CN114010842B - Microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate and preparation method thereof - Google Patents

Microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate and preparation method thereof Download PDF

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CN114010842B
CN114010842B CN202111264250.2A CN202111264250A CN114010842B CN 114010842 B CN114010842 B CN 114010842B CN 202111264250 A CN202111264250 A CN 202111264250A CN 114010842 B CN114010842 B CN 114010842B
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scaffold
polyhydroxyalkanoate
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microfiber
preparation
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CN114010842A (en
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赵远锦
过文泰
王小成
王月桐
商珞然
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Wenzhou Research Institute Of Guoke Wenzhou Institute Of Biomaterials And Engineering
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Abstract

The invention provides a microfluidic 3D printing bionic skin scaffold based on polyhydroxyalkanoate and a preparation method thereof, the method combines a microfluidic chip and a 3D printing technology to generate a continuous stable liquid flow containing polyhydroxyalkanoate, the liquid flow is dripped into an ethanol collecting phase, and a microfiber scaffold with uniform size is formed by diffusion and volatilization of an organic solvent in an ethanol water solution. The microfiber support prepared by the invention has hierarchical porous and extracellular matrix-imitated structures, the diameter of the microfiber can be accurately adjusted by changing preparation conditions (the flow velocity of liquid flow in a microfluidic chip or the moving speed of a 3D printer nozzle), the preparation method is simple and easy to implement, and the shape of the support can be adjusted by a preset 3D model pattern. The prepared polyhydroxyalkanoate scaffold with the bionic structure can effectively load various active cells to realize double-sided anisotropy so as to realize repair and regeneration of complicated skin wounds.

Description

Microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate and preparation method thereof
The technical field is as follows:
the invention belongs to the technical field of biological materials, and particularly relates to a microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate and a preparation method thereof.
Background art:
healing of skin wounds is a common but challenging clinical problem, placing a significant burden on the global healthcare system. Traditional wound dressings only cover the wound surface, absorb exudate and provide limited protection to the wound. Notably, tissue engineering scaffolds with a simulated extracellular matrix (ECM) structure have been extensively studied in wound healing applications. However, the existing tissue engineering scaffold has a simple structure and insufficient mechanical properties. For example, most of the reported electrospun tissue-skin scaffolds have an unordered fibrous structure, and the mechanical strength thereof reaches only 50 to 100kPa. In addition, these tissue engineering scaffolds often lose some of their biological activity with increasing strength, which may lead to adverse reactions such as tissue allergy and inflammation. Therefore, it is necessary to develop a tissue engineering scaffold material with high mechanical strength and bioactivity for wound healing.
Microfluidic technology is an effective tool for preparing uniform, continuous, controllable microfibers, and particularly in combination with 3D printing technology, provides a more effective choice for highly ordered customized 3D structural fibrous textiles. Furthermore, poly (3-hydroxybutyrate-4-hydroxybutyrate) is of increasing interest in tissue engineering due to its natural biocompatibility, flexible mechanical and degradation properties. More importantly, the degradation product 3-hydroxybutyric acid is one of the basic metabolic energy substances of the human body and can provide basic nutrients for cell growth and tissue regeneration. However, there are few reports of microfluidic 3D printed tissue engineering scaffolds based on polyhydroxyalkanoate materials for wound healing.
The invention content is as follows:
the invention aims to provide a microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate and a preparation method thereof aiming at the defects of the prior art so as to prepare a tissue engineering stent material with high mechanical strength and biological activity;
in order to achieve the technical purpose, the invention adopts the following technical scheme:
a preparation method of a microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate comprises the following steps:
s1, building a single-channel micro-fluidic chip, taking a mixed solution of polyhydroxyalkanoate and an organic solvent as an internal liquid, taking an ethanol water solution as a collection phase, and allowing the mixed solution of polyhydroxyalkanoate and the organic solvent to diffuse and volatilize in the ethanol water solution to form microfibers with uniform sizes;
s2, fixing the micro-fluidic chip built in the S1 on a nozzle of a 3D printer, presetting a 3D model pattern, starting the 3D printer, and forming a microfiber support with hierarchical porous and extracellular matrix-imitated structure under the directional movement of the nozzle;
and S3, sterilizing the microfiber scaffold prepared in the S2, and co-culturing the microfiber scaffold and active cells to form the double-sided anisotropic bionic skin scaffold loaded with the active cells.
