Preparation method of bioactive scaffold for promoting nerve vascularization bone regeneration

Abstract

The invention discloses a preparation method of a bioactive scaffold for promoting nerve vascularization bone regeneration. The preparation method comprises the following steps: mixing a synthetic high polymer material and / or a natural high polymer material with an osteogenesis promoting nano material to obtain printing ink; printing ink is used in a 3D printing mode, and the basic support is obtained. The preparation method comprises the following steps: preparing a piezoelectric high polymer material into a micro-nano fiber membrane through an electrostatic spinning technology, cutting the micro-nano fiber membrane into small blocks, soaking the small blocks in an ethanol solution, and dispersing and filtering the small blocks to obtain a micro-nano fiber / ethanol dispersion liquid; and immersing the basic scaffold in the micro-nano fiber / ethanol dispersion liquid for micro-nano fiber self-assembly, then removing ethanol, placing and drying to obtain the bioactive scaffold. The scaffold has a micro-nano fiber structure of a bionic natural extracellular matrix, and cell behaviors of adhesion, proliferation, differentiation and the like of cells are promoted. Cells in contact with the stent can exert a self-stimulation bioelectric effect driven by cell adhesion, so that efficient nerve vascularization bone regeneration can be realized, and the quality of bone defect repair is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a bioactive scaffold for promoting neurovascularization and bone regeneration. Background Art

[0002] In clinical practice, achieving effective repair of large segmental bone defects with structural and functional biomimetic properties remains a major challenge. Bone is a highly innervated and vascularized tissue. During bone formation and remodeling, nerve fibers and blood vessels closely accompany and interact with each other. The neural network within the bone tissue located in metabolically active areas plays a vital role in transmitting neurotrophic factors or neurotransmitters to regulate osteogenic differentiation, fracture healing, and bone homeostasis. In addition, sensory nerves play a key role in normal vascularization and ossification, and insufficient peripheral nerve innervation has been found to be associated with poor fracture healing or recurrent fractures. Therefore, bone repair scaffolds with neurovascularization induction function can achieve more effective and efficient bone regeneration by providing a biomimetic repair and metabolic microenvironment for osteoprogenitor cells. In recent decades, although many regenerative strategies and biomaterial scaffolds have been developed for bone tissue engineering, the key role of the nervous system is often overlooked, and few scaffolds can successfully reproduce the innervation and vascularization within the regenerated bone tissue, resulting in delayed or impaired bone healing.

[0003] In addition, bioelectric phenomena are an important part of the life system, playing a vital role in the development and physiological functions of tissues such as synapses, muscle contraction, bone and cartilage regeneration, and skin. With the development of biomedicine, electrical stimulation therapy based on bioelectric effects has been developed for tumor treatment, brain disease treatment, and repair and regeneration of various tissues. As an important physiological regulatory signal, electrical signals affect cell surface receptors, intracellular signaling pathways, and gene expression. However, current electrical stimulation therapy requires an external power supply or electrode, and later power supply replacement and electrode removal often bring patients the risk of wound infection and secondary surgery. Therefore, the complexity and inconvenience of electrical stimulation therapy have promoted the development of new piezoelectric biomaterials with self-powered capabilities. In recent years, with the rapid development of 3D printing technology, the use of 3D printed bone tissue engineering scaffolds to repair large bone defects is considered to be a feasible method. However, current 3D printed scaffolds often lack the micro-nano fiber structure of biomimetic natural extracellular matrix, and usually ignore the important role of bioelectric effects in the bone repair process, resulting in poor bone regeneration effects. Summary of the invention

[0004] The technical purpose of the present invention is to provide a method for preparing a bioactive scaffold that promotes neurovascularized bone regeneration, so as to solve the problem of the regeneration effect of current bone regeneration scaffolds.

