CN116173310A

CN116173310A - High molecular polymer/hydroxyapatite composite scaffold with highly ordered interconnected porous structure, and preparation method and application thereof

Info

Publication number: CN116173310A
Application number: CN202310163512.9A
Authority: CN
Inventors: 王卉; 邵云菲; 张克勤; 朱怡然
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2023-02-24
Filing date: 2023-02-24
Publication date: 2023-05-30

Abstract

The invention relates to a high molecular polymer/hydroxyapatite composite scaffold with a highly ordered interconnected porous structure, and a preparation method and application thereof. The preparation method comprises the following steps: placing the high-molecular polymer microspheres on a balance vibration table for vibration, and then heating to enable the microspheres to be fused and bonded to obtain a high-molecular polymer microsphere template; cooling and melting the high molecular polymer microsphere template, and soaking the template in a high molecular polymer/hydroxyapatite mixed solution; gel curing treatment is carried out under an electric field; removing the high molecular polymer microsphere template by using an organic solvent to obtain a composite bracket; and replacing the organic solvent in the composite scaffold with ethanol, replacing the ethanol in the composite scaffold with water, and finally freeze-drying to obtain the ordered high molecular polymer/hydroxyapatite composite scaffold. A solid, uniform, highly ordered and pore-size interconnected three-dimensional structure with large area and high quality can be obtained, and the scaffold is suitable for perfusion culture systems and has the potential for further clinical application.

Description

High molecular polymer/hydroxyapatite composite scaffold with highly ordered interconnected porous structure, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a high-molecular polymer/hydroxyapatite composite scaffold with a highly ordered interconnected porous structure, and a preparation method and application thereof.

Background

The tissue engineering three-dimensional scaffold material plays a very important role in bone tissue engineering research, and can simulate extracellular matrix in the bone tissue regeneration process so as to provide proper growth environment and mechanical support for cells. The high molecular polymer has good biocompatibility and simple processing and forming, and has been widely applied to the preparation of bone tissue engineering scaffolds. The Hydroxyapatite (HA) is taken as a main inorganic component in bone tissue, and is combined with a high polymer to form a composite material, so that the mechanical property of a composite material system can be enhanced, the biological activity of the composite material system can be endowed, and the bone regeneration capability of the composite material system can be enhanced. In different types of composite porous scaffolds, macropores (> 100 μm) tend to be more conducive to cell ingrowth and osteogenic differentiation, and the strong, uniform, interconnected porous structure of the scaffold is critical for guiding cell adhesion and penetration, providing a relatively stable growth environment for cell adhesion and proliferation. On the other hand, the culture of cells on porous scaffolds is a key step in bone tissue engineering, and in vivo, many cells are exposed to fluid shear stress generated by biological fluid systems, and these mechanical forces have important effects on the growth and morphology of cells. However, the conventional static cell culture mode limits the diffusion and flow of nutrients, and the growth of cells can only exist on the surface of cells, which is unfavorable for further proliferation. In an in-vitro bioreactor, the bracket is in a dynamic perfusion culture mode, and can simulate the fluid shear force environment similar to that in the body by adjusting the flow rate, so as to realize in-vitro long-term dynamic 3D cell culture and maintain the cell functionality. Such as endothelial cell culture, proper shear forces are beneficial in promoting its normalized arrangement; stem cell cultures require a low shear environment to avoid stimulating differentiation. The dynamic culture process promotes the entry and output of nutrients and waste more, which requires the composite scaffold to have a porous network that is interconnected, firm, ordered and uniform to ensure the effective diffusion of the substances and provide good conditions for further growth such as cell proliferation and differentiation.