Further, in S1, the polyhydroxy fatty acid ester is poly (3-hydroxybutyrate-4-hydroxybutyrate), and the concentration of the poly (3-hydroxybutyrate-4-hydroxybutyrate) in the mixed solution is 0.1-0.4 mg/mL; the organic solvent consists of dichloromethane and N, N-dimethylformamide; the volume ratio of dichloromethane to N, N-dimethylformamide is 3.
Further, in S1, the ethanol concentration in the collecting phase is 75-90 vol%, and the time for the microfiber solidification and formation can be adjusted by changing the ethanol concentration.
Furthermore, in S1, the diameter of the capillary tube of the microfluidic chip is 200-300 μm, and the diameter of the microfiber can be adjusted by changing the diameter of the capillary tube.
Further, in S2, the flow rate of liquid in the microfluidic chip is controlled to be 1-5 mL/h, the moving speed of the nozzle of the 3D printer is controlled to be 3-10 mm/S, and the size of the microfiber support can be adjusted by changing the flow rate of the liquid and the moving speed of the nozzle.
Further, in S2, the 3D model pattern may be designed according to the shape of the skin wound, specifically, a circular shape, a square shape, a spider web shape, a triangular shape, or an irregular shape.
Further, in S3, the active cells are one or more of fibroblasts, umbilical vein endothelial cells, and bone marrow mesenchymal stem cells.
Further, in S3, the microfiber scaffold is sterilized by one or more of ethylene oxide sterilization, ultraviolet radiation sterilization, or ethanol immersion sterilization.
Further, in S3, after the micro fiber scaffold is subjected to sterilization treatment, the micro fiber scaffold and active cells are subjected to co-culture for 24-72 hours to form the double-sided anisotropic bionic skin scaffold loaded with the active cells.
The invention also provides a microfluidic 3D printing bionic skin stent based on the polyhydroxyalkanoate, which is prepared by adopting the preparation method.
The invention has the beneficial effects that:
1) Compared with the traditional melt extrusion 3D printing technology, the invention provides a simple method for preparing the bionic scaffold by microfluidic 3D printing, a reticular three-dimensional structure with fibers tightly adhered to each other is formed by diffusion and volatilization of an organic solvent in an ethanol water solution, the requirement on reaction conditions is low, high temperature or high pressure is not required, and the preparation process is green and easy to implement;
2) The microfiber scaffold prepared by the invention has hierarchical porous and extracellular matrix-imitated structures, and the shape and size of the scaffold can be easily adjusted by changing preparation conditions and software setting;
3) The microfiber scaffold prepared by the invention adopts a polyhydroxyalkanoate material, has excellent mechanical properties and biocompatibility, and can effectively promote adhesion and proliferation of active cells.
