Micro-nano fiber patterned tissue engineering scaffold and solution electrostatic spinning preparation method thereof

Abstract

The invention belongs to the technical field of tissue engineering scaffolds, and relates to a micro-nano fiber patterned tissue engineering scaffold and a solution electrostatic spinning preparation method thereof, and the micro-nano fiber patterned tissue engineering scaffold is formed by randomly interlacing and compounding uniformly distributed micron-scale skeleton fibers and nano-scale filling fibers on a patterned receiving electrode, the diameter of the micron-scale framework fiber is 15-25 microns, and the diameter of the nano-scale filling fiber is 200-500 nm; the nano-scale filling fibers are distributed on the surfaces of the micron-scale skeleton fibers and among different micron-scale skeleton fibers, and the micron-scale skeleton fibers and the nano-scale filling fibers penetrate through each other to form a three-dimensional graded fiber network structure; the patterned receiving electrode is of a three-layer structure; during preparation, a spinning solution A and a spinning solution B are prepared respectively, the patterned receiving electrode is used as a receiving device, and the micro-nano fiber patterned tissue engineering scaffold is prepared by spraying the solutions through double nozzles for electrostatic spinning. The product provided by the invention can be better matched with the mechanical behavior of a target organization; the preparation method is simple.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering scaffold technology, and relates to a micro / nano fiber patterned tissue engineering scaffold and its solution electrospinning preparation method. Background Technology

[0002] The existing biodegradable tissue engineering scaffolds mainly have the following structural designs: (1) Nanofiber scaffolds, the 200~500nm fiber scale of nanofibers can imitate the extracellular matrix structure and promote the directional migration and proliferation of cells; (2) 3D printed scaffolds, which can construct fiber scaffolds with a scale of more than 10 micrometers, which can provide effective mechanical strength, and through the preset printing path, larger pore structures can be designed to achieve three-dimensional cell ingrowth; (3) Combining electrospinning with 3D printing to construct dual-scale tissue regeneration scaffolds, where micron fibers can provide long-lasting mechanical support, and nanofibers can provide high specific surface area to promote cell proliferation.

[0003] However, these biodegradable tissue-engineered scaffolds still have the following shortcomings:

[0004] (1) Pure nanofiber scaffolds have excessively dense pores and insufficient mechanical properties: scaffolds formed by the stacking of electrospun nanofibers typically have fiber diameters ranging from tens to hundreds of nanometers. The pore size between fibers is small, and the pore structure is compact, resulting in a highly dense membrane-like morphology. Such scaffolds can usually only provide cells with a two-dimensional or quasi-two-dimensional adhesion surface. Cells mainly migrate and proliferate on the surface or in the superficial region of the scaffold, making it difficult to achieve three-dimensional ingrowth and uniform distribution along the thickness direction of the scaffold. At the same time, due to the small diameter of the nanofibers and the limited load-bearing capacity of a single fiber, the overall mechanical properties of the scaffold are poor, with insufficient tensile strength and compressive stiffness. It is difficult to provide continuous mechanical support that matches the long-term tissue repair process, and there is a risk of early collapse and failure in in vivo applications.

[0005] (2) Insufficient ability of 3D-printed macroscopic scaffolds to mimic the extracellular matrix and limited interaction with cells: Scaffolds prepared by 3D printing technology typically have characteristic scales of support rods or strips on the order of hundreds of micrometers or even larger, achieving high overall mechanical strength and good macroscopic structural stability, and providing mechanical support to defective sites for a relatively long time. However, the diameter of the fibers or support units of this type of scaffold is much larger than the scale of collagen fiber bundles in the natural extracellular matrix, making it difficult to simulate the fine fiber network of the ECM at the microscopic level. Due to the lack of micro- and nano-scale topological structures and adhesion cues that match the extracellular matrix, the interfacial interaction between cells and scaffolds is weak, and the cell adhesion morphology, spreading mode, and migration behavior are difficult to effectively regulate, thereby weakening the induction and guidance effect on the tissue regeneration process.

[0006] (3) The integration of simple layered combination of electrospinning and 3D printing is limited and still belongs to a layered structure: Some existing solutions attempt to combine electrospinning and 3D printing. Generally, a scaffold skeleton with macroscopic support capacity is first obtained through 3D printing, and then one or more layers of electrospun nanofiber membranes are introduced on its surface or in the gap area in order to take into account both mechanical support and ECM-like microstructure. However, this type of printed skeleton + spun membrane mode is usually mainly based on layer stacking. The connection between micron and nanostructures is only achieved through physical adhesion or local entanglement. The layers are prone to degradation or delamination during in vivo degradation or stress, making it difficult to form a truly continuous and integral three-dimensional integrated structure. In addition, the 3D printed layer and the electrospun layer often play independent roles in terms of function. The structure is essentially still a layered combination, rather than a hierarchical fiber network with true spatial interpenetration and synergistic effect, which makes it difficult to give full play to the synergistic advantages between micro and macro multi-scale structures.

[0007] Furthermore, from the perspective of biomechanical requirements for tissue repair, an ideal tissue-engineered scaffold should possess mechanical properties similar to or controllable to damaged tissue, exhibiting high mechanical compliance and good biomimetic mechanical characteristics. Matching or gradually transitioning elastic modulus, tensile properties, and stiffness gradients with the host tissue helps reduce interfacial stress concentration, promotes benign integration between the scaffold and the host tissue, and avoids adverse reactions such as scar formation and fibrous capsule encapsulation caused by abrupt stiffness transitions. However, many existing tissue-engineered scaffolds are designed and fabricated primarily with an emphasis on improving strength and stability, resulting in materials with excessive overall stiffness and insufficient toughness and tensile properties. Moreover, the means to adjust structural parameters (such as fiber diameter, porosity, and hierarchical structure) are limited, making it difficult to achieve precise control over mechanical compliance and thus failing to simultaneously meet the requirements of long-term mechanical support and good biomechanical adaptability.

[0008] For example, the literature (Embedding aligned nanofibrous architectures within 3D-printed polycaprolactone scaffolds for directed cellular infiltration and tissue regeneration[J]. International Journal of Extreme Manufacturing, 2023,5(2).) utilizes 3D printing to prepare PCL frameworks and embeds oriented nanofiber porous structures within them to construct dual-scale tissue engineering scaffolds, successfully achieving pore orientation at the microscopic level and inducing anisotropic growth behavior of cells and tissues. However, its overall mechanical design still highly depends on the macroscopic PCL framework, and the anisotropic structure has not been transformed into direction-related effective modulus changes or compliance differences, and the fragile nanofiber pore structure is difficult to adapt to tissue movement changes.

[0009] Therefore, it is of great significance to study a micro / nano fiber patterned tissue engineering scaffold and its solution electrospinning preparation method to solve the problems existing in the prior art. Summary of the Invention

[0010] The purpose of this invention is to solve the problems existing in the prior art and to provide a micro / nano fiber patterned tissue engineering scaffold and its solution electrospinning preparation method.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] The micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology is composed of uniformly distributed micron-sized skeleton fibers and uniformly distributed nano-sized filler fibers randomly interwoven on a patterned receiving electrode. The diameter of the micron-sized skeleton fibers is 15~25μm, and the diameter of the nano-sized filler fibers is 200~500nm. The diameter difference between the micron-sized skeleton fibers and the nano-sized filler fibers is more than 50 times.

[0013] Nanoscale filler fibers are distributed on the surface of micron-scale skeleton fibers and between different micron-scale skeleton fibers. The micron-scale skeleton fibers and nanoscale filler fibers interpenetrate to form a three-dimensional hierarchical fiber network structure.