In recent years, researchers have developed a variety of manufacturing techniques for preparing three-dimensional composite scaffold materials, such as freeze-drying, particle leaching, and 3D printing. The growth of ice crystals in freeze-drying processes is random, and the porous structures obtained are generally disordered and have poor pore connectivity. The particle leaching method is to form a porous bracket by using a pore-forming agent, and based on the particle leaching method, researchers develop a template formed by closely-packed monodisperse microsphere crystal lattices to manufacture a traditional inverse opal bracket, but on the arrangement of microspheres, the existing self-assembly technologies such as a spin coating method, a convection method and the like are only suitable for the assembly of two-dimensional nano-particles, the arrangement of three-dimensional micro-large particles is easy to generate multilayer accumulation or defect phenomenon, and the three-dimensional arrangement of large microspheres with large area and high quality is still difficult to realize. Meanwhile, the traditional inverse opal method is easy to generate incomplete filling in the filling process, so that the formed porous structure of the bracket is easy to collapse, the integrity of the bracket structure is influenced, and the porous structure with large-area orderly interconnection cannot be constructed. 3D printing technology developed in recent years can accurately and rapidly manufacture 3D structures of arbitrary complex shapes, but this method is relatively costly and does not have such precise instrumentation for every laboratory. Therefore, the conventional method is difficult to prepare a firm, uniform, highly ordered and pore-size-interconnected porous scaffold, the emerging 3D printing technology is not available in every laboratory, and in order to solve the problem, the electric field induced inverse opal method is adopted to prepare a high-molecular polymer/hydroxyapatite composite scaffold with a highly ordered interconnected porous structure, so that the method is a feasible strategy.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a high-molecular polymer/hydroxyapatite composite scaffold with a highly ordered interconnected porous structure. The invention can be used for preparing high molecular polymer/hydroxyapatite by electric field induced inverse opal method in any conventional material laboratory

The scheme of the (HA) composite scaffold can obtain a solid, uniform, highly ordered and pore-size interconnected three-dimensional structure, and the scaffold is suitable for a dynamic perfusion culture system and HAs potential for further clinical application.

The first aim of the invention is to provide a preparation method of a high molecular polymer/hydroxyapatite composite scaffold with a highly ordered interconnected porous structure, which comprises the following steps:

(1) Placing the high polymer microspheres on a balance vibration table to vibrate for 1-3 h, and heating to melt and bond the high polymer microspheres to obtain a high polymer microsphere template;

(2) Cooling the high polymer microsphere template after melting and bonding in the step (1), and soaking the template in the high polymer/hydroxyapatite mixed solution; (the polymer/hydroxyapatite mixed solution is prepared by simply mixing a polymer with hydroxyapatite).

(3) Performing gel curing treatment on the high polymer microsphere template soaked in the step (2) under an electric field, and freeze-drying;

(4) Cutting off redundant high polymer/hydroxyapatite solids in the freeze-dried high polymer microsphere template, and removing the high polymer microsphere template by using an organic solvent to obtain a composite scaffold;

(5) And (3) replacing the organic solvent in the composite scaffold in the step (4) by ethanol, replacing the ethanol in the composite scaffold by water, and finally freeze-drying to obtain the high-molecular polymer/hydroxyapatite composite scaffold with the highly ordered interconnected porous structure.

In one embodiment of the present invention, in step (1), the high molecular polymer is selected from PMMA or PS.

In one embodiment of the present invention, in the step (1), the high molecular polymer is spherical and has a diameter of 10 μm to 1000 μm.

In one embodiment of the invention, in step (1), the diameter is 40 μm to 60 μm or 90 μm to 110 μm.

In one embodiment of the invention, in step (1), the diameter is 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or any value between any two values.

In one embodiment of the present invention, in the step (1), the heating temperature is 175 ℃ to 195 ℃ and the heating time is 3 hours to 5 hours.

In one embodiment of the present invention, in the step (2), the concentration of the hydroxyapatite in the high molecular polymer/hydroxyapatite mixed solution is 5 to 50wt%, and may be 5wt% to 10wt%, 10wt% to 15wt%,15wt% to 20wt%,25wt% to 30wt%,35wt% to 40wt%,45wt% to 50wt%; specifically, the concentration may be 5wt%, 10wt%, 15wt%, 20wt%,25wt%, 30wt%,35wt%, 40wt%,45wt%, 50wt%, or any concentration between any two values. The high molecular polymer in the high molecular polymer/hydroxyapatite mixed solution is selected from one or more of silk fibroin, sericin and carboxymethyl chitosan; silk fibroin and sericin are preferable. The high molecular polymer has good electric field gel performance.

In one embodiment of the present invention, in the step (2), the concentration of the silk fibroin in the high molecular polymer/hydroxyapatite mixed solution is 6 to 12wt%; the concentration is not particularly limited, and may be 6wt% to 7wt%, 7wt% to 8wt%, 8wt% to 9wt%, 9wt% to 10wt%, 10wt% to 11wt%, 11wt% to 12wt%, and may be particularly 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%; or any concentration value between any two values.

In one embodiment of the present invention, in the step (3), the specific operation of the gel curing treatment under the electric field: binding a high polymer microsphere template soaked with a high polymer/hydroxyapatite mixed solution on a conductive substrate plate A, then immersing the high polymer/hydroxyapatite mixed solution into another conductive substrate plate B, connecting the conductive substrate plate A with a positive electrode of a power supply, connecting the conductive substrate plate B with a negative electrode of the power supply, and electrifying; the conductive substrate plate A or the conductive substrate plate B is a graphite plate or an inert metal plate.

