally, the scaffold is soaked in a dichloromethane solution to remove PLA, and then the scaffold is dried in the air to prepare the porous tendon scaffold with an oriented structure.

In order to further illustrate the beneficial effects of the invention, the following application examples and comparative examples are specially set for testing:

application example 1

An oriented microchannel scaffold for promoting tissue regeneration, prepared according to the following steps:

step 1, preparing a fiber support framework; selecting a PLA material as a raw material, and preparing a fiber supporting framework by adopting a 3D printer; the PLA fiber angle was 15 °, and the fiber diameter was 120 μm.

Step 2, filling natural scaffold materials; immersing the fiber support skeleton prepared in the step 1 into a silk fibroin solution, freezing at-20 ℃, and freeze-drying for 48 hours by using a freeze dryer to obtain a composite scaffold (PLA-SF);

step 3, eluting the fiber support skeleton; soaking the composite scaffold (PLA-SF) prepared in the step 2 in absolute ethyl alcohol for 4 hours to promote silk fibroin denaturation, then taking out, transferring the composite scaffold to dichloromethane, washing for 48 hours, changing liquid every 12 hours, and then adding dichloro: the mixture solution of anhydrous methanol 1:1 was washed for 24h, and the solution was changed every 12h to sufficiently remove the polymer material. And finally, washing the sample by absolute ethyl alcohol, taking out the sample, and placing the sample into 75% alcohol for later use to obtain the natural material support with the controllable topological structure.

Application examples 2 to 18

Application examples 2-18 differ from application example 1 only in the manufacturing parameters, as detailed in table 1:

table 1 application examples 1-18 production parameter table

Comparative examples 1 to 2

To further illustrate the advantageous effects of the present invention, comparative examples 1 to 2 were different from application example 1 only in the production parameters.

The difference between comparative example 1 and application example 1 is that the scaffold was directly prepared from fibroin without preparing the scaffold in step 1, and the other operating parameters were the same.

Comparative example 2 was compared with comparative example 1, except that-196 deg.C (liquid nitrogen) was used as the freezing temperature.

In order to further illustrate the beneficial effects of the parameter selection of the present invention, the following experiments were specifically set up:

influence of the angle of the textile fibres

As shown in fig. 1, application examples 1 to 3 are compared with comparative example 1, and each of the channels of application examples 1 to 3 is hollow and connected to each other, and the periphery of the channel shows open pores. In addition, each hole in the corner bracket is connected to its adjacent hole or channel, which also reveals the establishment of a three-dimensional interconnect network.

It is noteworthy that the apparent angular intersection between microchannels can be observed from the figure. The pore diameter, the porosity of the pore column and the pore wall and the porosity of the stent in the application examples 1-3 have no obvious difference. While in comparative example 1, the disordered pores of 43.7. + -. 11.0. mu.m were randomly distributed throughout the stent, the pores were mostly closed and open when viewed from the same two directions, with only pores having a diameter of 16.0. + -. 4.3. mu.m being randomly distributed over the circular pores, significantly smaller than all the angular stents. Accordingly, the porosity of the control scaffold was significantly lower than the angle scaffold.

In addition, as shown in the mechanical test results of fig. 2, the young's modulus and the maximum compressive stress of the stent of comparative example 1 were significantly higher than those of the angle stent.

Influence of freezing temperature

As shown in fig. 3, in application examples 4 and 13, compared with comparative example 2, the pore structure of comparative example 2 is difficult to observe by Micro CT by controlling the temperature to regulate the size of the micropores of the matrix, and on the contrary, the pore diameter and channel structure can be obviously observed in application examples 4 and 13; the corresponding SEM shows that the structure of comparative example 2 is compact, while the pore diameter distribution is uniform in application example 4 and application example 13, and pores with different sizes are distributed on the pillars and the channel walls.

Meanwhile, the diameter of the strut hole in the application example 4 and the application example 13 is obviously higher than that of the channel wall hole although the diameters of the channels in the application example 4 and the application example 13 are similar, only the strut hole structure is distributed in the comparative example 2, and the pore diameter is 1.5 +/-0.4 mu m; and the pillar pore size and channel wall pore size of application example 13 were significantly higher than application example 4, and correspondingly, the porosity of application example 13 was significantly higher than application example 4. Therefore, the water absorption of application example 13 was also significantly higher than that of application example 4, and both were significantly higher than that of comparative example 2. It is shown that the gradient structure scaffold can be prepared by the matrix pore structure and the channel wall structure of the temperature control scaffold under the same channel diameter. With decreasing freezing temperature, the scaffold pore size decreases significantly. The pores of the matrix frozen at-20 ℃ were 40 μm and the pores of the matrix frozen with liquid nitrogen were 3 μm.

As shown in fig. 4, the final mechanical test found that the young's modulus and the compressive modulus of comparative example 2 were significantly higher than those of application examples 4 and 13, whereas the compressive modulus of application example 13 was significantly higher than that of application example 4.

Influence of fiber diameter

As shown in fig. 5, in application examples 11 and 14, compared with application example 17, the CT and SEM observations of fibroin scaffolds with different diameters prepared at the same freezing temperature confirmed that the pore size of the prepared scaffolds was controllable, and the SEM results showed that the microchannel structure had an interconnected gradient pore structure, in which the channel diameters were 120 μm, 280 μm and 450 μm, respectively, the matrix micropore size was 60 μm, and the pore size on the channel walls was about 30 μm; as the channel diameter increases, porosity increases; no significant difference in mechanics.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of an oriented microchannel scaffold for promoting tissue regeneration is characterized by comprising the following steps:

2. The method for preparing an oriented microchannel scaffold for promoting tissue regeneration according to claim 1, wherein in the step 1, the spinning technology adopts at least one of electrostatic spinning, wet spinning, melt spinning and 3D printing.

3. The method for preparing an oriented microchannel scaffold for promoting tissue regeneration according to claim 1, wherein in the step 1, the fiber arrangement angle of the fiber support skeleton is 15-90 °.

4. The method for preparing an oriented microchannel scaffold for promoting tissue regeneration as claimed in claim 1, wherein the fiber diameter is 120-280 μm in the step 1.

5. The method for preparing an oriented microchannel scaffold for promoting tissue regeneration according to claim 1, wherein in the step 1, the biodegradable polymer material is one or more of polycaprolactone, polyglycolic acid, polylactic acid, poly-L-lactic acid, polylactic acid-polyglycolic acid copolymer, poly-L-lactide-caprolactone, polysebacate, polyhydroxyalkanoate, poly-dioxanone, polyurethane, polyvinyl alcohol, and polyethylene glycol.

6. The method for preparing an oriented microchannel scaffold for promoting tissue regeneration according to claim 1, wherein the prepared fibrous support scaffold is heated to control the bonding between fibers in step 1.

7. The method for preparing an oriented microchannel scaffold for promoting tissue regeneration according to claim 1, wherein the scaffold material in the step 2 is a natural material.

8. The method for preparing the oriented microchannel scaffold for promoting tissue regeneration according to claim 1, wherein the solidification treatment in the step 2 is freeze drying at a temperature of-20 to-196 ℃.

9. An oriented microchannel scaffold for promoting tissue regeneration, prepared by the method of any one of claims 1 to 8.