Skull injury repair stent as well as preparation method and application thereof

Abstract

The invention provides a skull injury repair stent as well as a preparation method and application thereof. The stent is of an ordered fiber structure and is obtained by printing a high-molecular polymer serving as a base material through a near-field direct-writing melting technology. The orderly-arranged micro-nano fiber structure of the scaffold can well promote migration and proliferation of cells from surrounding tissues to damaged areas, so that the purpose of promoting tissue repair is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and more particularly to a skull injury repairing bracket and a preparation method and application thereof. Background Art

[0002] Bone tissue is an important tissue in the human body, which plays a role in protecting internal organs, supporting the body, promoting movement, producing blood cells, regulating blood pH, storing and releasing fat and minerals, etc.

[0003] Natural bone tissue has the ability to heal itself, but when the defect area reaches a critical threshold (5mm in the skull), surgical intervention is required. Autologous transplantation is the "gold standard" for bone repair, but donors are limited and can cause complications related to the donor site; allogeneic transplantation has problems such as fibrous nonunion, inflammation, and microcrack expansion, leading to implant failure. Therefore, the development of tissue engineering scaffolds in the field of bone regeneration has received increasing attention in recent years.

[0004] There are many ways to prepare artificial scaffolds, such as electrospinning, 3D printing, freeze drying, and hydrogel method. The nanofiber scaffolds prepared by electrospinning technology have a large specific surface area and high porosity. Electrospinning methods include solution electrospinning and melt electrospinning. Among them, the organic solvents used in solution electrospinning are toxic and volatile, and solvent residues will occur, which is not suitable for applications with high safety requirements; it is also unfriendly to the environment and has high solvent recovery costs. Compared with traditional solution electrospinning, melt electrospinning has the advantages of being solvent-free and environmentally friendly. Electrospinning technology is still difficult to meet the needs of special structural design. 3D printing is a process of accurately stacking printing materials layer by layer from 3D model data to quickly generate structures of any shape. It has the advantages of rapid prototyping, structural designability and precise manufacturing, but its resolution is limited to the microscale, which is not enough to construct a bionic functional scaffold with a nanoscale multi-level structure. In addition, the scaffolds prepared by traditional 3D printing methods have a relatively thick diameter, basically 300 to 400 μm, which is not conducive to cell adhesion.

[0005] Skull injury is a common clinical problem. Especially for the repair of defects with a certain depth, it is difficult for general scaffolds to promote the surrounding tissues or cells to recruit cells to the center to achieve better treatment effects. Summary of the invention

[0006] To solve the above problems, the present invention provides a skull injury repair scaffold and a preparation method thereof. The scaffold of the present invention is an orderly arranged micro-nano fiber structure, which can better promote the migration and proliferation of cells from surrounding tissues to the damaged area, thereby achieving the purpose of promoting tissue repair.

[0007] Firstly, one of the purposes of the present invention is to provide a skull injury repair bracket.

[0008] Specifically, the scaffold provided by the present invention is a polymer-based material, and an ordered fiber structure is obtained by printing through near-field direct writing melting technology (i.e., near-field melt electrospinning printing technology). The present invention can print any scaffold that meets the practical shape structure according to the structure designed by the software. For example, the scaffold provided by the present invention can be a polygonal structure, a circular structure, or a spider web structure formed by a plurality of orderly arranged and connected fibers.

[0009] Preferably, the stent of the present invention is a spider web structure, and the spider web structure is formed by connecting a plurality of concentric circles through a plurality of radially arranged fibers; wherein the size of the spider web structure, the number of concentric circles, and the number of radially arranged fibers can be adjusted according to actual practical situations. For example, the radially arranged fibers can be regarded as dividing the concentric circles into equal circumferences; or a smaller number of radially arranged fibers makes the gaps between the spider web structures larger; or a larger number of radially arranged fibers makes the spider web structure more compact.

[0010] Furthermore, the thickness of the skull injury repair scaffold of the present invention is 100 to 5000 μm; the diameter of the orderly connected fibers in the scaffold is 1 to 200 μm.

[0011] Secondly, the second object of the present invention is to provide a method for preparing the skull injury repair scaffold which is one of the objects of the present invention.

[0012] Specifically, the preparation method of the stent comprises the following steps:

[0013] The base materials are blended and then transferred to a near-field direct writing melt electrospinning machine (i.e., a near-field melt electrospinning printer) for printing to obtain a scaffold with an ordered fiber structure.

[0014] Preferably, the base material comprises a high molecular polymer, wherein the high molecular polymer is an aliphatic polyester; more preferably, it is one or a combination of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-glycolic acid-caprolactone copolymer, and degradable polyurethane.