[0014] During deposition, coarser microfibers preferentially construct a three-dimensional support framework spanning larger scales. Fine-scale nanofibers, instead of simply aligning parallel to these microfibers, intertwine and crisscross between and on the surface of the microfibers, filling and refining the pores between the framework. This forms a three-dimensional hierarchical fiber network structure where microfibers and nanofibers interpenetrate. This hierarchical structure, composed of microfibers and nanofibers, retains the large pores formed by the microfibers, facilitating the penetration and exchange of cells, nutrients, and metabolites within the scaffold and providing sufficient overall mechanical support. Furthermore, the deposited microfibers no longer form traditional dense two-dimensional planar membranes but instead construct a three-dimensional interpenetrating network with larger pore sizes. Combined with the significantly increased specific surface area and number of micropores from nanofibers, this provides more interfacial sites for cell adhesion and migration, thus achieving a dual biomimetic effect on both the extracellular matrix microenvironment and macroscopic mechanical properties.

[0015] The patterned receiving electrode has a three-layer structure. The inner layer is a rotatable metal roller connected to an electrostatic generator, which provides a stable and continuous collection substrate. The middle layer is an insulating material layer covering the outer surface of the rotatable metal roller, which electrically isolates the high potential of the inner layer from the outer conductive template. The outer layer is a patterned receiving template covering the outer surface of the insulating material layer. The patterned receiving template is a partially perforated conductive copper plate, which serves as the actual fiber deposition electrode exposed to the outside. The fiber deposition principle is that the combined effect of the electrostatic attraction of the patterned receiving electrode with zero potential difference and the positive repulsion of the unpatterned region breaking down the insulating layer induces positively charged fibers to deposit on the patterned electrode. By introducing a hollowed-out pattern structure onto a basic fiber scaffold, the local cross-sectional area and stress distribution can be altered, optimizing the overall mechanical compliance of the scaffold. Furthermore, the design of the pattern shape and arrangement direction (such as anisotropic patterns like rhombuses, regular hexagons, or concave honeycomb shapes) can impart differentiated mechanical responses to the scaffold in different directions, thereby obtaining a scaffold with anisotropic mechanical properties. This improves its biomimetic mechanical performance under tensile and compressive conditions, better matching the mechanical behavior of the target tissue itself.

[0016] As a preferred technical solution:

[0017] As described above, the micro / nanofiber patterned tissue engineering scaffold based on solution electrospinning technology has a mass ratio of micron-scale skeleton fibers to nano-scale filler fibers of 3.5~5:0.08~1.6.

[0018] As described above, the micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology has a hollowed-out area on the patterned receiving template that is circular, square, rhomboid, regular hexagonal, or has a concave honeycomb structure.

[0019] The micro / nanofiber patterned tissue engineering scaffold based on solution electrospinning technology, as described above, has a thickness of 200~2000μm, an average pore size of 10~300μm, an elongation at break ≥50%, a tensile strength of 2.5~17.6MPa, an elastic modulus of 10~50MPa, and a bursting strength of 30~50kPa.

[0020] The compliance index of the micro / nano fiber patterned tissue engineering scaffold is 0.9~1.1, and the anisotropy index is 0.2~0.8. The compliance index refers to the ratio of the initial elastic modulus of the micro / nano fiber patterned tissue engineering scaffold to the initial elastic modulus of the tissue at the repair site. The anisotropy index refers to the ratio of the horizontal elastic modulus to the vertical elastic modulus of the micro / nano fiber patterned tissue engineering scaffold.

[0021] Cell culture was performed using micro / nanofiber patterned tissue engineering scaffolds, with an average single-cell spreading area of ​​180–300 μm. 2 / mm (Unactivated cells are usually round or spherical, while activated cells will spread out over a large area, refer to YY / T 1577-2017); The cell proliferation rate is 1.2~2.4 times after 3 days of cell seeding (the ratio of the number of cells on the third day to the number of cells on the first day after culture, refer to GB / T 16886.5-2017, YY / T 1562-2017), the Ki-67 positivity rate is 30~48% (positive results indicate that the cells are activated and proliferating, negative results indicate that the cells are not activated and proliferating), and the average infiltration depth is 120~240μm (characterizing the depth at which cells migrate from the upper surface of the scaffold to the lower surface, refer to YY / T 1744-2020).

[0022] The degradation time of micro-nano fiber patterned tissue engineering scaffolds is 6 to 24 months. After 3 months of degradation, the mechanical properties (tensile strength, burst strength, elongation at break and elastic modulus) of micro-nano fiber patterned tissue engineering scaffolds remain at 50 to 80%.

[0023] The present invention also provides a method for preparing a micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology as described in any of the preceding claims, wherein spinning solution A and spinning solution B are prepared respectively, a patterned receiving electrode is used as a receiving device, and a micro / nano fiber patterned tissue engineering scaffold is prepared by electrospinning of the solution by dual nozzles.

[0024] The concentration of spinning solution A is 30~55wt%, the solute is polycaprolactone, polylactic acid, polyglycolic acid or polydioxanone, and the solvent is dichloromethane, chloroform, hexafluoroisopropanol or acetone. Under these conditions of solute, solvent and solution concentration, the viscosity of spinning solution A is 3~6 Pa·s. The solute can be stirred evenly in the solution, avoiding the situation where it is impossible to stir evenly due to the solution viscosity being too high.

[0025] The dielectric constants of these solvents are 4 to 12, the boiling points are 38 to 70°C, or the vapor pressures are 100 to 300 mmHg (approximately 15 to 45 kPa). When the vapor pressure equals atmospheric pressure, the lower the boiling point, the stronger the volatility. A boiling point in the range of 38 to 70°C ensures timely evaporation of the solvent and avoids excessively rapid evaporation that could clog the spinning needle. Alternatively, a vapor pressure range of 100 to 300 mmHg can also meet the requirement for rapid solvent evaporation into fibers without causing the solution to rapidly evaporate and solidify at the spinning needle, thus preventing needle blockage.

[0026] The concentration of spinning solution B is 10~20wt%, the solute is polycaprolactone, polyvinyl alcohol, polylactic acid, polypyrrolidone, polyhydroxybutyrate, polyvinylidene fluoride, polylactic acid-hydroxyacetic acid copolymer, poly-L-lactide-caprolactone, polyglycolic acid or polydioxanone, and the solvent is N,N-dimethylformamide, dimethyl sulfoxide, chloroform, formic acid, methanol, acetic acid, hexafluoroisopropanol or acetone;

[0027] The spinning temperature is 2-8°C lower than the boiling point of the solvent in spinning solution A, and more than 20°C lower than the melting point of the solute in spinning solution A and the solute in spinning solution B.

[0028] Under high concentration, high injection rate, and low voltage process conditions, the high viscosity of the solution leads to a coarser diameter in the stable jet formed by the Taylor cone. At this point, the solute ratio in the solution is high, while the relative content of the conductive solvent is low. This results in the jet requiring a longer time to accumulate sufficient charge during flight to overcome surface tension and form filaments. The unstable whipping jets formed by these filaments eventually deposit on the receiving platform to form fibers. However, under these process conditions, as the charge accumulation time increases, the length of the stable jet also increases. Due to the relatively weak charge repulsion, the number of jets formed by filaments is relatively small, leading to a significant increase in the fiber diameter of the unstable whipping jet. This is one of the direct reasons for obtaining coarser fibers. However, with a fixed receiving distance, an increase in the stable jet length corresponds to a decrease in the unstable jet length. Simultaneously, the larger diameter of the formed jet results in lower solvent evaporation efficiency during the whipping process, preventing complete evaporation (after filamentation, the specific surface area increases, improving evaporation efficiency; if the fibers are coarse and there are few filaments, the specific surface area is small, resulting in lower evaporation efficiency). When fibers are deposited on the receiving platform, they are still in a high solvent content or even close to a solution state, which makes them prone to adhesion, collapse and morphological distortion, resulting in poor fiber forming effect and difficulty in obtaining large-diameter microfibers with complete structure and clear outline of 15μm or more.