Description of the drawings:
fig. 1 is a schematic diagram of a process for preparing a microfluidic 3D printed microfiber scaffold according to the present invention;
FIG. 2 is a graph of the relationship between the liquid flow rate inside the microfluidic chip and the microfiber diameter;
FIG. 3 is an optical, general and cross-sectional electron micrographs of microfibers and scaffolds prepared according to example 1, wherein FIG. a is an optical, general and cross-sectional electron micrograph, respectively, and FIG. b is an optical, general and enlarged electron micrograph, respectively, of a hierarchical porous structure scaffold;
FIG. 4 is an optical view of differently shaped stents customized to the shape of a skin wound;
FIG. 5 is a graph representing the flexibility of a microfiber scaffold prepared in example 1;
FIG. 6 is a fluorescent staining pattern of co-culture of the microfiber scaffold prepared in example 1 with living cells, wherein a is a fluorescent staining pattern of proliferation of living cells in the absence of the scaffold, and b is a fluorescent staining pattern of adhesion and proliferation of living cells on the surface of the scaffold;
FIG. 7 is a cell viability diagram of co-culture of the microfiber scaffold prepared in example 1 with viable cells.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention provides a preparation method of a microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate, which comprises the following steps:
s1, building a single-channel microfluidic chip, wherein the diameter of a capillary tube of the microfluidic chip is 200-300 mu m; the method comprises the following steps of taking a mixed solution of polyhydroxyalkanoate and an organic solvent as an internal liquid, taking an ethanol water solution as a collection phase, enabling the mixed solution of polyhydroxyalkanoate and the organic solvent to be diffused and volatilized in the ethanol water solution to form microfibers with uniform sizes, and enabling the diameters of the microfibers to be adjustable by changing the pipe diameters of tips of capillary glass tubes; the polyhydroxy fatty acid ester is poly (3-hydroxybutyrate-4-hydroxybutyrate), and the concentration of the poly (3-hydroxybutyrate-4-hydroxybutyrate) in the mixed solution is 0.1-0.4 mg/mL; the organic solvent consists of dichloromethane and N, N-dimethylformamide; the volume ratio of the dichloromethane to the N, N-dimethylformamide is 3; and in the collecting phase, the ethanol concentration is 75-90 vol%, and the time for curing and forming the microfibers can be adjusted by changing the ethanol concentration.
S2, fixing the micro-fluidic chip built in the S1 on a nozzle of a 3D printer, presetting a 3D model pattern, starting the 3D printer according to the shape design of a skin wound, and forming a microfiber bracket with hierarchical porous and extracellular matrix-simulated structure under the directional movement of the nozzle; the flow rate of liquid in the micro-fluidic chip is controlled to be 1-5 mL/h, the moving speed of the nozzle of the 3D printer is controlled to be 3-10 mm/s, and the size of the microfiber support can be adjusted by changing the flow rate of the liquid and the moving speed of the nozzle.
S3, sterilizing the microfiber scaffold prepared in the S2, and co-culturing the microfiber scaffold and active cells for 24-72 hours to form a double-sided anisotropic bionic skin scaffold loaded with the active cells; the active cells are one or more of fibroblasts, umbilical vein endothelial cells and bone marrow mesenchymal stem cells; the sterilization mode of the microfiber scaffold is one or more of ethylene oxide sterilization, ultraviolet radiation sterilization or ethanol soaking sterilization.
Example 1
The embodiment provides a preparation method of a microfluidic 3D printing bionic skin scaffold based on polyhydroxyalkanoate, the preparation flow is shown in fig. 1, and the preparation method specifically comprises the following steps:
(1) And (3) constructing a single-channel microfluidic chip, gradually lengthening the tip of the glass capillary tube with the original tube diameter of 580 mu m and subjected to hydrophilic and hydrophobic treatment, grinding the tip to the tube diameter of 300 mu m, and fixing the tip on a glass slide by using epoxy resin.
(2) Dissolving 0.5mg of poly (3-hydroxybutyrate-4-hydroxybutyrate) powder in 2.5mL of a mixed organic solvent of dichloromethane and N, N-dimethylformamide (v 1/v2= 5/5), magnetically stirring overnight at 1200rpm under a dark condition, and taking the obtained mixed liquid as an internal liquid; an aqueous ethanol solution (75 vol% concentration) was selected as the collection phase.
(3) A micro-injection pump is used for uniformly pumping a mixed liquid of poly (3-hydroxybutyrate-4-hydroxybutyrate) and an organic solvent into a micro-fluidic chip, a glass capillary extends below the liquid level of a collecting phase, and the micro-fiber with uniform size is formed by rapid diffusion and volatilization of a dichloromethane and N, N-dimethylformamide mixed organic solvent in an ethanol water solution, and FIG. 2 is a curve graph of the relation between the liquid flow rate and the micro-fiber diameter in the micro-fluidic chip.