[0015] Preferably, the base material also includes a bioactive substance; preferably an active substance ceramic; more preferably one or a combination of calcium phosphate, calcium sulfate, hydroxyapatite, calcium peroxide, and black phosphorus nanosheets.

[0016] More preferably, the amount of the bioactive substance added is 0 to 40% of the mass of the high molecular weight polymer; more preferably 0 to 30%; particularly preferably 0 to 25%; and even more particularly preferably 10 to 20%.

[0017] More specifically, the method for preparing the stent of the present invention comprises the following steps:

[0018] A certain mass of high molecular weight polymer, or high molecular weight polymer and bioactive substance are weighed and blended, the evenly mixed pellets are added into a barrel for near-field direct writing melt electrospinning, the parameters of the spinning machine are set, and a scaffold with an ordered fiber structure is printed.

[0019] Preferably, the present invention designs the ordered fiber structure to be prepared, for example, a spider web structure, by conventional 3D modeling software (including but not limited to EFL-Potato E software, Blender software, Shapr3D), imports the model and starts printing.

[0020] Again, the third object of the present invention is to provide an application of the skull injury repair stent which is one of the objects of the present invention.

[0021] Specifically, the scaffold of the present invention has an orderly arranged micro-nano fiber structure, which can better promote the migration and proliferation of cells from the surrounding tissue to the damaged area, thereby achieving the purpose of promoting tissue repair. In particular, the scaffold of the present invention can be designed into any applicable structure, among which the spider web structure scaffold is more suitable for skull injury repair.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention adopts near-field direct writing melt electrospinning technology (i.e., near-field melt electrospinning printing technology) to prepare a scaffold with an ordered micro-nano fiber structure. On the one hand, it avoids the use of solvents, thereby avoiding solvent volatilization and toxic pollution; on the other hand, the near-field direct writing melt electrospinning machine printing along the stable movement of the platform can achieve orderly deposition of fibers, thereby obtaining an orderly arranged micro-nano fiber structure, thereby developing a multi-scale scaffold to simulate the complexity and hierarchical structure of natural bone tissue.

[0024] 2. The near-field direct writing technology (i.e., near-field melt electrospinning printing technology) adopted by the present invention prints electrospun micro-nano fibers. Compared with the scaffold obtained by traditional electrospinning, the fiber diameter, arrangement, and spacing of the scaffold of the present invention are controllable. For example, the fiber diameter can be regulated by adjusting the distance between the print nozzle and the platform, as well as the printing voltage and air pressure; the fiber arrangement and spacing can be regulated by conventional 3D modeling software design codes. The fiber diameter in the scaffold provided by the present invention is 1 to 200 μm, which makes it easy for cells to climb on it and facilitate bone growth. At the same time, the present invention can also flexibly adjust the fiber arrangement and spacing according to different inoculated cells and the required repair tissue.

[0025] 3. The high molecular polymers used in the present invention include polycaprolactone (PCL) and the like as the basic materials of the scaffold, which have good safety; bioactive substances including hydroxyapatite and the like are doped inside the high molecular polymer, which has reliable safety. In addition, the fiber diameter of the scaffold provided by the present invention is relatively thin, which is easy for stem cells to adhere and migrate. The biomimetic designed scaffold microstructure and microenvironment can make stem cells differentiate in different directions on different structures of the scaffold, and finally achieve the effect of promoting skull repair. Therefore, the scaffold provided by the present invention has good biological safety and the characteristics of promoting stem cell differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the near-field direct writing electrospinning printing platform of the present invention;

[0027] Figure 2 A model diagram of a spider web structure designed by the EFL-Potato E software used in the present invention;

[0028] Figure 3 This is a scanning electron microscope image of the PCL scaffold prepared in Example 1;

[0029] Figure 4 This is a scanning electron microscope image of the PCL-HAP scaffold prepared in Example 2;

[0030] Figure 5 This is the cell migration image of the scaffold prepared in Example 1;

[0031] Figure 6 This is the cell migration image of the scaffold prepared in Example 2;

[0032] Figure 7 This is the cell migration image of the scaffold prepared in Example 3;

[0033] Figure 8 This is the cell migration image of the scaffold prepared in Example 4;

[0034] Fig. 9 This is a graph of RUNX-2 gene expression in osteogenic differentiation of cells in the scaffolds prepared in Examples 1 to 4;

[0035] Fig.10 This is a graph of OPN gene expression levels of osteogenic differentiation cells of the scaffolds prepared in Examples 1 to 4;

[0036] Fig.11 This is a graph of OCN gene expression levels of osteogenic differentiation cells of the scaffolds prepared in Examples 1 to 4;

[0037] Among them, Figures 9-11 In the table, “*” means P<0.05, “**” means P<0.01, and “***” means P<0.001, indicating statistically significant differences between the two groups. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with specific drawings and embodiments. It is necessary to point out that the following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by those skilled in the art based on the content of the present invention still fall within the scope of protection of the present invention.