[0029] The key to this invention's ability to stably obtain coarse-diameter microfibers ≥15μm under solution electrospinning lies in its unconventional synergistic strategy of reducing fiber splitting and enhancing solvent evaporation. Firstly, it selects biodegradable polymers with low crystallinity and controlled molecular weight, maintaining the spinning solution viscosity within a high viscosity window of 3-6 Pa·s to ensure stable fiber formation and the formation of coarser, stable jet segments even at high polymer concentrations. Simultaneously, it prioritizes solvent combinations with low boiling points / high vapor pressures and low dielectric constants (4-12) to accelerate drying during the unstable jet whipping process and suppress excessive fiber splitting, thus addressing both coarse diameter and forming integrity from the outset. Furthermore, the spinning temperature is set slightly below the solvent boiling point by 2-8°C while maintaining a safety margin with the polymer melting point to further promote timely evaporation of residual solvents and reduce the risk of adhesion. By grounding the receiving roller at the receiving end to maintain a zero potential difference, premature attraction to the positively charged jet is weakened, and the residence time in the air is extended. This avoids adhesion and morphological collapse of coarse fibers during deposition due to incomplete solvent removal, ultimately achieving solution electrospinning to obtain microfibers ≥15μm in diameter. Using patterned receiving electrodes as the receiving device, large-diameter microfibers and nanofibers are combined using solution electrospinning to obtain micro / nanofiber patterned tissue engineering scaffolds.

[0030] As a preferred technical solution:

[0031] The specific steps of the fabrication method for micro / nano fiber patterned tissue engineering scaffolds based on solution electrospinning technology, as described above, are as follows:

[0032] (1) Spinning solution A and spinning solution B were prepared by dissolving the solute in the solvent respectively;

[0033] (2) Spinning solution A and spinning solution B are loaded into different syringes, and two positive high voltage electrostatic generators are connected to a spinning nozzle. The rotatable metal roller of the patterned receiving electrode is connected to a third positive high voltage electrostatic generator. The grounding wire of the rotatable metal roller maintains a potential difference of 0. There is an insulating material layer between the rotatable metal roller with voltage and the patterned electrode with a potential of 0 maintained by the grounding wire, so that the two do not interfere with each other. The shape of the insulating layer of the roller should be consistent with the shape of the patterned receiving template, because the positive charge of the roller needs to generate positive charge in the hollow part of the patterned template, and generate charge repulsion with the positively charged spinning jet, so that it is deposited on the patterned receiving platform with a potential of 0.

[0034] (3) Turn on two positive high voltage electrostatic generators to provide positive high voltage electrostatics to the spinning nozzle, turn on the third positive high voltage electrostatic generator to provide positive high voltage electrostatics to the hollow area of ​​the patterned receiving template, set the spinning voltage, receiving distance, the angle between the radial direction of the spinning nozzle and the upper tangent of the receiving roller, and the speed of the spinning liquid injection, and then perform electrostatic spinning, and use the patterned receiving electrode to receive the micron-scale skeleton fiber and nano-scale filling fiber formed by the whip jet of the unstable section.

[0035] (4) According to the thickness and pattern requirements, obtain micro- and nano-fiber patterned tissue engineering scaffolds with continuous, uniform distribution of micron-level skeleton fibers and nano-level filling fibers, without obvious beading or adhesion, and dry them in a vacuum oven at 37°C for 24 hours.

[0036] In the preparation method of micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology as described above, the specifications of the two spinning nozzles in step (2) are both 20G~24G.

[0037] As described above, in the preparation method of micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology, in step (3), for micron-sized skeleton fibers, the spinning voltage (voltage of positive high voltage electrostatic generator) is 8~16kV, the receiving distance is 16~20cm, the sliding table moving speed is 0.5~3cm / s, the angle between the radial direction of the spinning nozzle and the upper tangent of the rotatable metal roller is 0°~20°, and the spinning solution injection speed is 6~10mL / h;

[0038] For nanoscale filler fibers, the spinning voltage (voltage of the positive high voltage electrostatic generator) is 16~20kV, the receiving distance is 15cm, the sliding table moving speed is 0.5~10cm / s (the size of the pores formed by the micro and nanofibers can be adjusted by changing the moving speed of the sliding table; a faster relative speed of the sliding table during nanofiber forming results in larger pores, while a slower relative speed results in denser pores), the angle between the radial direction of the spinning nozzle and the upper tangent of the rotatable metal roller is 15°~45°, and the spinning solution injection speed is 0.5~2mL / h.

[0039] The spinning temperature for micron-sized skeleton fibers and nano-sized filler fibers is 35~60℃, and the relative humidity for spinning is 18~30%.

[0040] The rotatable metal drum has a rotation speed of 200~500rpm and a voltage of 1~8kV.

[0041] Invention principle:

[0042] In existing micro / nano composite fiber scaffolds, the diameter difference between microfibers and nanofibers is only about 2 to 30 times. Under an electric field, microfibers and nanofibers exhibit similar stretching and orientation behaviors, and their jet trajectories and stress states during spinning are similar, ultimately resulting in a quasi-parallel arrangement on the collector. At this point, the overall scaffold structure is closer to a simple superposition of two single-scale fiber membranes, still primarily planar layered with parallel orientation, making it difficult to construct a hierarchical network structure with significant three-dimensional interpenetrating characteristics. However, attempting to increase the diameter difference between the two fibers by further reducing the nanofiber diameter is not advisable. When the nanofiber diameter is reduced to approximately 50-100 nm, although its size increases the diameter ratio with the microfiber, excessively fine fibers can easily cause "scratching" local mechanical stimulation on the cell membrane surface upon contact with cells. This not only fails to provide effective guidance and support but may also damage cells, reduce cell adhesion and activity, and hinder tissue regeneration. Therefore, simply reducing the nanofiber diameter to increase the diameter difference does not meet the requirements of biomimetic scale and also poses potential risks to cell compatibility.

[0043] This invention utilizes a dual-nozzle electrospinning technique to simultaneously obtain composite tissue engineering scaffolds of microfibers and nanofibers. The microfibers have a diameter of 15-25 μm, while the nanofibers have a diameter of 200-500 nm, resulting in a diameter difference of approximately 50-125 times. This significantly alters the deposition morphology of the system in an electrostatic field: on one hand, the coarser microfibers preferentially form a larger-scale three-dimensional support framework on the collector, constructing a main channel and mechanical load-bearing frame with greater spatial span; on the other hand, the smaller nanofibers, still within the reasonable 200-500 nm range, no longer simply align parallel to the microfibers, but instead exhibit a crisscrossing, entangled, and bridging distribution between and on the surface of the microfibers, filling and refining the pores between the framework, ultimately forming a three-dimensional hierarchical fiber network structure where microfiber framework fibers and nanofiber filler fibers interpenetrate and support each other.

[0044] This invention employs patterned receiving electrodes to receive the formed microfibers and nanofibers. The patterned receiving electrode, from the inside out, comprises a rotatable metal roller, an insulating material layer, and a partially perforated conductive copper plate. By introducing a perforated pattern structure onto the basic fiber scaffold, on the one hand, the local cross-sectional area and stress distribution can be altered, optimizing the overall mechanical compliance of the scaffold. This results in the ratio of the initial elastic modulus of the microfiber-patterned tissue engineering scaffold to the initial elastic modulus of the tissue at the repair site being 0.9 to 1.1. On the other hand, through the design of the pattern shape and arrangement direction (e.g., anisotropic patterns such as rhombuses, regular hexagons, or concave honeycomb shapes), the scaffold can be endowed with differentiated mechanical responses in different directions, thereby obtaining a scaffold with anisotropic mechanical properties. The ratio of its horizontal elastic modulus to its vertical elastic modulus is 0.2 to 0.8, improving its biomimetic mechanical performance under tensile and compressive conditions and better matching the mechanical behavior of the target tissue itself.