(4) And the microfluidic chip is fixed on a nozzle of the 3D printer, and a simple and convenient microfluidic 3D printing device is built. The 3D model pattern is preset to be square, the 3D printer is started, the flow rate of liquid in the microfluidic chip is set to be 1.5mL/h, the moving speed of a nozzle of the 3D printer is set to be 5mm/s, the printing time is set to be 20 minutes, and the printing filling rate is set to be 80%. The microfiber scaffold with hierarchical porosity and a simulated extracellular matrix structure is formed under the directional movement of the nozzle.
The shape and size of the microfiber support can be adjusted by varying the liquid flow rate and the nozzle travel speed, and the height of the microfiber support can be adjusted by varying the printing time.
As shown in fig. 3, fig. 3 is a light microscopic image and an electron microscopic image of the prepared microfibers and the scaffold, wherein a is a light microscopic image, a general electron microscopic image and a cross-sectional electron microscopic image of the microfibers, respectively, and b is a light microscopic image, a general electron microscopic image and an enlarged electron microscopic image of the scaffold having a hierarchical porous structure, respectively.
The resulting microfiber scaffold has excellent flexibility, as shown in fig. 5, and can be stretched, twisted, folded, and rapidly restored to an original shape after an external force is removed.
(5) The above-prepared microfiber scaffold was immersed in 75vol% ethanol aqueous solution and irradiated with ultraviolet rays for 2 hours for sterilization, followed by washing three times with PBS and pre-culturing by immersing it in a cell complete medium. Then the scaffolds were placed on the bottom of a 24-well plate, and the cells were added to the plate at a cell density of 10 5 The umbilical vein endothelial cells and the bone marrow mesenchymal stem cell suspension are cultured together for 24 hours to form a bracket loaded with active cells, and the double-sided anisotropic bionic skin bracket is formed through self-assembly of the bracket.
Cells were then fluorescently stained using F-actin/DAPI stain and visualized by confocal laser microscopy. FIG. 6 is a fluorescent staining pattern of co-culture of the microfiber scaffold and the living cells, wherein a is a fluorescent staining pattern of the living cells proliferating without the scaffold, and b is a fluorescent staining pattern of the living cells adhering to and proliferating on the surface of the scaffold. FIG. 7 is a diagram of cell viability in co-culture of the microfiber scaffold with viable cells, in which the cell density of the control group at days 1, 3, and 5 was 1.00, 4.04, and 6.51 times the initial density (initial density of cells was 10) 5 /mL), fineness of the stent groupThe cell density is 1.37, 5.19 and 7.00 times of the initial density respectively.
Through detection, the mechanical strength of the bionic skin stent prepared by the embodiment can reach 2.78MPa, and the bionic skin stent has high mechanical strength and good biological activity, and meets the requirements of materials for wound healing.
Example 2
The embodiment provides a preparation method of a microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate, which specifically comprises the following steps:
(1) And (3) building a single-channel microfluidic chip, gradually elongating the tip of the glass capillary tube with the original tube diameter of 580 mu m and subjected to hydrophilic and hydrophobic treatment, polishing the tip to the tube diameter of 200 mu m, and fixing the tip on a glass slide by using epoxy resin.
(2) Dissolving 0.25mg of poly (3-hydroxybutyrate-4-hydroxybutyrate) powder and 0.25mg of polycaprolactone powder in 2.0mL of a mixed organic solvent of dichloromethane and N, N-dimethylformamide (v 1/v2= 7/3), magnetically stirring at 1000rpm under a dark condition overnight, and taking the obtained mixed liquid as an internal liquid; an aqueous ethanol solution (90 vol% concentration) was selected as the collection phase.