[0039] In the following examples and comparative examples, the raw materials are all derived from commercially available products.

[0040] Example 1

[0041] This example is used to illustrate the preparation of a skull injury repair scaffold, and is composed of a pure PCL scaffold. The specific steps are as follows:

[0042] Weigh 10g of polycaprolactone PCL (macklin Mn45000) and put it directly into the barrel. Then raise the barrel temperature and the nozzle temperature to 90°C and keep it for 10 minutes to make the PCL pellets fully melt in the barrel. The spider web structure model (such as Figure 2 As shown), a micro-nano scaffold with a spider web structure (such as Figure 1 shown).

[0043] Parameter settings: The maximum diameter of the spider web is 5mm, the voltage is 3.7KV, the air pressure is 5KV, the high voltage spacing is 2.5mm, and the number of printing layers is 50. Finally, a spider web structure bracket with a fiber diameter of 5μm is obtained. The electron microscope scanning image of the bracket is as follows Figure 3 shown.

[0044] Example 2

[0045] This example is used to illustrate the preparation of a skull injury repair scaffold, and is composed of a PCL-HAP (10% hydroxyapatite) scaffold. The specific steps are as follows:

[0046] Weigh 10g of polycaprolactone PCL particles (macklin Mn45000) and 1g of hydroxyapatite HAP (macklinH875582) and mix them evenly; add the mixed raw materials into the barrel of a near-field direct writing melt electrospinning machine (i.e., near-field melt electrospinning printing technology), heat it to 90°C to melt the material, and import the spider web structure model designed by the EFL-PotatoE software into the near-field melt electrospinning printer printing bracket.

[0047] Parameter settings: The size of the spider web structure is 10mm, the voltage is 5KV, the air pressure is 7Kpa, the high voltage spacing is 2mm, and the number of printing layers is 100. Finally, a spider web structure bracket with a fiber diameter of 10μm is obtained. The electron microscope scanning image of the bracket is as follows Figure 4 shown.

[0048] Example 3

[0049] This example is used to illustrate the preparation of a skull injury repair scaffold, and is composed of a PCL-HAP (30% hydroxyapatite) scaffold. The specific steps are as follows:

[0050] Weigh 10g of polycaprolactone PCL particles (macklin Mn45000) and 3g of hydroxyapatite HAP (macklinH875582) and mix them evenly; add the mixed raw materials into the barrel of a near-field direct writing melt electrospinning machine (i.e., a near-field melt electrospinning printer), heat it to 90°C to melt the materials, and import the spider web structure model designed by the EFL-PotatoE software into the near-field melt electrospinning printer printing bracket.

[0051] Parameter settings: Set the maximum diameter of the spider web to 15mm, the voltage to 5KV, the air pressure to 10Kpa, the high voltage spacing to 2mm, and the number of printing layers to 30. Finally, a spider web structure bracket with a fiber diameter of 15μm was obtained.

[0052] Example 4

[0053] This example is used to illustrate the preparation of a skull injury repair scaffold, and is composed of a PCL and 20% hydroxyapatite (HAP) scaffold. The specific steps are as follows:

[0054] Weigh 10g of polycaprolactone PCL particles (macklin Mn45000) and 2g of hydroxyapatite HAP (macklinH875582) and mix them evenly; add the evenly mixed materials into the barrel of a near-field direct writing melt electrospinning machine (i.e., a near-field melt electrospinning printer), heat it to 90°C to melt the materials, and import the spider web structure model designed by the EFL-PotatoE software into the near-field melt electrospinning printer printing bracket.

[0055] Parameter settings: Set the spider web structure size to 10mm, voltage to 5KV, air pressure to 10Kpa, high voltage spacing to 2mm, and the number of printing layers to 80. Finally, a spider web structure bracket with a fiber diameter of 11μm was obtained.

[0056] Depend on Figures 5 to 8It can be seen that the spider web structure scaffold prepared by Examples 1 to 4 was used for the BMSC cell migration experiment. The scaffold was placed in the middle of a 24-well plate, and then the middle part of the scaffold was covered with polydimethylsiloxane (PDMS) to provide space for subsequent cell migration. The cells were then planted at the edge of the scaffold. After 24 hours, the PDMS was removed, and the results of immunofluorescence photography were taken three days later to characterize the migration ability of the cells. The results showed that the radial spider web structure was more conducive to promoting cell migration along the scaffold. This method can better simulate the real cells in the body migrating from the surrounding tissue along the scaffold to the middle defect area.