[0005] To solve the above problems, the technical solution of the present invention is: A method for preparing a bioactive scaffold for promoting neurovascular bone regeneration comprises the following steps: Prepare printing ink, mix synthetic polymer materials and / or natural polymer materials with osteogenic nanomaterials, and achieve mixing by heating, hot melting or solvent dissolution to obtain printing ink required for 3D printing; preparing a basic scaffold, using a biological 3D printer, adjusting printing parameters, and printing ink in a 3D printing manner to obtain a basic scaffold; Prepare monodisperse micro-nano fibers, prepare piezoelectric polymer materials into micro-nano fiber membranes through electrospinning technology, and cut the obtained micro-nano fiber membranes into small pieces and soaking them in an ethanol solution, dispersing and filtering them after soaking to obtain a single fiber dispersed micro-nano fiber / ethanol dispersion; The basic scaffold is immersed in a micro-nano fiber / ethanol dispersion to perform micro-nano fiber self-assembly, and then the ethanol is removed and the scaffold is left to dry, thereby obtaining a bioactive scaffold.

[0006] Wherein, in the step of preparing the printing ink, the synthetic polymer material is selected from at least one of PCL, PLA, PLGA, PLLA, PLCL, PEG and PEEK; The natural polymer material is selected from at least one of collagen, gelatin, alginate, hyaluronic acid, chitosan, starch and decellularized matrix; The bone-promoting nanomaterial is selected from at least one of hydroxyapatite, β-tricalcium phosphate, calcium phosphate, bioactive glass, mesoporous silica and lithium metasilicate.

[0007] Specifically, in the step of preparing the printing ink, if the mixing is achieved by solvent dissolution, the mixture needs to be dissolved in a solvent and adjusted to a desired concentration, and the solvent is selected from at least one of water, chloroform, dichloromethane, tetrahydrofuran, hexafluoroisopropanol, acetone, 1,4-dioxane and DMSO.

[0008] Among them, in the step of preparing the basic bracket, the printing parameters are set as follows: the printing temperature is adjusted according to the thermal properties of the printing ink, a printing needle with a specification of 23G-30G is selected, the barrel air pressure is set to 200-3000kPa during printing, the high-voltage module voltage is set to -50 to +50 kV, the distance from the printing needle to the receiving plate is set to 1-5 mm, the printed wire diameter is 10-1000 μm, the structure of the basic bracket is determined by the printing path, the fiber interlacing angle is 0°-180°, and the fiber spacing is 50-1000μm.

[0009] The printed basic support is vacuum dried at room temperature, specifically, placed in an environment of 20 to 30° and a pressure of 5 to 30 Pa for 1 to 4 hours.

[0010] The preparation of monodisperse micro-nano fibers specifically includes the following steps: The piezoelectric polymer material is prepared into a micro-nano fiber membrane by electrospinning technology, and the obtained micro-nano fiber membrane is cut into small pieces and soaked in an ethanol solution, the mass ratio of micro-nano fibers to ethanol is 0.5-5wt%. After soaking for 24 hours, the micro-nano fibers are dispersed in an ice water bath using a high-speed homogenizer, the speed of the homogenizer is 10000-20000 rpm, the homogenization time is 0.5-3 hours, and then filtered through 20 mesh, 40 mesh, 60 mesh, 80 mesh, and 100 mesh filter screens in turn to obtain a single fiber dispersed micro-nano fiber / ethanol dispersion.

[0011] Among them, in the process of preparing monodisperse micro-nano fibers, the piezoelectric polymer material is selected from at least one of polylactic acid, polyvinylidene fluoride, polyvinylidene fluoride-trifluoroethylene copolymer, polyhydroxybutyrate, polybutylene succinate, bacterial cellulose, and chitosan.

[0012] Wherein, the micro-nano fiber self-assembly specifically includes the following steps: The dried base scaffold is immersed in a micro-nanofiber / ethanol dispersion at a preset flow rate, wherein the preset flow rate is 0.1-30 m / s and the immersion time is 1-60 s, and then taken out and immersed in deionized water for 30-50 s to remove the ethanol, and then placed in a room temperature vacuum environment for drying, specifically, at 20 to 30° and a pressure of 5 to 30 Pa for 6 to 8 hours to obtain a bioactive scaffold.

