CN110772663A

CN110772663A - Bionic support with micro-nano hierarchical structure and preparation method thereof

Info

Publication number: CN110772663A
Application number: CN201910845516.9A
Authority: CN
Inventors: 陈亮; 顾勇; 唐锦程; 毛剑楠; 吴亮; 崔文国
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2019-09-06
Filing date: 2019-09-06
Publication date: 2020-02-11

Abstract

The invention provides a micron-nanoscale hierarchical structure bionic scaffold, which is strengthened from the inside of pores and a framework of a micron-scale silk fibroin porous scaffold by self-assembled collagen nanofibers and bioactive glass nanoparticles, and the inside and outside double-strengthened micro-nanoscale hierarchical structure bionic scaffold is established.

Description

Bionic support with micro-nano hierarchical structure and preparation method thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a micro-nano hierarchical structure bionic scaffold and a preparation method thereof.

Background

Currently, two basic strategies are mainly surrounded with regard to the construction of biological scaffolds to promote bone regeneration: namely, active ingredients are added into the scaffold or the structural form of the scaffold is regulated. With the progress of bioactive factor synthesis technology and stem cell treatment technology, the biomaterial scaffold can obtain good bone regeneration effect by adding bioactive factors, inorganic salts and stem cells with regeneration capacity. However, problems such as uncontrolled regeneration, potential cancer risk, etc. result from the use of exogenous bioactive components. In contrast, although some studies have been made, the structural factor promoting osteogenesis has not been known to contribute to the bone effect, and the mechanism of bone formation is not known, and thus it is still difficult to use.

The natural bone tissue is stacked in the fiber by the fiber collagen phase and the hydroxyapatite according to a complex sequence, and shows extremely strong mechanical stability and regeneration capacity. When a fracture occurs, the formation of new bone begins with the infiltration of granulation tissue and the formation of callus, which provides not only a temporary fixation effect, but also the nutrients and calcium required for the next repair. Thereafter, the body's own stem cell secretion produces an extracellular matrix network, an abundant nanofiber matrix network serving as the basis for cell-matrix interactions and as a template for hydroxyapatite deposition. Finally, further mineralization and reconstruction of these nanofiber networks, while supporting gradual tissue degradation. It can be seen that the natural healing process of human bone is a highly coordinated and hierarchical process. Compared with an artificial bracket, the fracture repair complex composed of the micron-sized supporting tissue and the nanofiber matrix network can promote bone regeneration through the specific micro-nano structure of the fracture repair complex. Inspired by the natural process, the natural repair process of human skeleton is simulated, and the bone regeneration is promoted through the microstructure of the bracket, which is the key point of the current research.

Collagen is the most abundant protein in the organic phase of natural bone, and has been widely reported in the biomimetic reconstruction of bone by its superior biological properties. However, the existing material science method for constructing the collagen bionic extracellular matrix network structure is single, such as an electrospinning bracket and self-assembled hydrogel, and the prepared bracket has poor mechanical property and limited application. On the other hand, the Silk Fibroin (SF) porous scaffold, as one of the traditional raw materials of bone tissue engineering scaffold, cannot provide a bionic microenvironment like extracellular matrix. Some researchers tried to add inorganic salt such as Hydroxyapatite (HA) and Bioactive Glass (BG) to silk fibroin to make scaffold have good osteoconductivity and mechanical properties, and this enhancement effect is related to ionic bonds between calcium ions in inorganic components and carboxyl and hydroxyl groups abundant in silk fibroin. However, the addition of inorganic salts does not alter the mode of interaction of this material with the cells. Therefore, the bionic scaffold which has good mechanical property and high biological activity is provided, and the bionic scaffold has important significance.

Disclosure of Invention

In view of the above, the technical problem to be solved by the invention is to provide a micro-nano hierarchical structure bionic scaffold and a preparation method thereof.