[0057] Depend on Figures 9-11 It can be seen that the spider web structure scaffolds prepared in Examples 1 to 4 were used in the BMSC cell osteogenic differentiation experiment. After 21 days of differentiation, the genes of different components were extracted and PCR detection was performed. The results showed that the expression of RUNX-2, OCN, and OPN genes of the prepared scaffolds gradually increased with the increase of HAP content, which further demonstrated that the prepared scaffolds were beneficial to promoting osteogenic differentiation in in vitro cell experiments.

[0058] Example 5

[0059] This example is used to illustrate the preparation of a skull injury repair scaffold, and is composed of a PLGA-HAP (40% hydroxyapatite) scaffold. The specific steps are as follows:

[0060] Weigh 10g of polylactic acid-glycolic acid copolymer (PLGA) particles (50:50) and 4g of hydroxyapatite HAP (macklin H875582) and mix them evenly; add the materials into the barrel of a near-field direct writing melt electrospinning machine (i.e., a near-field melt electrospinning printer), heat it to 90°C to melt the materials, and import the spider web structure model designed by Blender software into the near-field melt electrospinning printer to print the bracket.

[0061] Parameter settings: Set the spider web structure size to 20mm, voltage to 5KV, air pressure to 10KPa, high voltage spacing to 2mm, and number of printing layers to 30. Finally, a spider web structure scaffold with a fiber diameter of 20μm was obtained.

[0062] Example 6

[0063] This example is used to illustrate the preparation of a skull injury repair scaffold, and is composed of a PCL and calcium peroxide (30%) scaffold. The specific steps are as follows:

[0064] Weigh 10g of polycaprolactone PCL particles (macklin Mn45000) and 3g of calcium peroxide and mix them evenly; add the materials into the barrel of a near-field direct writing melt electrospinning machine (i.e., a near-field melt electrospinning printer), heat it to 90°C to melt the materials, and import the spider web structure model designed by Shapr3D software into the near-field melt electrospinning printer to print the bracket.

[0065] Parameter setting: Set the maximum diameter of the spider web to 20mm, the voltage to 5KV, the air pressure to 10KPa, the high voltage spacing to 2mm, and the number of printing layers to 30. The spider web structure with a fiber diameter of 20μm is finally obtained. The above describes in detail the preferred embodiment of the present invention, but the present invention is not limited to this. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A skull injury repair stent, characterized in that: The scaffold is a scaffold with an ordered fiber structure obtained by printing a macromolecular polymer as a basic material through a near-field direct writing melting technology.

2. The skull injury repair stent according to claim 1, characterized in that: The ordered fiber structure is a polygonal structure, a circular structure or a spider web structure formed by a plurality of ordered arranged and connected fibers.

3. The skull injury repair stent according to claim 1, characterized in that: The thickness of the support is 100-5000 μm; the diameter of the fiber in the ordered fiber structure is 1-200 μm.

4. The skull injury repair bracket according to claim 2, characterized in that: The spider web structure is formed by connecting a plurality of concentric circles through a plurality of radially arranged fibers.

5. The method for preparing a skull injury repair scaffold according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: After the base materials are blended, they are transferred to a near-field direct writing melt electrospinning machine for printing to obtain a scaffold with an ordered fiber structure.

6. The method for preparing a skull injury repair scaffold according to claim 5, characterized in that: The basic material includes a high molecular polymer, or a high molecular polymer and a bioactive substance.

7. The method for preparing a skull injury repair scaffold according to claim 6, characterized in that: The high molecular polymer is an aliphatic polyester; preferably one or a combination of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-glycolic acid-caprolactone copolymer, and degradable polyurethane.

8. The method for preparing a skull injury repair scaffold according to claim 6, characterized in that: The bioactive substance is an active material ceramic; preferably one or a combination of calcium phosphate, calcium sulfate, hydroxyapatite, calcium peroxide, black phosphorus nanosheets, etc.

9. The method for preparing a skull injury repair scaffold according to claim 6, characterized in that: The added amount of the bioactive substance is 0-40% of the mass of the high molecular weight polymer; preferably 0-30%; more preferably 0-25%.

10. Use of the skull injury repair stent according to any one of claims 1 to 4, characterized in that: The scaffold is used for promoting tissue repair; preferably for repairing skull injuries.

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