In one embodiment of the invention, the energizing voltage is 15V to 30V.

In one embodiment of the present invention, in step (4), the organic solvent is selected from one or more of acetone, toluene, chloroform and tetrahydrofuran.

The second object of the present invention is to provide an ordered high molecular polymer/hydroxyapatite composite scaffold obtained by the preparation method.

The third object of the invention is to provide the application of the ordered high molecular polymer/hydroxyapatite composite scaffold in an in-vitro cell perfusion system.

Compared with the prior art, the technical scheme of the invention has the following advantages:

1. the three-dimensional high-order interconnected porous high-molecular polymer/hydroxyapatite composite scaffold is obtained, has the integrity, is firm and is not easy to collapse.

2. The invention provides flowing nutrient substances for cells in the bracket in a dynamic perfusion culture system, promotes substance exchange and is beneficial to cell proliferation.

Further explanation of the invention:

1. action of high molecular Polymer (PMMA, PS) microspheres: as a template for preparing the ordered structure of the scaffold, high molecular polymers (PMMA, PS) are arranged into a opal structure, and then SF/HA solution is filled, so that the highly ordered interconnected porous structure of inverse opal is formed.

2. Action of electric field: the SF/HA solution filled in the gaps of the high molecular polymer (PMMA, PS) microspheres is gelled, so as to strengthen the bracket.

3. The finally obtained SF/HA bracket HAs a firm and uniform three-dimensional structure with highly ordered pore diameters which can ensure that nutrient substances and oxygen in the bracket are better transmitted and waste is better output, thereby showing superiority in a dynamic perfusion culture system and being beneficial to migration, growth and proliferation of cells to holes in the bracket during dynamic culture. (meanwhile, the traditional butanol SF/HA composite scaffold is adopted as comparison to make data of cell dynamic perfusion culture, and as a result, cells gradually die and decrease on the butanol SF/HA composite scaffold).

4. Compared with the traditional inverse opal composite scaffold, the composite scaffold has the advantages of good filling property and strong scaffold integrity, and can be used for preparing inverse opal ordered interconnected porous structures with high quality and large area, and the prepared highly ordered porous structures can promote normal growth of cells under a dynamic perfusion system, so that good cell activity is maintained.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which,

FIG. 1 is a schematic diagram of the construction of an ordered SF/HA composite scaffold;

FIG. 2 is a schematic diagram of a cell dynamic perfusion culture system;

FIG. 3 is an SEM image of an anti-opal composite scaffold of the invention with about 50 μm SF/10wt% HA and about 100 μm SF/20wt% HA;

FIG. 4 is a schematic X-ray microscope image and three-dimensional reconstructed HA (particle)/SF (solid) profile of a composite scaffold of about 100 μm SF/20wt% HA inverse opal according to the present invention;

FIG. 5 is an infrared and XRD of the 100 μm or so SF/20wt% HA inverse opal composite scaffold of the present invention;

FIG. 6 is the mechanical properties of a 100 μm or so SF/20wt% HA inverse opal composite scaffold of the present invention;

FIG. 7 is a perfusion cell culture confocal image of a butanol SF/HA composite scaffold of the present invention, and a SF/20wt% HA inverse opal composite scaffold of about 100 μm;

FIG. 8 is a graph showing MTT results of perfusion cell culture of butanol SF/HA composite scaffolds of the present invention, and SF/20wt% HA inverse opal composite scaffolds of about 100 μm.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Example 1

The embodiment provides a preparation method of an ordered silk fibroin/hydroxyapatite composite scaffold, which comprises the following specific steps:

1. placing PMMA microspheres with the diameter of 90-110 mu m on a balance vibration table to vibrate for 2 hours;

2. transferring the well-vibrated PMMA colloidal crystal into an oven at 190-195 ℃ for about 3 hours, so that the PMMA microspheres are melted and bonded;

3. cooling, taking out the PMMA template, placing the PMMA template in an SF/20wt% HA mixed solution, and soaking for 2 days;