The invention provides a bionic scaffold with a micro-nano hierarchical structure, which is obtained by reinforcing a micron-sized silk fibroin porous scaffold by using self-assembled collagen nanofibers and bioactive glass nanoparticles. The invention also provides a preparation method of the bionic scaffold with the micro-nano hierarchical structure, which comprises the following steps:

1) mixing bioactive glass nanoparticles with a silk fibroin solution, carrying out ultrasonic oscillation, adding glycerol, pre-freezing, and carrying out freeze drying to obtain a bioactive glass nanoparticle-reinforced silk fibroin porous scaffold;

2) and (2) dripping the collagen solution on the silk fibroin porous scaffold reinforced by the bioactive glass nano particles prepared in the step 1), and placing the silk fibroin porous scaffold in a proper environment for self-assembly to obtain the bionic scaffold for promoting osteogenesis.

Preferably, the concentration of the silk fibroin solution is 4-8%, and more preferably 6%.

Preferably, the mass ratio of the silk fibroin to the bioactive glass nanoparticles is (15-30) to 1.

Preferably, glycerol is further added in the step 1) for reaction, and the mass ratio of the silk fibroin to the glycerol is 1: 0.2-0.3.

Preferably, the bioactive glass nanoparticles are prepared according to the following method:

and (2) in a Tris-HCl buffer solution, adding a calcium source, a phosphorus source and a silicon source into hexadecyl ammonium bromide serving as a template agent for reaction to obtain the bioactive glass nanoparticles.

Preferably, the self-assembly temperature is 35-40 ℃.

Preferably, the collagen solution is prepared according to the following method:

dissolving the I-type collagen in 0.05-0.2M acetic acid solution to obtain a collagen-containing solution, then diluting the collagen-containing solution with ten times of concentration of phosphate buffer solution in an ice bath, and dropwise adding NaOH aqueous solution to adjust the pH to 7.0 to obtain the collagen solution to be self-assembled.

Compared with the prior art, the invention provides a bionic scaffold with a micro-nano hierarchical structure, which is obtained by reinforcing a micron-sized silk fibroin porous scaffold by using self-assembled collagen nanofibers and bioactive glass nanoparticles; the invention establishes the inside and outside double-reinforced micro-nano hierarchical structure bionic scaffold, and experiments show that the bionic scaffold obtained by the invention not only has good mechanical property, but also stably integrates three components of collagen nanofiber, bioactive glass and silk fibroin, and can better promote the osteogenesis of stem cells through the bionic structure of the scaffold.

Drawings

FIG. 1 is a schematic diagram of the construction of a biomimetic scaffold;

FIG. 2 is a schematic diagram of the shape and composition structure of the bionic scaffold;

fig. 3 is a schematic structural diagram of the obtained bionic scaffold, an enlarged illustration of the interaction between silk fibroin, collagen fibers and bioglass particles, and a corresponding SEM view;

FIG. 4 is a graph representing the mechanical properties and mineralization promoting capacity of a scaffold;

FIG. 5 is a graph showing the results of cell adhesion and proliferation assays for biomimetic scaffolds;

FIG. 6 is a representation of biomimetic scaffold adhesion patterns and integrin expression;

FIG. 7 shows the results of the study on the mechanism of promoting osteogenesis by using a bionic structure of micro-nano scale;

FIG. 8 shows the in vivo performance evaluation results of the biomimetic scaffolds obtained in the examples;

fig. 9 is a histological analysis of a bone defect model.

Detailed Description

The invention provides a bionic scaffold with a micro-nano hierarchical structure, which is obtained by reinforcing a micron-sized silk fibroin porous scaffold by using self-assembled collagen nanofibers and bioactive glass nanoparticles. Wherein the collagen is preferably type I collagen; the mass ratio of the micron-sized silk fibroin to the bioactive glass nanoparticles is preferably (15-30) to 1, and more preferably (20-25) to 1; the amount of collagen used in the present invention is not particularly limited, and those skilled in the art can select an appropriate amount according to the common general knowledge in the art. Researches show that the bionic scaffold with the micro-nano hierarchical structure, which is obtained by the application, establishes a bone factor-stem cell-release scaffold reinforced by a hierarchical structure through the coordination of a bioglass reinforced microporous scaffold and a nano collagen network, realizes the active interaction among silk fibroin, collagen nanofibers and bioglass nanoparticles, endows the silk fibroin, collagen nanofibers and bioglass nanoparticles with excellent mechanical properties and mineralization promoting activity, and promotes cell adhesion, diffusion and osteogenic differentiation of progenitor cells.