[0045] Therefore, this invention enables the simultaneous deposition of micro / nano dual-scale fibers on the aforementioned patterned receiving template, achieving the following simultaneously within the same scaffold: At the microscopic level, a three-dimensional hierarchical fiber network resembling the extracellular matrix is ​​constructed through the joint construction of nanofibers and microfibers, effectively guiding cell adhesion, migration, and three-dimensional ingrowth (structural mimicry of ECM); At the macroscopic level, the overall stiffness, compliance, and anisotropy of the scaffold are controlled through patterned hollow structures, obtaining a mechanical response that is more closely matched to the target tissue (mechanical biomimicry).

[0046] It should be noted that the diameter of microfibers that can be stably prepared by electrospinning using existing technologies is typically no more than 10 μm. In this invention, there is a sufficiently large diameter difference between microfibers and nanofibers, and their filamentation behaviors differ significantly during fiber formation. The diameter of the microfibers is 15–25 μm, which ultimately determines the pore structure and micro / nano interwoven morphology of the scaffold. Firstly, due to the high viscosity and surface tension of the spinning solution, the number of whipping jets formed by the jetting droplets in the unstable phase is relatively small; often only a few jets participate in the stretching process. Simultaneously, these jets have a larger whipping amplitude and a wider oscillation range, resulting in relatively large spacing between fibers and insufficient coverage after deposition on the receiving platform. In other words, single-layer deposition of a microfiber scaffold often fails to achieve suitable porosity and fiber density in a single step, typically requiring 2–3 repeated depositions to form a more stable pore structure and fiber network before constructing the next layer. In contrast, nanofibers correspond to solutions with lower viscosity and lower surface tension. The jetting liquid is more easily stretched and split into filaments under the influence of an electric field, and the accumulated charge more readily overcomes surface tension, causing the jet to split into hundreds or thousands of tiny jets in the whipping phase. Therefore, nanofibers are often deposited on the receiving platform with a higher fiber density, forming a finer network structure. During the alternating or simultaneous deposition of both, a "bridging" overlap occurs: after the first deposition of microfibers, one end of some nanofibers rests on the receiving platform, while the other end overlaps the already deposited microfibers, creating an angle between these nanofibers and the horizontal plane. During the subsequent second deposition of microfibers, although the overall microfibers are still deposited in the same planar area, some directly cover the surface of the previously deposited nanofibers on the platform, thus forming a crisscrossing network of nanofibers within the microfiber pores on the same plane. Furthermore, some nanofibers have a gap of one microfiber diameter between their ends, while others have a gap of two to four, with a larger fiber angle. This ultimately results in a crisscrossing, entangled, and bridged fiber structure.

[0047] Beneficial effects:

[0048] (1) The present invention provides a micro-nano fiber patterned tissue engineering scaffold based on solution electrospinning technology. By using dual-nozzle spray solution electrospinning technology, micron-fibers and nanofibers are simultaneously deposited on a customized patterned receiving platform to form a composite tissue engineering scaffold with specific geometric patterns and multi-scale hierarchical structures. The deposited fibers are evenly distributed and the edges of the hollowed-out parts of the scaffold are clear.

[0049] (2) The present invention provides a method for preparing a micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology. The micro / nano dual-scale fibers are simultaneously deposited on the above-mentioned patterned receiving template. This method can simultaneously achieve the following within the same scaffold: at the micro level, a three-dimensional hierarchical fiber network similar to the extracellular matrix is ​​constructed by nanofibers and microfibers, which effectively guides cell adhesion, migration and three-dimensional ingrowth (structure mimicking ECM); at the macro level, the overall stiffness, compliance and anisotropy of the scaffold are controlled by the patterned hollow structure, so as to obtain a mechanical response that is more matched with the target tissue.

[0050] (3) The present invention provides a micro-nano fiber patterned tissue engineering scaffold based on solution electrospinning technology. The scaffold has differentiated mechanical responses in different directions, thereby obtaining a scaffold with anisotropic mechanical properties, improving its mechanical biomimetic effect under tensile, compressive and other working conditions, and better matching the mechanical behavior of the target tissue itself. Attached Figure Description

[0051] Figure 1 This is a SEM image of the micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology in Example 1. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0053] The test methods involved in the performance indicators in the embodiments and comparative examples of this invention are as follows:

[0054] Elongation at break: The prepared tissue engineering scaffold was used as a sample, and the elongation at break was tested in accordance with the standard GB / T 3923.1-2013 "Textiles - Tensile properties of fabrics - Part 1: Determination of breaking strength and elongation at break (strip method)".

[0055] Bursting strength: The prepared tissue engineering scaffold was used as a sample, and the bursting strength was measured in accordance with the standard GB / T 19976-2005 "Textiles - Determination of bursting strength - Steel ball method".

[0056] Tensile strength: The prepared composite tissue engineering scaffold was used as a sample, and the tensile strength was measured in accordance with the standard GB / T 1040.1-2018 "Determination of tensile properties of plastics - Part 1: General".

[0057] Elastic modulus: The prepared composite tissue engineering scaffold was used as a sample, and the elastic strength of the scaffold was measured in accordance with the standard GB / T 1040.1-2018 "Determination of tensile properties of plastics - Part 1: General".

[0058] Degradation test: The prepared tissue engineering scaffold was used as a sample and then tested according to the standard YY / T 1806.1—2021 "Evaluation method for in vitro degradation performance of biomedical materials - Part 1: Degradable polyesters". The degradation time was determined when the material mass loss reached 100%.

[0059] Compliance index: The prepared tissue engineering scaffold and the intact tissue of the site to be repaired are used as samples. The initial elastic modulus of the scaffold and the tissue are measured according to the standard GB / T 1040.1-2018 "Determination of tensile properties of plastics - Part 1: General". The ratio of the initial elastic modulus of the scaffold to the initial elastic modulus of the tissue is calculated to obtain the compliance index of the material.

[0060] Anisotropy index: The prepared tissue engineering scaffold was used as a sample. The elastic modulus of the scaffold in the horizontal and vertical directions was measured according to the standard GB / T 1040.1-2018 "Determination of tensile properties of plastics - Part 1: General". The ratio of the elastic modulus of the horizontal direction to the elastic modulus of the vertical direction of the scaffold was calculated to obtain the anisotropy index of the material.

[0061] Example 1

[0062] The fabrication method of micro / nano fiber patterned tissue engineering scaffolds based on solution electrospinning technology includes the following specific steps:

[0063] (1) Polycaprolactone (manufacturer: Shanghai Maclean Biochemical Technology Co., Ltd., number average molecular weight: 80,000) was dissolved in dichloromethane to prepare a spinning solution A with a concentration of 50 wt%.

[0064] The dichloromethane has a boiling point of 40℃ and a vapor pressure of 429.53 mmHg; its dielectric constant is 8.9 F / m; and the viscosity of spinning solution A is 5.2 Pa·s.

[0065] (2) Polycaprolactone (manufacturer: Shanghai Maclean Biochemical Technology Co., Ltd., number average molecular weight: 80,000) was dissolved in a mixed solvent of chloroform: N,N-dimethylformamide = 7:3 (v:v) to prepare a spinning solution B with a concentration of 10wt%.

[0066] (3) Load spinning solution A and spinning solution B into different syringes respectively, and connect two positive high voltage electrostatic generators to a spinning nozzle with a specification of 21G respectively. Connect the rotatable metal roller of the patterned receiving electrode to the third positive high voltage electrostatic generator. The grounding wire of the rotatable metal roller maintains a potential difference of 0.