[0013] Due to the adoption of the above technical solution, the present invention has the following advantages and positive effects compared with the prior art: The present invention modifies the surface of a 3D-printed scaffold by dispersing micro-nano fibers with high-speed homogenization having a piezoelectric effect in a self-assembly manner driven by fluid shear stress. On the one hand, the 3D-printed scaffold can have a micro-nano fiber structure that mimics the natural extracellular matrix, which can promote cell behaviors such as cell adhesion, proliferation and differentiation. On the other hand, cells in contact with the scaffold can exert a self-stimulating bioelectric effect driven by cell adhesion, promote the directional differentiation of nerve cells, vascular endothelial cells and bone-related cells, achieve efficient neurovascular bone regeneration, and improve the quality of bone defect repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0015] Figure 1 A flow chart of a method for preparing a bioactive scaffold for promoting neurovascular bone regeneration according to the present invention; Figure 2 It is a schematic diagram of a three-dimensional model during the preparation process of the bioactive scaffold of the present invention. DETAILED DESCRIPTION

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings and other implementation methods can be obtained based on these drawings without creative work.

[0017] In order to simplify the drawings, only the parts related to the present invention are schematically shown in each figure, and they do not represent the actual structure of the product. In addition, in order to simplify the drawings and facilitate understanding, in some figures, only one of the parts with the same structure or function is schematically drawn or marked. In this article, "one" not only means "only one", but also means "more than one".

[0018] The following is a further detailed description of a method for preparing a bioactive scaffold for promoting neurovascularized bone regeneration proposed by the present invention in conjunction with the accompanying drawings and specific embodiments. The advantages and features of the present invention will become more apparent from the following description and claims.

[0019] See also Figure 1 , this embodiment provides a method for preparing a bioactive scaffold that promotes neurovascular bone regeneration. The bioactive scaffold can construct a bionic electrophysiological microenvironment, promote the directional differentiation of nerve cells, vascular endothelial cells and bone-related cells, and is a potential treatment method for achieving neurovascular bone regeneration. At the same time, a variety of cells can achieve a self-driven bioelectric effect mediated by cell adhesion on the bioactive scaffold, and can maintain a high survival rate, a good adhesion and spreading morphology, and excellent anisotropic differentiation ability. In addition, the neurotrophic factors and neurotransmitters secreted by nerve cells and the nutrient supply of blood vessels can provide a good osteogenic induction microenvironment for bone regeneration cells, further enhancing the effect of neurovascular bone regeneration. Therefore, the 3D printed bioactive scaffold with bionic electrical effect in this embodiment provides a new treatment idea for the efficient repair of large bone defects. The preparation method of the bioactive scaffold is now described, which specifically includes the following steps: First, it is necessary to prepare printing ink, mix synthetic polymer materials (such as one or more combinations of PCL, PLA, PLGA, PLLA, PLCL, PEG and PEEK) and / or natural polymer materials (such as one or more combinations of collagen, gelatin, alginate, hyaluronic acid, chitosan, starch and decellularized matrix) with an appropriate amount (1-20%) of osteogenic nanomaterials (such as one or more combinations of hydroxyapatite, β-tricalcium phosphate, calcium phosphate, bioactive glass, mesoporous silica and lithium magnesium silicate), and mix by heating hot melting or solvent dissolution to obtain the printing ink required for 3D printing. Preferably, if the solvent dissolution method is used to achieve mixing, the mixture needs to be dissolved in a solvent and adjusted to the required concentration. The solvent is selected from one or more combinations of water, chloroform, dichloromethane, tetrahydrofuran, hexafluoroisopropanol, acetone, 1,4-dioxane and DMSO.

[0020] Here is an example of ink preparation: In the hot melt method, assuming that PCL is selected from the synthetic polymer material, hydroxyapatite (10wt%) is selected as the bone-promoting nanomaterial, and it is heated to 60°C for hot melt dissolution. In the solvent dissolution method, assuming that PLGA is selected from the synthetic polymer material, mesoporous silica (2.5wt%) is selected as the bone-promoting nanomaterial and added to the hexafluoroisopropanol (HFIP) solution to prepare a printing ink with a mass volume ratio of 20%.