The invention provides a preparation method of a micro-nano hierarchical structure bionic scaffold, which comprises the following steps:

1) mixing bioactive glass nano particles with silk fibroin solution, carrying out ultrasonic oscillation, pre-freezing, and carrying out freeze drying to obtain a bioactive glass nano particle reinforced silk fibroin porous scaffold;

2) and (2) dripping a collagen solution onto the silk fibroin porous scaffold reinforced by the bioactive glass nanoparticles prepared in the step 1), and placing the silk fibroin porous scaffold in a proper environment for self-assembly to obtain the micro-nano hierarchical structure bionic scaffold.

According to the invention, bioactive glass nano-particles and silk fibroin solution are mixed and ultrasonically reacted to obtain a silk fibroin porous scaffold reinforced by bioactive glass nano-particles; wherein the mass concentration of the silk fibroin solution is preferably 4-8 w/w%, more preferably 6-7 w/w%; the mass ratio of the silk fibroin to the bioactive glass nanoparticles is preferably (15-30) to 1, and more preferably 20 to 1; in the invention, in order to add glycerol to the step 1) for reaction, the silk protein porous scaffold reinforced by bioactive glass nanoparticles is obtained; wherein the mass ratio of the silk fibroin to the glycerol is preferably 1: 0.2-0.3, and more preferably 1: 0.25; the temperature of the ultrasonic reaction is room temperature reaction; the time of the ultrasonic reaction is 0.5-2 hours, preferably 1-1.5 hours; and after the ultrasonic reaction is finished, freezing and drying the obtained product to obtain the silk fibroin porous scaffold reinforced by bioactive glass nano particles.

In the invention, the silk fibroin solution is prepared by the following method:

boiling silk fibroin in an alkaline aqueous solution, and cleaning to obtain the silk fibroin of the degummed silk; mixing the silk fibroin of the degummed silk with a lithium bromide solution, dialyzing, and dehydrating to obtain a silk fiber protein solution; wherein the alkaline aqueous solution is preferably sodium carbonate aqueous solution or potassium carbonate aqueous solution, and more preferably sodium carbonate aqueous solution; the mass concentration of the alkaline aqueous solution is preferably 0.3-0.7%, and more preferably 0.5-0.6%; the dehydrating agent for dehydration is polyethylene glycol powder, and more preferably polyethylene glycol powder with the number average molecular weight of 20000.

In the invention, the bioactive glass nanoparticles are prepared according to the following method: and (2) in a Tris-HCl buffer solution, adding a calcium source, a phosphorus source and a silicon source into hexadecyl ammonium bromide serving as a template agent for reaction to obtain the bioactive glass nanoparticles. Wherein the reaction temperature is 50-70 ℃, and more preferably 60-65 ℃; the reaction time is preferably 20 to 30 hours, and more preferably 24 to 26 hours. In the invention, after the reaction is finished, CTBA is preferably calcined from the reaction product at 500-600 ℃ to obtain the bioactive glass nano-particles.

According to the invention, the collagen solution is dropped on the silk fibroin porous scaffold reinforced by the bioactive glass nano particles prepared in the step 1) for self-assembly, so as to obtain the bionic scaffold for promoting osteogenesis, wherein the self-assembly temperature is 35-40 ℃, and preferably 37-38 ℃; the self-assembling time is 30-50 min.

In the invention, the collagen solution is prepared according to the following method:

dissolving I type collagen in 0.05-0.2M acetic acid solution to obtain collagen-containing solution, then diluting the collagen-containing solution with ten times of concentration of phosphate buffer solution, and then dropwise adding NaOH aqueous solution to adjust the pH value to 7.0 to obtain collagen solution. Wherein the concentration of the acetic acid solution is preferably 0.1-0.15M; the invention has no special requirement on the source of the type I collagen, and can be purchased or made by oneself.