(3) The mixed liquid is uniformly pumped into the microfluidic chip by a micro-injection pump, the glass capillary extends below the liquid level of the collection phase, and the microfiber with uniform size is formed by rapid diffusion and volatilization of the organic solvent in the ethanol water solution.
(4) And the microfluidic chip is fixed on a nozzle of the 3D printer, and the simple and convenient microfluidic 3D printing device is built. The 3D model pattern is preset to be circular, the 3D printer is started, the flow rate of liquid in the microfluidic chip is set to be 3mL/h, the moving speed of a nozzle of the 3D printer is set to be 10mm/s, the printing time is set to be 10 minutes, and the printing filling rate is set to be 60%. The microfiber scaffold with hierarchical porosity and a simulated extracellular matrix structure is formed under the directional movement of the nozzle.
(5) The above-prepared microfiber scaffold was immersed in 90vol% ethanol aqueous solution and irradiated with ethylene oxide for sterilization, followed by washing three times with PBS and immersing in a cell complete mediumAnd (4) pre-culturing. Then the scaffolds were placed on the bottom of a 24-well plate, and the cells were added at a cell density of 10 4 And (3) culturing the suspension of the fibroblast and umbilical vein endothelial cell suspension for 48 hours to form a scaffold loaded with active cells, and self-assembling the scaffold to form the double-sided anisotropic bionic skin scaffold. Cells were then fluorescently stained using F-actin/DAPI stain and visualized by confocal laser microscopy.
Through detection, the bionic skin stent prepared by the embodiment has the mechanical strength of 2.65kPa and good biological activity, and meets the requirements of materials for wound healing.
Example 3
The embodiment provides a preparation method of a microfluidic 3D printing bionic skin scaffold based on polyhydroxyalkanoate, which specifically comprises the following steps:
(1) And (3) building a single-channel microfluidic chip, gradually elongating the tip of the glass capillary tube with the original tube diameter of 580 mu m and subjected to hydrophilic and hydrophobic treatment, polishing the tip to the tube diameter of 150 mu m, and fixing the tip on a glass slide by using epoxy resin.
(2) Dissolving 0.5mg of polycaprolactone powder in 4.0mL of a mixed organic solvent of dichloromethane and N, N-dimethylformamide (v 1/v2= 3/7), and magnetically stirring at 1500rpm under a dark condition overnight to obtain a mixed liquid as an internal liquid; an aqueous ethanol solution (50 vol% concentration) was selected as the collection phase.
(3) The mixed liquid is uniformly pumped into the microfluidic chip by a micro-injection pump, the glass capillary extends below the liquid level of the collection phase, and the micro fiber with uniform size is formed by rapid diffusion and volatilization of the organic solvent in the ethanol water solution.
(4) And the microfluidic chip is fixed on a nozzle of the 3D printer, and a simple and convenient microfluidic 3D printing device is built. Presetting a 3D model pattern to be a triangle, starting a 3D printer, setting the flow rate of liquid in the microfluidic chip to be 0.6mL/h, setting the moving speed of a nozzle of the 3D printer to be 3mm/s, setting the printing time to be 30 minutes, and setting the printing filling rate to be 70%. The microfiber scaffold with hierarchical porosity and a simulated extracellular matrix structure is formed under the directional movement of the nozzle.
(5) The above-prepared microfiber scaffold was immersed in a 50vol% ethanol aqueous solution and irradiated with ultraviolet rays for sterilization, followed by washing three times with PBS and pre-culturing by immersing it in a cell complete medium. Then the scaffolds were placed on the bottom of a 24-well plate, and the cells were added to the plate at a cell density of 10 3 The preparation method comprises the following steps of co-culturing a fibroblast suspension and a bone marrow mesenchymal stem cell suspension for 72 hours to form a scaffold loaded with active cells, and forming the double-sided anisotropic bionic skin scaffold through self-assembly of the scaffold. Cells were then fluorescently stained using F-actin/DAPI stain and visualized by confocal laser microscopy.