[0021] Then, the basic scaffold is prepared and a three-dimensional model of the scaffold to be prepared is constructed (such as Figure 2 As shown), the bio-3D printer is then used to adjust the printing parameters, and the printing ink is printed by 3D printing to obtain the basic scaffold. The basic scaffold obtained by printing needs to be vacuum dried at room temperature, specifically, placed in an environment of 20 to 30° and a pressure of 5 to 30pa for 1 to 4 hours. Among them, the printing parameters are set as follows: the printing temperature is adjusted according to the thermal properties (melting point) of the printing ink (-30-400°C), the printing needle is selected in the specification range of 23G-30G, the barrel pressure is set to 200-3000kPa during printing, the high-voltage module voltage is set to -50 to +50 kV, the distance from the printing needle to the receiving plate is set to 1-5 mm, the printed wire diameter is 10-1000 μm, and the structure of the basic scaffold is determined by the printing path (including rotation angle, fiber spacing, etc.), the fiber interlacing angle is 0°-180°, and the fiber spacing is 50-1000μm.

[0022] Then, prepare monodisperse micro-nano fibers, and prepare piezoelectric polymer materials into micro-nano fiber membranes through electrospinning technology. Electrospinning technology is a special fiber manufacturing process. Through the jet spinning of polymer solution or melt in a strong electric field, under the action of the electric field, the droplet at the needle tip will change from a sphere to a cone (i.e., "Taylor cone"), and extend from the tip of the cone to obtain fiber filaments. This method can produce polymer filaments with nanometer diameters. The obtained micro-nano fiber membrane is cut into The micro-nano fibers are separated into small pieces and soaked in an ethanol solution, the mass ratio of micro-nano fibers to ethanol is 0.5-5wt%, and after soaking for 24 hours, the micro-nano fibers are dispersed in an ice water bath using a high-speed homogenizer, the homogenizer speed is 10000-20000 rpm, and the homogenization time is 0.5-3 hours. Then, the micro-nano fibers are filtered through 20 mesh, 40 mesh, 60 mesh, 80 mesh, and 100 mesh filter screens in turn to obtain a single fiber dispersed micro-nano fiber / ethanol dispersion. Among them, the piezoelectric polymer material is selected from at least one material of polylactic acid, polyvinylidene fluoride, polyvinylidene fluoride-trifluoroethylene copolymer, polyhydroxybutyrate, polybutylene succinate, bacterial cellulose, and chitosan.

[0023] Finally, the dried base scaffold is immersed in a micro-nanofiber / ethanol dispersion at a certain flow rate (0.1-30 m / s) for micro-nanofiber self-assembly. The immersion time is 1-60 s, and then it is quickly taken out and immersed in deionized water for 30-50 s to remove ethanol. The scaffold is then placed in an environment with a temperature of 20 to 30° and a pressure of 5 to 30 Pa to achieve drying. It is placed for 6 to 8 hours to obtain a bioactive scaffold. After drying, it is stored in a dry place.

[0024] When the bone defect needs to be repaired, the bioactive scaffold with bionic electroactivity is implanted into the bone defect, so that the scaffold promotes cell adhesion and exerts a self-stimulating bioelectric effect driven by cell adhesion, thereby promoting the directional differentiation of nerve cells, vascular endothelial cells and bone-related cells, and achieving efficient neurovascular bone regeneration.

[0025] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, if these changes fall within the scope of the claims of the present invention and their equivalents, they still fall within the protection scope of the present invention.

Claims

1. A method for preparing a bioactive scaffold for promoting neurovascular bone regeneration, characterized in that: The steps include: Prepare printing ink, mix synthetic polymer materials and / or natural polymer materials with osteogenic nanomaterials, and achieve mixing by heating, hot melting or solvent dissolution to obtain the printing ink required for 3D printing; Prepare a basic scaffold, use a biological 3D printer, adjust printing parameters, and print the printing ink in a 3D printing manner to obtain the basic scaffold; Prepare monodisperse micro-nano fibers, prepare piezoelectric polymer materials into micro-nano fiber membranes by electrospinning technology, and cut the obtained micro-nano fiber membranes into small pieces and soaking them in an ethanol solution, dispersing and filtering them after soaking to obtain a single fiber dispersed micro-nano fiber / ethanol dispersion; The basic scaffold is immersed in the micro-nano fiber / ethanol dispersion to perform micro-nano fiber self-assembly, and then the ethanol is removed and the scaffold is left to dry, thereby obtaining the bioactive scaffold.