More specifically, the construction process of the biomimetic scaffold provided by the invention is shown in fig. 1, and fig. 1 is a construction diagram of the biomimetic scaffold provided by the invention, wherein (a) a schematic diagram of the construction process of the biomimetic scaffold with a micro-nano hierarchical structure; (B) schematic diagram of the type I collagen molecule level self-assembly process; (C) comparing the regeneration mode schematic diagrams of the micro-stent and the micro-nano stent.

The invention provides a bionic scaffold for promoting osteogenesis, which is obtained by self-assembling collagen on a silk fibroin porous scaffold reinforced by bioactive glass nano particles; the bionic scaffold has the advantages that the silk fibroin microporous scaffold can be formed by adopting the biological glass nano-structure, the coordination of the self-assembly nano collagen network is adopted, the bone factor-stem cell-release scaffold reinforced by the hierarchical structure is established, experiments show that the bionic scaffold obtained by the application has good mechanical property and good stability, and the bionic scaffold obtained by the application can better promote the osteogenesis of stem cells.

The following will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) Preparing silk fibroin solution and mesoporous bioactive glass nano-particles.

In Na ₂CO ₃Boiling raw silk in water solution (0.5% w/w) for 1 hour, repeatedly washing the boiled raw silk thoroughly 8 times to remove sericin sufficiently, drying the obtained degummed silk at 60 ℃, and then adding the degummed silk under heating in oil bath at 60 ℃ by using 9.3M LiBr solution and stirring magnetically for 4 hours to dissolve the degummed silk. The resulting solution was filled into dialysis bags and dialyzed against deionized water, with the dialysis water being replaced twice a day for 4 days. Finally, the dialyzed silk fibroin solution was dehydrated by polyethylene glycol powder (molecular weight 20,000), concentrated and purified, and finally obtained silk fibroin solution with 6% w/w concentration for further experiments.

The preparation reaction of bioactive glass nanoparticles was performed in Tris-HCl buffer solution (ph8.0) using CTAB (cetyltrimethylammonium bromide) as a template. Respectively adding calcium, phosphorus and silicon, reacting for 24 hours at 60 ℃, respectively washing the obtained nanoparticles with ethanol and deionized water for 3 times, and then calcining at 500 ℃ to remove CTAB, so as to obtain bioactive glass nanoparticles, wherein the diameter of the bioactive glass nanoparticles is 200-300 nm (nanometer level).

(2) And (5) preparing the micron-sized bracket.

Bioactive glass nanoparticle (MBG) reinforced Silk Fibroin (SF) scaffolds were prepared by lyophilization. MBG (mass ratio of SF to MBG: 20: 1) and glycerol (mass ratio of SF to glycerol: 80: 20) were added to a 6% (w/w) SF solution, the resulting solution was subjected to ultrasonic treatment in a water bath at room temperature for 1 hour, then the solution was placed in a mold, the mold was rapidly placed in an environment at-80 ℃ for prefreezing, and then freeze-dried for 48 hours to obtain an MBG-reinforced SF porous scaffold.

The detection shows that the pore diameter of the silk fibroin scaffold reinforced by the bioactive glass nano particles is 72.2 +/-5.5 microns.

(3) And (5) constructing a micro-nano hierarchical structure.

Preparation of micro-nano-scale structure the MBG-reinforced SF porous scaffold obtained in the above step is on a micron-scale basis. Dissolving type I collagen (Col I) from rat tail in 0.1M acetic acid solution, then, performing self-assembly of collagen in an ice bath, firstly diluting collagen solution with phosphate buffer solution (PBS, 10x) with ten times concentration in a ratio of 6: 1, (wherein, the diameter of collagen nanofiber in the solution is 200-300 nm (nanometer), then, dropwise adding 0.5M NaOH aqueous solution to adjust the pH to 7.0, then, dripping 25 microliter of the neutralized collagen solution on a cylindrical porous scaffold with the diameter of 6mm and the height of 1mm, ensuring that the collagen solution permeates into pores of the porous scaffold, then, incubating the scaffold at 37 ℃ for 30 minutes to complete the self-assembly process, and washing the obtained scaffold with deionized water for three times to obtain the micro-nano hierarchical structure bionic scaffold.