Through detection, the bionic skin stent prepared by the embodiment has the mechanical strength of 2.71kPa and good biological activity, and meets the requirements of materials for wound healing.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention, it should be noted that, for those skilled in the art, several modifications and decorations without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims (9)

1. A preparation method of a microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate is characterized by comprising the following steps:
s1, building a single-channel micro-fluidic chip, taking a mixed solution of polyhydroxyalkanoate and an organic solvent as an internal liquid, taking an ethanol water solution as a collection phase, and allowing the mixed solution of polyhydroxyalkanoate and the organic solvent to diffuse and volatilize in the ethanol water solution to form microfibers with uniform sizes;
s2, fixing the micro-fluidic chip built in the S1 on a nozzle of a 3D printer, presetting a 3D model pattern, starting the 3D printer, and forming a microfiber support with hierarchical porous and extracellular matrix-imitated structure under the directional movement of the nozzle;
s3, sterilizing the microfiber scaffold prepared in the S2, and co-culturing the microfiber scaffold and active cells to form a double-sided anisotropic bionic skin scaffold loaded with the active cells;
in S1, the polyhydroxy fatty acid ester is poly (3-hydroxybutyrate-4-hydroxybutyrate), and the concentration of the poly (3-hydroxybutyrate-4-hydroxybutyrate) in the mixed solution is 0.1-0.4 mg/mL; the organic solvent consists of dichloromethane and N, N-dimethylformamide, and the volume ratio of the dichloromethane to the N, N-dimethylformamide is 3.
2. The preparation method of the polyhydroxyalkanoate-based microfluidic 3D printing bionic skin stent according to claim 1, wherein in S1, the ethanol concentration in the collection phase is 75-90 vol%, and the time for microfiber solidification and formation can be adjusted by changing the ethanol concentration.
3. The preparation method of the microfluidic 3D printing bionic skin scaffold based on the polyhydroxyalkanoate of claim 1, wherein in S1, the diameter of a capillary tube of a microfluidic chip is 200-300 μm, and the diameter of the microfiber can be adjusted by changing the diameter of the capillary tube.
4. The preparation method of the microfluidic 3D printing bionic skin scaffold based on the polyhydroxyalkanoate of claim 1, wherein in S2, the flow rate of liquid in the microfluidic chip is controlled to be 1-5 mL/h, the moving speed of a nozzle of a 3D printer is controlled to be 3-10 mm/S, and the size of the microfiber scaffold can be adjusted by changing the flow rate of the liquid and the moving speed of the nozzle.
5. The method for preparing the polyhydroxyalkanoate-based microfluidic 3D printing bionic skin scaffold, according to claim 1, wherein in S2, the 3D model pattern can be designed according to the shape of a skin wound, specifically, a circle, a square, a spider web, a triangle or an irregular shape.
6. The preparation method of the polyhydroxyalkanoate-based microfluidic 3D printing biomimetic skin scaffold according to claim 1, wherein in S3, the active cells are one or more of fibroblasts, umbilical vein endothelial cells and bone marrow mesenchymal stem cells.
7. The preparation method of the microfluidic 3D printing bionic skin scaffold based on the polyhydroxyalkanoate of claim 1, wherein in S3, the sterilization mode of the microfiber scaffold is one or more of ethylene oxide sterilization, ultraviolet radiation sterilization or ethanol soaking sterilization.
8. The preparation method of the microfluidic 3D printing bionic skin scaffold based on the polyhydroxyalkanoate of claim 1, wherein in S3, after the microfiber scaffold is subjected to sterilization treatment, the microfiber scaffold and active cells are subjected to co-culture for 24-72 hours to form a double-sided anisotropic bionic skin scaffold loaded with the active cells.
9. A microfluidic 3D printing bionic skin scaffold based on polyhydroxyalkanoate, which is characterized by being prepared by the preparation method of any one of claims 1-8.
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