[0067] (4) Turn on two positive high voltage electrostatic generators to provide positive high voltage electrostatics to the spinning nozzle, turn on the third positive high voltage electrostatic generator to provide positive high voltage electrostatics to the hollow area of ​​the patterned receiving template, set the spinning voltage, receiving distance, the angle between the radial direction of the spinning nozzle and the upper tangent of the receiving roller, and the speed of the spinning liquid injection, perform electrostatic spinning, and receive the formed micron fibers and nano fibers.

[0068] Spinning process parameters for micron-sized fibers: spinning voltage 12kV, receiving distance 16cm, angle between the radial direction of the spinning nozzle and the upper tangential surface of the rotatable metal roller 15°, spinning solution injection speed 8mL / h, spinning temperature 38℃, and spinning relative humidity 18%.

[0069] The spinning process parameters for nanofibers are as follows: spinning voltage is 18kV, receiving distance is 15cm, sliding table moving speed is 5cm / s, the angle between the radial direction of the spinning nozzle and the upper tangential surface of the receiving roller is 30°, the spinning solution injection speed is 1mL / h, the spinning temperature is 38℃, and the spinning relative humidity is 18%.

[0070] The rotatable metal drum has a rotation speed of 250 rpm and a voltage of 8 kV.

[0071] (5) Based on the requirements of 1000μm thickness and circular hollow pattern, a micro-nano fiber patterned tissue engineering scaffold was obtained and dried in a vacuum oven at 37℃ for 24h.

[0072] The final fabricated micro / nanofiber patterned tissue engineering scaffold consists of uniformly distributed micron-sized framework fibers and uniformly distributed nano-sized filler fibers. The diameter of the micron-sized framework fibers is 23 μm, and the diameter of the nano-sized filler fibers is 300 nm. The mass ratio of micron-sized framework fibers to nano-sized filler fibers in the micro / nanofiber patterned tissue engineering scaffold is 5:0.5. The nano-sized filler fibers are distributed on the surface of the micron-sized framework fibers and between different micron-sized framework fibers. The micron-sized framework fibers and nano-sized filler fibers interpenetrate to form a three-dimensional hierarchical fiber network structure. The patterned receiving electrode has a three-layer structure: the inner layer is a rotatable metal roller connected to the electrostatic generator; the middle layer is an insulating material layer covering the outer surface of the rotatable metal roller; and the outer layer is a patterned receiving template covering the outer surface of the insulating material layer. The receiving template is a partially circular, perforated conductive copper plate; the micro / nanofiber patterned tissue engineering scaffold has a thickness of 1000 μm, an average pore size of 150 μm, an elongation at break of 600%, a tensile strength of 5 MPa, a bursting strength of 45 kPa, a horizontal elastic modulus of 10 MPa, and a vertical elastic modulus of 50 MPa; the degradation time of the micro / nanofiber patterned tissue engineering scaffold is 24 months. After 3 months of degradation, the tensile strength, bursting strength, elongation at break, and elastic modulus of the micro / nanofiber patterned tissue engineering scaffold are all maintained at 80%; the compliance index is 0.9; the anisotropy index is 0.2; cell culture using the micro / nanofiber patterned tissue engineering scaffold shows an average single-cell spreading area of ​​240 μm. 2 / mm; Three days after cell seeding, the cell proliferation rate was 2.1-fold, the Ki-67 positivity rate was 42%, and the average infiltration depth was 240μm.

[0073] like Figure 1 As shown, the microfibers and nanofibers are uniformly and randomly distributed, and the nanofibers are interwoven in the gaps formed by the microfibers. It can be seen that the complex electric field environment does not affect the deposition of microfibers and nanofibers.

[0074] Comparative Example 1

[0075] The preparation method of micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology is basically the same as in Example 1, except that the diameter of the micron-scale skeleton fiber is 8μm, that is, the concentration of spinning solution A in step (1) is 24wt%.

[0076] The final micro / nanofiber composite tissue engineering scaffold has a thickness of 200 μm, an average pore size of 2 μm, an elongation at break of ≥300%, a tensile strength of 2 MPa, a bursting strength of 20 kPa, and an elastic modulus of 25 MPa. Cell culture was performed using the micro / nanofiber patterned tissue engineering scaffold, with an average infiltration depth of 80 μm.

[0077] Comparing Comparative Example 1 and Example 1, it can be found that the mechanical strength of Comparative Example 1 is reduced and the cell infiltration depth is decreased. This is because the mechanical effect of the micro / nanofiber patterned tissue engineering scaffold is mainly provided by the microfibers. When the fiber diameter is reduced, the microfibers are more likely to break under stress. In addition, when the concentration of the spinning solution is reduced, the voltage is more likely to overcome the surface tension of the solution to form more jets. The increased jets result in higher deposition efficiency on the roller, so the microfibers will be deposited more densely, leading to the formation of smaller micropores. In addition, the deposition of nanofibers in the micropores is insufficient to form a pore structure suitable for cell penetration. Therefore, cells can only grow on the surface of the scaffold, and the cell infiltration effect is reduced.

[0078] Comparative Example 2

[0079] A method for preparing a micro / nanofiber composite tissue engineering scaffold is basically the same as in Example 1, except that the diameters of the micron-scale skeleton fibers and the nano-scale filler fibers differ by 40 times. That is, the concentration of spinning solution B in step (2) is 20wt%, forming fibers with a diameter of 500nm; at the same time, the concentration of spinning solution A in step (1) is 45wt%, forming fibers with a diameter of 20μm.

[0080] The final micro-nanofiber composite tissue engineering scaffold was used for cell culture, with a Ki-67 positivity rate of 30% and an average infiltration depth of 80 μm.

[0081] Comparing Comparative Example 2 with Example 1, it can be found that the Ki-67 positivity rate and average infiltration depth are relatively lower in Comparative Example 2. This is because the diameter difference between microfibers and nanofibers is reduced, and the slope of the overlap between microfibers during nanofiber deposition is reduced, which prevents cells from growing longitudinally along the guidance of microfibers, thereby reducing cell infiltration efficiency.

[0082] Comparative Example 3

[0083] A method for preparing a micro / nanofiber composite tissue engineering scaffold is basically the same as in Example 1, except that: no patterned spinning structure is constructed, that is, the patterned receiving template in step (4) has no hollow area.

[0084] The final micro / nanofiber composite tissue engineering scaffold had a compliance index of 2 and an anisotropy index of 1.

[0085] Comparing Comparative Example 3 with Example 1, it can be found that both the compliance and anisotropy indices are improved in Comparative Example 3. This is because the patterned structure design mainly serves to optimize the compliance and anisotropy of the scaffold. The scaffold without a patterned structure design is a homogeneous isotropic structure with increased stiffness and an initial elastic modulus greater than that of the tissue, resulting in an improved compliance index. The elastic moduli in the vertical and horizontal directions are equal, resulting in an anisotropy index of 1.

[0086] Example 2

[0087] The fabrication method of micro / nano fiber patterned tissue engineering scaffolds based on solution electrospinning technology includes the following specific steps:

[0088] (1) Polylactic acid (manufacturer: Shanghai Maclean Biochemical Technology Co., Ltd., number average molecular weight: 80,000) was dissolved in tetrahydrofuran to prepare a spinning solution A with a concentration of 55 wt%.

[0089] Among them, tetrahydrofuran has a boiling point of 66℃ and a vapor pressure of 162.18 mmHg; the dielectric constant of tetrahydrofuran is 7.6 F / m; and the viscosity of spinning solution A is 4.4 Pa·s.

[0090] (2) Polyvinyl alcohol (manufacturer: Shanghai Titan Co., Ltd., type 1799) was dissolved in water to prepare a spinning solution B with a concentration of 20 wt%;

[0091] (3) Load spinning solution A and spinning solution B into different syringes respectively, and connect two positive high voltage electrostatic generators to a spinning nozzle with a specification of 20G respectively. Connect the rotatable metal roller of the patterned receiving electrode to the third positive high voltage electrostatic generator. The grounding wire of the rotatable metal roller keeps the potential difference at 0.