The morphology and structure of the obtained biomimetic scaffold were characterized, and the results are shown in fig. 2-3:

FIG. 2 is a schematic diagram of the morphology and composition structure of the biomimetic scaffold, wherein (A) is a Scanning Electron Microscope (SEM) image of various scaffolds; (B) fourier infrared variation spectrograms (FTIR) before and after addition of MBG; (C) FTIR spectrograms before and after adding ColI and comparing with pure collagen; the surface morphology is observed by a scanning electron microscope, and thus bioactive glass nanoparticles (MBG) and nanofiber collagen are successfully integrated in the silk fibroin scaffold, and FTIR also verifies that the MBG and the collagen are still stably integrated in the scaffold after the scaffold is washed by the liquid crystal material.

Fig. 3 is a schematic structural diagram of the obtained bionic scaffold, an enlarged illustration of the interaction between silk fibroin, collagen fibers and bioglass particles, and a corresponding SEM view; wherein, (a) a schematic view of a layered scaffold, (B) an enlarged illustration of the interaction between silk fibroin, collagen fibers and bioglass particles, (C) a corresponding SEM view.

After fully characterizing the physicochemical properties of the biomimetic scaffold, the study on the biological reaction of co-culture of bone marrow mesenchymal stem cells (BMSCs) and the scaffold also reveals the potential mechanism, and the study on the regeneration mode of the biological scaffold by adopting an SD rat critical dimension bone defect model and adopting radiology and histology methods discusses the reconstruction details of the bone defect, which is shown in the specific figures 4-9:

FIG. 4 is a graph representing the mechanical properties and mineralization promoting capacity of a scaffold, wherein (A) is a TEM image of assembled collagen nanofibers; (B) characterization of rheological properties of the self-assembled collagen hydrogel; (C) the stress-strain curve and the compression modulus of the stent under the uniaxial compression test are shown; (D) the rheological property of the micro-nano bracket and the micron bracket is shown; (E) SEM images of scaffolds after different times of mineralization in SBF. (F) The detection result of the concentration of calcium and phosphorus ions in SBF is obtained; (G) XRD images of the stent before and after mineralization; it can be seen from the figure that the mechanical properties of the scaffold are greatly improved (four times) by adding MBG and collagen, and the mineralization promoting capability of the material is obviously enhanced.

FIG. 5 is a graph showing the results of cell adhesion and proliferation assays for biomimetic scaffolds; wherein (a) is an SEM image of BMSCs adhered to the scaffold (represented by white triangles); (B) proliferation results of BMSCs after 1, 3, 5, 7 days of co-culture on scaffolds; (C) is the result of the release of silicon ions in SBF; (D) results for alizarin red staining for 14 days and 21 days; (E) results for ALP staining on day 7 and day 14; (F) for respective quantification, it can be seen from the detection result of fig. 5 that the biomimetic scaffold can significantly promote osteogenic differentiation of mesenchymal stem cells.

FIG. 6 is a representation of adhesion patterns and integrin expression of a biomimetic scaffold, wherein (A) is a result graph of cytoskeleton (Phalloidin red) and nucleus (DAPI blue) morphology observed after BMSCs and the scaffold are co-cultured for 3 days by using a laser confocal microscope (CLSM), (B) is a schematic graph of adhesion patterns of BMSCs on the scaffold, (C) is a result of β -immunofluorescence staining (CLSM) of integrin after BMSCs are cultured on the scaffold for 24 hours in a common culture medium, (D) is a result of quantification of cell spreading areas on different scaffolds, (E) is a western blot detection and semiquantification of β -integrin, and it can be seen from the graph that cells present a better spreading state on the surface of the biomimetic scaffold with a micro-nano hierarchical structure, the spreading area is significantly larger than that of the scaffold, and the expression of integrin is significantly increased, which indicates that the cells have a better state under the biomimetic structure.