4. after the soaking is finished, carrying out gel curing treatment on a template containing SF/20wt% of HA filling solution under an electric field, and freeze-drying after the treatment; wherein the gel curing treatment step comprises the following steps: binding PMMA microsphere templates soaked with a silk fibroin/hydroxyapatite mixed solution on a graphite plate A, immersing the PMMA microsphere templates into an SF/20wt% HA filling solution, inserting another graphite plate B, connecting a conductive substrate plate A with a positive electrode of a power supply, connecting the conductive substrate plate B with a negative electrode of the power supply, and enabling the electrifying voltage to be 30V;

5. removing redundant SF/HA solids on the freeze-dried sample, transferring the sample to tetrahydrofuran, and removing PMMA globule templates;

6. firstly using ethanol to replace tetrahydrofuran in the SF/HA scaffold, then using ultrapure water to soak the ethanol in the replacement scaffold, and performing freeze drying again to obtain the three-dimensional highly ordered SF/HA composite scaffold. The synthetic materials were characterized and the results are shown in figures 3-6. As can be seen from FIG. 3, the SF/HA scaffold prepared by the method of the invention HAs a highly ordered porous structure, and from the graph, the scaffold HAs uniform pore diameter and good formability, pores are communicated with each other, and from the graph, the integrity of the composite scaffold can be seen. As can be seen from FIG. 4, the uniform order of the three-dimensional structure inside the SF/HA scaffold prepared by the present invention, in order to clearly observe the HA distribution in the SF scaffold, the split reconstruction is performed by the difference of brightness during the material imaging, as can be seen from the figureThe HA is shown to be in a granular state and distributed uniformly. As can be seen from FIG. 5, the SF/HA scaffold prepared by the present invention was at 1621cm ^-1 Has obvious absorption peak corresponding to the amide I conformation of SF, which indicates that SF in the bracket is mainly beta-folded secondary structure and is 1033cm at the same time ^-1 、604cm ^-1 And 560cm ^-1 Absorption peaks corresponding to PO in HA were observed at the positions of (2) ₄ ^3- The XRD patterns also demonstrate successful SF/HA complexation, with characteristic XRD diffraction peaks at 26 °, 31.8 °, 32.3 °, 33 °, 34.2 °, 46.8 ° and 49.5 ° corresponding to the (002), (211), (112), (300), (202), (222) and (213) crystal planes, respectively, of the HA crystals. As can be seen from FIG. 6, the SF/HA scaffold prepared by the method HAs a maximum compressive stress of 3.5+ -0.5 MPa and an elastic modulus of 1.5+ -0.3 MPa, which all meet the requirements of cancellous bone.

Example 2

1. placing PMMA microspheres with the diameter of 40-60 mu m on a balance vibration table to vibrate for 1h;

2. transferring the well-vibrated PMMA colloidal crystal into an oven at 180-185 ℃ for about 4 hours, so that the PMMA microspheres are melted and bonded;

3. cooling, taking out the PMMA template, placing the PMMA template in an SF/50wt% HA mixed solution, and soaking for 3 days;

4. after the soaking is finished, carrying out gel curing treatment on a template containing SF/50wt% of HA filling solution under an electric field, and freeze-drying after the treatment; wherein the gel curing treatment step comprises the following steps: binding PMMA microsphere templates soaked with a silk fibroin/hydroxyapatite mixed solution on a graphite plate A, then immersing the PMMA microsphere templates into an SF/50wt% HA filling solution, and then inserting another graphite plate B, connecting a conductive substrate plate A with a positive electrode of a power supply, connecting the conductive substrate plate B with a negative electrode of the power supply, and enabling the electrifying voltage to be 15V;

5. removing redundant SF/HA solids on the freeze-dried sample, transferring the sample to toluene, and removing PMMA (polymethyl methacrylate) pellet templates;

6. firstly using ethanol to replace toluene in the SF/HA scaffold, then using ultrapure water to soak the ethanol in the replacement scaffold, and freeze-drying again to obtain the three-dimensional highly ordered SF/HA composite scaffold.

Example 3

1. placing PS microspheres with the diameter of 90-110 mu m on a balance vibration table to vibrate for 2 hours;

2. transferring the well-vibrated PS colloidal crystal into an oven at 190-195 ℃ for about 5 hours, so that the PS microspheres are melted and bonded;

3. cooling, taking out the PS template, placing the PS template in an SF/10wt% HA mixed solution, and soaking for 2 days;

4. after the soaking is finished, carrying out gel curing treatment on a template containing SF/10wt% of HA filling solution under an electric field, and freeze-drying after the treatment; wherein the gel curing treatment step comprises the following steps: binding a PS microsphere template soaked with a silk fibroin/hydroxyapatite mixed solution on a graphite plate A, then immersing the graphite plate A in an HA filling solution containing SF/10wt%, and then inserting another graphite plate B, connecting a conductive substrate plate A with a positive electrode of a power supply, connecting the conductive substrate plate B with a negative electrode of the power supply, and enabling the electrifying voltage to be 20V;

5. removing redundant SF/HA solids on the freeze-dried sample, transferring the sample into acetone, and removing the PS pellet template;

6. firstly using ethanol to replace acetone in the SF/HA scaffold, then using ultrapure water to soak the ethanol in the replacement scaffold, and freeze-drying again to obtain the three-dimensional highly ordered SF/HA composite scaffold.