[0092] (4) Turn on two positive high voltage electrostatic generators to provide positive high voltage electrostatics to the spinning nozzle, turn on the third positive high voltage electrostatic generator to provide positive high voltage electrostatics to the hollow area of ​​the patterned receiving template, set the spinning voltage, receiving distance, the angle between the radial direction of the spinning nozzle and the upper tangent of the receiving roller, and the speed of the spinning liquid injection, perform electrostatic spinning, and receive the formed micron fibers and nano fibers.

[0093] Spinning process parameters for micron-sized fibers: spinning voltage 8kV, receiving distance 17cm, angle between the radial direction of the spinning nozzle and the upper tangential surface of the rotatable metal roller 5°, spinning solution injection speed 7mL / h, spinning temperature 60℃, and spinning relative humidity 20%.

[0094] The spinning process parameters for nanofibers are as follows: spinning voltage is 16kV, receiving distance is 15cm, sliding table moving speed is 10cm / s, the angle between the radial direction of the spinning nozzle and the upper tangential plane of the receiving roller is 45°, the spinning solution injection speed is 2mL / h, the spinning temperature is 60℃, and the spinning relative humidity is 20%.

[0095] The rotatable metal drum has a rotation speed of 200 rpm and a voltage of 1 kV.

[0096] (5) Based on the thickness requirement of 2000μm and the square hollow pattern requirement, a micro-nano fiber patterned tissue engineering scaffold was obtained and dried in a vacuum oven at 37℃ for 24h.

[0097] The final fabricated micro / nanofiber patterned tissue engineering scaffold consists of uniformly distributed micron-sized framework fibers and uniformly distributed nano-sized filler fibers. The diameter of the micron-sized framework fibers is 25 μm, and the diameter of the nano-sized filler fibers is 500 nm. The mass ratio of micron-sized framework fibers to nano-sized filler fibers in the micro / nanofiber patterned tissue engineering scaffold is 5:1.6. The nano-sized filler fibers are distributed on the surface of the micron-sized framework fibers and between different micron-sized framework fibers, and the micron-sized framework fibers and nano-sized filler fibers interpenetrate to form a three-dimensional hierarchical fiber network structure. The patterned receiving electrode has a three-layer structure: the inner layer is a rotatable metal roller connected to the electrostatic generator; the middle layer is an insulating material layer covering the outer surface of the rotatable metal roller; and the outer layer is a patterned receiving template covering the outer surface of the insulating material layer. The receiving template is a partially square, perforated conductive copper plate; the micro / nanofiber patterned tissue engineering scaffold has a thickness of 2000 μm, an average pore size of 10 μm, an elongation at break of 100%, a tensile strength of 6.5 MPa, a bursting strength of 50 kPa, a horizontal elastic modulus of 16 MPa, and a vertical elastic modulus of 44 MPa; the degradation time of the micro / nanofiber patterned tissue engineering scaffold is 18 months. After 3 months of degradation, the tensile strength retention rate is 76%, the bursting strength retention rate is 74%, the elongation at break retention rate is 75%, and the elastic modulus retention rate is 70%; the compliance index is 1; the anisotropy index is 0.36; cell culture using the micro / nanofiber patterned tissue engineering scaffold shows an average single-cell spreading area of ​​270 μm². 2 / mm; Three days after cell seeding, the cell proliferation rate was 1.6-fold, the Ki-67 positivity rate was 48%, and the average infiltration depth was 120μm.

[0098] Example 3

[0099] The fabrication method of micro / nano fiber patterned tissue engineering scaffolds based on solution electrospinning technology includes the following specific steps:

[0100] (1) Polydioxanone with a number average molecular weight of 50,000 was dissolved in chloroform to prepare a spinning solution A with a concentration of 45 wt%;

[0101] The boiling point of chloroform is 61℃, and its vapor pressure is 194.23 mmHg; the dielectric constant of chloroform is 4.8 F / m; and the viscosity of spinning solution A is 3 Pa·s.

[0102] (2) Polyvinylidene fluoride (manufacturer: Beijing Mairuida Technology Co., Ltd., CAS No.: 24937-79-9) was dissolved in a mixed solvent of N,N-dimethylformamide:acetone = 8:2 (v:v) to prepare a spinning solution B with a concentration of 15wt%.

[0103] (3) Load spinning solution A and spinning solution B into different syringes respectively, and connect the positive high voltage electrostatic generator to the spinning nozzle with a specification of 22G. Connect the rotatable metal roller of the patterned receiving electrode to the third positive high voltage electrostatic generator. The rotatable metal roller ground wire keeps the potential difference at 0.

[0104] (4) Turn on two positive high voltage electrostatic generators to provide positive high voltage electrostatics to the spinning nozzle, turn on the third positive high voltage electrostatic generator to provide positive high voltage electrostatics to the hollow area of ​​the patterned receiving template, set the spinning voltage, receiving distance, the angle between the radial direction of the spinning nozzle and the upper tangent of the receiving roller, and the speed of the spinning liquid injection, perform electrostatic spinning, and receive the formed micron fibers and nano fibers.

[0105] Spinning process parameters for micron-sized fibers: spinning voltage 12kV, receiving distance 18cm, angle between the radial direction of the spinning nozzle and the upper tangential surface of the rotatable metal roller 10°, spinning solution injection speed 8mL / h, spinning temperature 54℃, and spinning relative humidity 24%.

[0106] The spinning process parameters for nanofibers are as follows: spinning voltage is 20kV, receiving distance is 15cm, sliding table moving speed is 6cm / s, the angle between the radial direction of the spinning nozzle and the upper tangential plane of the rotatable metal roller is 15°, the spinning solution injection speed is 1.5mL / h, the spinning temperature is 54℃, and the spinning relative humidity is 24%.

[0107] The rotatable metal drum has a rotation speed of 500 rpm and a voltage of 8 kV.

[0108] (5) Based on the thickness requirement of 800 μm and the requirement of diamond-shaped hollow pattern, a micro-nano fiber patterned tissue engineering scaffold was obtained and dried in a vacuum oven at 37℃ for 24 h.

[0109] The final fabricated micro / nanofiber patterned tissue engineering scaffold consists of uniformly distributed micron-sized framework fibers and uniformly distributed nano-sized filler fibers. The diameter of the micron-sized framework fibers is 20 μm, and the diameter of the nano-sized filler fibers is 250 nm. The mass ratio of micron-sized framework fibers to nano-sized filler fibers in the micro / nanofiber patterned tissue engineering scaffold is 4.5:1. The nano-sized filler fibers are distributed on the surface of the micron-sized framework fibers and between different micron-sized framework fibers, and the micron-sized framework fibers and nano-sized filler fibers interpenetrate to form a three-dimensional hierarchical fiber network structure. The patterned receiving electrode has a three-layer structure: the inner layer is a rotatable metal roller connected to the electrostatic generator; the middle layer is an insulating material layer covering the outer surface of the rotatable metal roller; and the outer layer is a patterned receiving template covering the outer surface of the insulating material layer. The receiving template is a partially diamond-shaped perforated conductive copper plate; the micro / nanofiber patterned tissue engineering scaffold has a thickness of 800 μm, an average pore size of 100 μm, an elongation at break of 500%, a tensile strength of 4 MPa, a bursting strength of 35 kPa, a horizontal elastic modulus of 19.2 MPa, and a vertical elastic modulus of 24 MPa; the degradation time of the micro / nanofiber patterned tissue engineering scaffold is 8 months. After 3 months of degradation, the tensile strength retention rate is 60%, the bursting strength retention rate is 58%, the elongation at break retention rate is 56%, and the elastic modulus retention rate is 68%; the compliance index is 0.95; the anisotropy index is 0.8; cell culture using the micro / nanofiber patterned tissue engineering scaffold shows an average single-cell spreading area of ​​210 μm². 2 / mm; Three days after cell seeding, the cell proliferation rate was 1.2 times, the Ki-67 positivity rate was 30%, and the average infiltration depth was 150μm.