2. The method for preparing a bioactive scaffold for promoting neurovascularization and bone regeneration according to claim 1, characterized in that: In the step of preparing the printing ink, the synthetic polymer material is selected from at least one of PCL, PLA, PLGA, PLLA, PLCL, PEG and PEEK; The natural polymer material is selected from at least one of collagen, gelatin, alginate, hyaluronic acid, chitosan, starch and decellularized matrix; The bone-promoting nanomaterial is selected from at least one of hydroxyapatite, β-tricalcium phosphate, calcium phosphate, bioactive glass, mesoporous silica and lithium metasilicate.

3. The method for preparing a bioactive scaffold for promoting neurovascularization and bone regeneration according to claim 1, characterized in that: In the step of preparing the printing ink, if the mixing is achieved by solvent dissolution, the mixture needs to be dissolved in a solvent and adjusted to a desired concentration, and the solvent is selected from at least one of water, chloroform, dichloromethane, tetrahydrofuran, hexafluoroisopropanol, acetone, 1,4-dioxane and DMSO.

4. The method for preparing a bioactive scaffold for promoting neurovascularization and bone regeneration according to claim 1, characterized in that: In the step of preparing the basic support, the printing parameters are set as follows: the printing temperature is adjusted according to the thermal properties of the printing ink, a printing needle with a specification of 23G-30G is selected, the barrel air pressure is set to 200-3000kPa during printing, the high-voltage module voltage is set to -50 to +50 kV, the distance from the printing needle to the receiving plate is set to 1-5 mm, the printed wire diameter is 10-1000 μm, the structure of the basic support is determined by the printing path, the fiber interlacing angle is 0°-180°, and the fiber spacing is 50-1000μm.

5. The method for preparing a bioactive scaffold for promoting neurovascularization and bone regeneration according to claim 1 or 4, characterized in that: The printed basic support is vacuum dried at room temperature, specifically, placed in an environment of 20 to 30° and a pressure of 5 to 30 Pa for 1 to 4 hours.

6. The method for preparing a bioactive scaffold for promoting neurovascularization and bone regeneration according to claim 1, characterized in that: The preparation of monodisperse micro-nano fibers specifically comprises the following steps: The piezoelectric polymer material is prepared into a micro-nano fiber membrane by electrospinning technology, and the obtained micro-nano fiber membrane is cut into small pieces and soaked in an ethanol solution, the mass ratio of micro-nano fibers to ethanol is 0.5-5wt%. After soaking for 24 hours, the micro-nano fibers are dispersed in an ice water bath using a high-speed homogenizer, the speed of the homogenizer is 10000-20000 rpm, the homogenization time is 0.5-3 hours, and then filtered through 20 mesh, 40 mesh, 60 mesh, 80 mesh, and 100 mesh filter screens in sequence to obtain the micro-nano fiber / ethanol dispersion with single fiber dispersion.

7. The method for preparing a bioactive scaffold for promoting neurovascularization and bone regeneration according to claim 6, characterized in that: In the process of preparing monodisperse micro-nano fibers, the piezoelectric polymer material is selected from at least one of polylactic acid, polyvinylidene fluoride, polyvinylidene fluoride-trifluoroethylene copolymer, polyhydroxybutyrate, polybutylene succinate, bacterial cellulose, and chitosan.

8. The method for preparing a bioactive scaffold for promoting neurovascularization and bone regeneration according to claim 1, characterized in that: The micro-nano fiber self-assembly specifically includes the following steps: The dried base scaffold is immersed in the micro-nano fiber / ethanol dispersion at a preset flow rate, wherein the preset flow rate is 0.1-30 m / s and the immersion time is 1-60 s, and then taken out and immersed in deionized water for 30-50 s to remove ethanol, and then placed in a room temperature vacuum environment for drying, specifically, placed at 20 to 30° and a pressure of 5 to 30 Pa for 6 to 8 hours to obtain the bioactive scaffold.

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