Test example 2

The test example provides an application test experiment of the three-dimensional highly ordered SF/HA composite stent obtained in the example 1

Cell seeding was performed on the SF/HA composite scaffolds obtained in example 1, and after 24 hours of static adhesion, the cells were transferred to a perfusion cell culture system for cell culture. The SF/HA composite scaffold prepared from conventional butanol was used as a comparative sample and the results are shown in FIGS. 7-8. From FIG. 7, it can be seen that there is substantially no difference in cell adhesion on the two composite scaffolds at 1D of perfusion culture. Along with the growth of perfusion culture time, cells gradually decrease on the SF/HA scaffold prepared from butanol, which indicates that the cells die, and cells gradually increase on the SF/HA scaffold prepared from the invention, and almost cover the whole scaffold by 7 days, which proves that the SF/HA scaffold prepared from the invention provides a more suitable environment for the cells and is beneficial to the dynamic perfusion culture and growth of the cells. FIG. 8 further quantifies the proliferation of cells, and the absorbance of SF/HA scaffolds MTT prepared from butanol is inversely related to time, which indicates that cells cannot proliferate, while the absorbance of MTT on SF/HA scaffolds prepared from butanol is positively related to time, and the number of cells is significantly higher than that of SF/HA scaffolds prepared from butanol, which further proves that the scaffolds obtained from the invention have the advantages of highly ordered and communicated porous structures in perfusion cell culture systems, and have good dynamic culture advantages.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims

1. The preparation method of the high molecular polymer/hydroxyapatite composite scaffold with the highly ordered interconnected porous structure is characterized by comprising the following steps of:

(2) Cooling the high polymer microsphere template after melting and bonding in the step (1), and soaking the template in the high polymer/hydroxyapatite mixed solution;

(5) And (3) replacing the organic solvent in the composite scaffold in the step (4) by ethanol, replacing the ethanol in the composite scaffold by water, and finally freeze-drying to obtain the ordered high-molecular polymer/hydroxyapatite composite scaffold.

2. The method according to claim 1, wherein in the step (1), the high molecular polymer in the high molecular polymer microsphere is selected from polymethyl methacrylate and polystyrene.

3. The method according to claim 2, wherein in the step (1), the polymer microspheres are spherical and have a diameter of 10 μm to 1000. Mu.m.

4. The method according to claim 1, wherein in the step (1), the heating temperature is 175 to 195 ℃ and the heating time is 3 to 5 hours.

5. The method according to claim 1, wherein in the step (2), the concentration of hydroxyapatite in the polymer/hydroxyapatite mixed solution is 5 to 50wt%; the high molecular polymer in the high molecular polymer/hydroxyapatite mixed solution is selected from one or more of silk fibroin, sericin and carboxymethyl chitosan.

6. The method according to claim 1, wherein in the step (2), the concentration of the polymer in the polymer/hydroxyapatite mixed solution is 6 to 12wt%.

7. The method according to claim 1, wherein in the step (3), the specific operation of the gel curing treatment under the electric field: binding a high polymer microsphere template soaked with a high polymer/hydroxyapatite mixed solution on a conductive substrate plate A, then immersing the high polymer/hydroxyapatite mixed solution into another conductive substrate plate B, connecting the conductive substrate plate A with a positive electrode of a power supply, connecting the conductive substrate plate B with a negative electrode of the power supply, and electrifying; the conductive substrate plate A or the conductive substrate plate B is a graphite plate or an inert metal plate.

8. The method according to claim 7, wherein the energizing voltage is 15V to 30V.

9. A highly ordered interconnected porous high molecular polymer/hydroxyapatite composite scaffold obtained by the method of any one of claims 1 to 6.

10. Use of the highly ordered interconnected porous high molecular polymer/hydroxyapatite composite scaffold according to claim 7 in an in vitro cell perfusion system.