FIG. 7 shows the results of the study on the mechanism of promoting osteogenesis by using a bionic structure of micro-nano scale; immunofluorescent staining of (A) vinculin (vinculin) and (B) YAP (Yes-associatedprotein) in BMSCs was observed using CLSM after 7 days by co-culturing with scaffolds in normal medium; (C) western blot of vinculin and YAP and semi-quantitative analysis thereof (D) after co-culture in osteogenic medium for 7 days, detection of the Western blot of RUNX2 and Osteocalcin (OCN) in BMSCs and semi-quantitative analysis thereof; as can be seen from the figure, the cells cultured on the surface of the micro-nano hierarchical structure bionic scaffold express vinculin and YAP proteins more than the cells of the micron-sized scaffold, that is, the bionic scaffold can promote the expression of the vinculin and YAP proteins to be increased through the micro-nano hierarchical structure and the higher mechanical properties of the micro-nano hierarchical structure; by carrying out western blot detection on two osteogenesis markers, namely RUNX2 and OCN, the bionic scaffold can be obtained, and the bionic scaffold can better promote the osteogenesis of stem cells compared with a micron scaffold.

FIG. 8 shows the in vivo performance evaluation results of the biomimetic scaffolds obtained in the examples; wherein, (A) is the preparation of critical bone defect model and the picture of stent implantation; (B) the results of bone morphology three-dimensional analysis of defect regions at 8 and 12 weeks after operation; (C) three-dimensional reconstructed images of defect regions filled for different stents; as can be seen from the figure, the micro CT shows that the bionic scaffold with the micro-nano hierarchical structure obtains good healing effect in 8 weeks and 12 weeks after operation, and is obviously superior to other micron-sized scaffolds.

FIG. 9 is a histological analysis of a bone defect model; wherein, (A) is the result of H & E staining of the defect area under high power and low power microscope at 4 weeks and 12 weeks after the operation; (B) the results were trichrome staining of the defect area at 4 and 12 weeks post-surgery; (C) the result of I-type collagen immunohistochemical staining in defect areas at 4 and 12 weeks after operation; as can be seen from the figure, the pathology examination showed: the micro-nano hierarchical structure bionic scaffold can better promote bone tissue to grow in 4 weeks after operation, and the healing effect in 12 weeks after operation is obviously superior to that of other micron-sized porous scaffold groups.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A bionic scaffold with micro-nano hierarchical structure is prepared by reinforcing the porous scaffold of micron-class silk fibroin with self-assembled nano fibres of collagen and nano particles of bioactive glass.

2. The bionic scaffold with the micro-nano hierarchical structure as claimed in claim 1, wherein the mass ratio of the micro-scale silk fibroin to the bioactive glass nanoparticles is (15-30) to 1.

3. The biomimetic scaffold with a nano-scale structure according to claim 1, wherein the mass ratio of the micron-sized silk fibroin to the bioactive glass nanoparticles is (20-25) to 1.

4. A preparation method of a bionic scaffold with a micro-nano hierarchical structure comprises the following steps:

2) and (2) dripping the collagen solution on the silk fibroin porous scaffold reinforced by the bioactive glass nano particles prepared in the step 1) for self-assembly to obtain the micro-nano hierarchical structure bionic scaffold.

5. The preparation method of claim 4, wherein the silk fibroin solution is prepared by the following method:

boiling raw silk in an alkaline aqueous solution, and cleaning to obtain silk fiber without sericin;

and (3) dissolving the silk fiber without sericin in a lithium bromide solution, and dialyzing and dehydrating to obtain a silk fibroin solution.

6. The method of claim 4, wherein the bioactive glass nanoparticles are prepared by:

7. The preparation method of claim 4, wherein glycerol is further added in the step 1) to crosslink the silk fibroin, so as to obtain the glycerol crosslinked bioactive glass nanoparticle-reinforced silk fibroin porous scaffold.

8. The preparation method of claim 4, wherein the mass ratio of the silk fibroin to the glycerol is 1: 0.2-0.3.

9. The method according to claim 4, wherein the self-assembly temperature is 35 to 40 ℃.

10. The method according to claim 4, wherein the collagen solution is prepared by the following method:

dissolving the I-type collagen in 0.05-0.2M acetic acid solution to obtain a solution containing collagen, then diluting the solution containing collagen with phosphate buffer solution with ten times of concentration in ice bath, and dropwise adding NaOH aqueous solution to adjust the pH value to 7.0 to obtain a collagen solution to be self-assembled.