[0110] Example 4

[0111] The fabrication method of micro / nano fiber patterned tissue engineering scaffolds based on solution electrospinning technology includes the following specific steps:

[0112] (1) Polyglycolic acid with a number average molecular weight of 100,000 was dissolved in hexafluoroisopropanol to prepare a spinning solution A with a concentration of 38 wt%.

[0113] Among them, the boiling point of hexafluoroisopropanol is 58℃, the vapor pressure is 155.26 mmHg, the dielectric constant of hexafluoroisopropanol is 12 F / m, and the viscosity of spinning solution A is 6 Pa·s.

[0114] (2) Polydioxanone (manufacturer: Shanghai McLean Biochemical Technology Co., Ltd., CAS No.: 31621-87-1) was dissolved in hexafluoroisopropanol to prepare a spinning solution B with a concentration of 12wt%;

[0115] (3) Load spinning solution A and spinning solution B into different syringes respectively, and connect two positive high voltage electrostatic generators to a spinning nozzle with a specification of 23G respectively. Connect the rotatable metal roller of the patterned receiving electrode to the third positive high voltage electrostatic generator. The grounding wire of the rotatable metal roller keeps the potential difference at 0.

[0116] (4) Turn on two positive high voltage electrostatic generators to provide positive high voltage electrostatics to the spinning nozzle, turn on the third positive high voltage electrostatic generator to provide positive high voltage electrostatics to the hollow area of ​​the patterned receiving template, set the spinning voltage, receiving distance, the angle between the radial direction of the spinning nozzle and the upper tangent of the receiving roller, and the speed of the spinning liquid injection, perform electrostatic spinning, and receive the formed micron fibers and nano fibers.

[0117] Spinning process parameters for micron-sized fibers: spinning voltage 14kV, receiving distance 19cm, angle between the radial direction of the spinning nozzle and the upper tangential surface of the rotatable metal roller 15°, spinning solution injection speed 9mL / h, spinning temperature 52℃, and spinning relative humidity 28%;

[0118] The spinning process parameters for nanofibers are as follows: spinning voltage is 17kV, receiving distance is 15cm, sliding table moving speed is 0.5cm / s, the angle between the radial direction of the spinning nozzle and the upper tangential plane of the receiving roller is 20°, the spinning solution injection speed is 0.8mL / h, the spinning temperature is 52℃, and the spinning relative humidity is 28%.

[0119] The rotatable metal drum has a rotation speed of 350 rpm and a voltage of 5 kV.

[0120] (5) Based on the thickness requirement of 400μm and the requirement of regular hexagonal hollow pattern, a micro-nano fiber patterned tissue engineering scaffold was obtained and dried in a vacuum oven at 37℃ for 24h.

[0121] The final fabricated micro / nanofiber patterned tissue engineering scaffold consists of uniformly distributed micron-sized framework fibers and uniformly distributed nano-sized filler fibers. The diameter of the micron-sized framework fibers is 18 μm, and the diameter of the nano-sized filler fibers is 400 nm. The mass ratio of micron-sized framework fibers to nano-sized filler fibers in the micro / nanofiber patterned tissue engineering scaffold is 3.8:0.08. The nano-sized filler fibers are distributed on the surface of the micron-sized framework fibers and between different micron-sized framework fibers. The micron-sized framework fibers and nano-sized filler fibers interpenetrate to form a three-dimensional hierarchical fiber network structure. The patterned receiving electrode has a three-layer structure: the inner layer is a rotatable metal roller connected to the electrostatic generator; the middle layer is an insulating material layer covering the outer surface of the rotatable metal roller; and the outer layer is a patterned receiving template covering the outer surface of the insulating material layer. The receiving template is a partially hexagonal perforated conductive copper plate; the micro / nanofiber patterned tissue engineering scaffold has a thickness of 400 μm, an average pore size of 300 μm, an elongation at break of 60%, a tensile strength of 8.8 MPa, a burst strength of 44 kPa, a horizontal elastic modulus of 20 MPa, and a vertical elastic modulus of 40 MPa; the degradation time of the micro / nanofiber patterned tissue engineering scaffold is 6 months, and after 3 months of degradation, the tensile strength, burst strength, elongation at break, and elastic modulus of the micro / nanofiber patterned tissue engineering scaffold retain 50%, 50%, and 80% respectively; the compliance index is 1.1; the anisotropy index is 0.5; cell culture using the micro / nanofiber patterned tissue engineering scaffold shows an average single-cell spreading area of ​​180 μm. 2 / mm; Three days after cell seeding, the cell proliferation rate was 2.4 times, the Ki-67 positivity rate was 48%, and the average infiltration depth was 240μm.

[0122] Example 5

[0123] The fabrication method of micro / nano fiber patterned tissue engineering scaffolds based on solution electrospinning technology includes the following specific steps:

[0124] (1) Polycaprolactone with a number average molecular weight of 80,000 was dissolved in acetone to prepare a spinning solution A with a concentration of 30 wt%.

[0125] Among them, the boiling point of acetone is 56℃, the vapor pressure is 229.52 mmHg, the dielectric constant of acetone is 12 F / m, and the viscosity of spinning solution A is 5 Pa·s.

[0126] (2) Polylactic acid (manufacturer: Shanghai Maclean Biochemical Technology Co., Ltd., number average molecular weight: 80,000) was dissolved in hexafluoroisopropanol to prepare a spinning solution B with a concentration of 18 wt%.

[0127] (3) Load spinning solution A and spinning solution B into different syringes respectively, and connect the positive high voltage electrostatic generator to the spinning nozzle with a specification of 24G. Connect the rotatable metal roller of the patterned receiving electrode to the third positive high voltage electrostatic generator. The grounding wire of the rotatable metal roller keeps the potential difference at 0.

[0128] (4) Turn on two positive high voltage electrostatic generators to provide positive high voltage electrostatics to the spinning nozzle, turn on the third positive high voltage electrostatic generator to provide positive high voltage electrostatics to the hollow area of ​​the patterned receiving template, set the spinning voltage, receiving distance, the angle between the radial direction of the spinning nozzle and the upper tangent of the receiving roller, and the speed of the spinning liquid injection, perform electrostatic spinning, and receive the formed micron fibers and nano fibers.

[0129] Spinning process parameters for micron-sized fibers: spinning voltage 16kV, receiving distance 20cm, angle between the radial direction of the spinning nozzle and the upper tangential surface of the rotatable metal roller 20°, spinning solution injection speed 10mL / h, spinning temperature 48℃, and spinning relative humidity 30%.

[0130] The spinning process parameters for nanofibers are as follows: spinning voltage is 19kV, receiving distance is 15cm, sliding table speed is 4cm / s, the angle between the radial direction of the spinning nozzle and the upper tangential plane of the receiving roller is 30°, the receiving roller speed is 400rpm, the spinning solution injection rate is 0.5mL / h, the spinning temperature is 48℃, and the relative humidity of spinning is 30%.

[0131] The rotatable metal drum has a rotation speed of 400 rpm and a voltage of 6 kV.

[0132] (5) Based on the thickness requirement of 200 μm and the requirement of hollow pattern of concave honeycomb structure, a micro-nano fiber patterned tissue engineering scaffold was obtained and dried in a vacuum oven at 37℃ for 24 h.

[0133] The final fabricated micro / nanofiber patterned tissue engineering scaffold consists of uniformly distributed micron-sized framework fibers and uniformly distributed nano-sized filler fibers. The diameter of the micron-sized framework fibers is 15 μm, and the diameter of the nano-sized filler fibers is 450 nm. The mass ratio of micron-sized framework fibers to nano-sized filler fibers in the micro / nanofiber patterned tissue engineering scaffold is 3.5:0.4. The nano-sized filler fibers are distributed on the surface of the micron-sized framework fibers and between different micron-sized framework fibers. The micron-sized framework fibers and nano-sized filler fibers interpenetrate to form a three-dimensional hierarchical fiber network structure. The patterned receiving electrode has a three-layer structure: the inner layer is a rotatable metal roller connected to the electrostatic generator; the middle layer is an insulating material layer covering the outer surface of the rotatable metal roller; and the outer layer is a patterned receiving template covering the outer surface of the insulating material layer. The patterned receiving electrode... The template is a partially concave honeycomb structure with perforated conductive copper plate. The micro / nanofiber patterned tissue engineering scaffold has a thickness of 200 μm, an average pore size of 150 μm, an elongation at break of 300%, a tensile strength of 2.5 MPa, a burst strength of 30 kPa, a horizontal elastic modulus of 18 MPa, and a vertical elastic modulus of 46 MPa. The degradation time of the micro / nanofiber patterned tissue engineering scaffold is 22 months. After 3 months of degradation, the tensile strength retention rate is 72%, the burst strength retention rate is 70%, the elongation at break retention rate is 68%, and the elastic modulus retention rate is 50%. The compliance index is 1.05, and the anisotropy index is 0.39. Cell culture using the micro / nanofiber patterned tissue engineering scaffold showed an average single-cell spreading area of ​​300 μm². 2 / mm; Three days after cell seeding, the cell proliferation fold was 2-fold, the Ki-67 positivity rate was 36%, and the average infiltration depth was 210μm.

Claims

1. A micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology, characterized in that: It is composed of uniformly distributed micron-sized skeleton fibers and uniformly distributed nano-sized filler fibers randomly interwoven on a patterned receiving electrode. The diameter of the micron-sized skeleton fibers is 15~25μm, and the diameter of the nano-sized filler fibers is 200~500nm. The diameters of the micron-sized skeleton fibers and the nano-sized filler fibers differ by more than 50 times. Nanoscale filler fibers are distributed on the surface of micron-scale skeleton fibers and between different micron-scale skeleton fibers. The micron-scale skeleton fibers and nanoscale filler fibers interpenetrate to form a three-dimensional hierarchical fiber network structure. The patterned receiving electrode has a three-layer structure: the inner layer is a rotatable metal roller connected to the electrostatic generator, the middle layer is an insulating material layer covering the outer surface of the rotatable metal roller, and the outer layer is a patterned receiving template covering the outer surface of the insulating material layer. The patterned receiving template is a partially perforated conductive copper plate.

2. The micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology according to claim 1, characterized in that, In micro / nano fiber patterned tissue engineering scaffolds, the mass ratio of micron-scale scaffold fibers to nano-scale filler fibers is 3.5~5:0.08~1.

6.

3. The micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology according to claim 1, characterized in that, The hollowed-out areas on the patterned receiving template can be circular, square, rhomboid, regular hexagonal, or have a concave honeycomb structure.

4. The micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology according to claim 1, characterized in that, The micro / nano fiber patterned tissue engineering scaffold has a thickness of 200~2000μm, an average pore size of 10~300μm, an elongation at break of ≥50%, a tensile strength of 2.5~17.6MPa, an elastic modulus of 10~50MPa, and a bursting strength of 30~50kPa. The compliance index of the micro / nano fiber patterned tissue engineering scaffold is 0.9~1.1, and the anisotropy index is 0.2~0.

8. The compliance index refers to the ratio of the initial elastic modulus of the micro / nano fiber patterned tissue engineering scaffold to the initial elastic modulus of the tissue at the repair site. The anisotropy index refers to the ratio of the horizontal elastic modulus to the vertical elastic modulus of the micro / nano fiber patterned tissue engineering scaffold. The degradation time of micro-nano fiber patterned tissue engineering scaffolds is 6 to 24 months, and the mechanical properties of micro-nano fiber patterned tissue engineering scaffolds are maintained at 50 to 80% after 3 months of degradation.

5. The method for preparing a micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology as described in any one of claims 1 to 4, characterized in that: Spinning solution A and spinning solution B were prepared separately. Using a patterned receiving electrode as a receiving device, micro-nano fiber patterned tissue engineering scaffolds were prepared by electrospinning the solutions with dual nozzles. The concentration of spinning solution A is 30~55wt%, the solute is polycaprolactone, polylactic acid, polyglycolic acid or polydioxanone, and the solvent is dichloromethane, chloroform, hexafluoroisopropanol or acetone. The concentration of spinning solution B is 10~20wt%, the solute is polycaprolactone, polyvinyl alcohol, polylactic acid, polypyrrolidone, polyhydroxybutyrate, polyvinylidene fluoride, polylactic acid-hydroxyacetic acid copolymer, poly-L-lactide-caprolactone, polyglycolic acid or polydioxanone, and the solvent is N,N-dimethylformamide, dimethyl sulfoxide, chloroform, formic acid, methanol, acetic acid, hexafluoroisopropanol or acetone; The spinning temperature is 2-8°C lower than the boiling point of the solvent in spinning solution A, and more than 20°C lower than the melting point of the solute in spinning solution A and the solute in spinning solution B.

6. The method for preparing a micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology according to claim 5, characterized in that, The specific steps are as follows: (1) Spinning solution A and spinning solution B were prepared by dissolving the solute in the solvent respectively; (2) The spinning solution A and the spinning solution B are respectively loaded into different syringes, and the two positive high voltage electrostatic generators are respectively connected to a spinning nozzle. The rotatable metal roller of the patterned receiving electrode is connected to the third positive high voltage electrostatic generator. The grounding wire of the rotatable metal roller is kept at a potential difference of 0. (3) Turn on two positive high voltage electrostatic generators to provide positive high voltage electrostatics to the spinning nozzle, turn on the third positive high voltage electrostatic generator to provide positive high voltage electrostatics to the hollow area of ​​the patterned receiving template, set the spinning voltage, receiving distance, the angle between the radial direction of the spinning nozzle and the upper tangent of the receiving roller, and the speed of the spinning liquid injection, then perform electrospinning and receive the micron-scale skeleton fibers and nano-scale filling fibers formed. (4) According to the thickness and pattern requirements, a micro-nano fiber patterned tissue engineering scaffold is obtained and dried in a vacuum oven at 37°C for 24 hours.

7. The method for preparing a micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology according to claim 6, characterized in that, In step (2), the specifications of the two spinning nozzles are both 20G~24G.

8. The method for preparing a micro / nano fiber patterned tissue engineering scaffold based on solution electrospinning technology according to claim 6, characterized in that, In step (3), for micron-sized skeleton fibers, the spinning voltage is 8~16kV, the receiving distance is 16~20cm, the sliding table moving speed is 0.5~3cm / s, the angle between the radial direction of the spinning nozzle and the upper tangent of the rotatable metal roller is 0°~20°, and the spinning solution injection speed is 6~10mL / h. For nanoscale filled fibers, the spinning voltage is 16~20kV, the receiving distance is 15cm, the sliding table moving speed is 0.5~10cm / s, the angle between the radial direction of the spinning nozzle and the upper tangent of the rotatable metal drum is 15°~45°, and the spinning solution injection speed is 0.5~2mL / h. The spinning temperature for micron-sized skeleton fibers and nano-sized filler fibers is 35~60℃, and the relative humidity for spinning is 18~30%. The rotatable metal drum has a rotation speed of 200~500rpm and a voltage of 1~8kV.

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