Biodegradable film having three-dimensional ordered porous structure, and preparation method therefor and use thereof

Abstract

The present invention provides a biodegradable film having a three-dimensional ordered porous structure, and a preparation method therefor and a use thereof. The biodegradable film has good mechanical properties, and an upper surface and a lower surface of the biodegradable film can be provided with the same or different ordered porous micro-nano structures, so that repair of the same or different tissues is guided. Moreover, degradation time of a corresponding structural layer can be regulated and controlled by means of structural difference and material selection, so as to adapt to repair periods of different tissues, thereby improving the repair effect. Further, the configuration of an intermediate disordered fiber layer endows the selective passability of a tissue engineering scaffold, and ensures that a first ordered layer and a second ordered layer are repaired without mutual interference, thereby expanding the application range of the tissue engineering scaffold. The preparation method of the present invention employs a near-field direct-writing 3D printing method, and by regulating and controlling printing parameters of different structural layers to control the structure of each layer, a product is endowed with expected performance. The preparation method is simple, convenient to operate, and easy to implement.

Description

A biodegradable membrane with three-dimensional ordered porous structure and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of medical tissue engineering, and in particular relates to a biodegradable membrane with a three-dimensional ordered porous structure, and a preparation method and application thereof. Background Art

[0002] Tissue engineering is a recently emerging discipline that primarily applies the principles and techniques of life science and engineering to research and develop biological substitutes for the repair, maintenance, and improvement of the function and morphology of damaged tissues and organs. As the effectiveness of tissue engineering technology continues to be validated through research and application, its application in repairing damaged tissues is attracting increasing attention.

[0003] An ideal tissue engineering scaffold should not only have excellent biocompatibility, but also have a three-dimensional structure similar to the extracellular matrix of different tissues in the human body, so as to facilitate cell adhesion and guide the directional growth of cells, thereby better promoting autologous tissue regeneration.

[0004] Tissue engineering scaffolds manufactured by existing traditional technologies such as electrospinning and freeze-drying cannot form an ordered three-dimensional porous structure, and thus cannot effectively promote the adhesion and growth of cells on their surface. Near-field direct writing 3D printing is a unique technology that can complete the manufacture of ordered structures. Based on its advantage of being able to prepare ordered three-dimensional structures, it has been widely used in the field of tissue engineering. However, the tissue engineering scaffolds currently manufactured by near-field direct writing on the market are all aimed at homogeneity, and are mostly continuously stacked disordered structures or have only one side as an ordered structure. Such structures are all aimed at homogeneity and symmetry, and cannot achieve synchronous repair of different locations of tissues in the body. For example, when performing guided bone regeneration surgery in the mouth, there is a problem that the gums cannot close. Either tension-reducing sutures or autologous gum transplants are used for repair, but these two methods are prone to cause postoperative pain and discomfort, increase the risk of postoperative infection, and are not aesthetically pleasing.

[0005] Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a biodegradable membrane with a three-dimensional ordered porous structure and a preparation method thereof. The biodegradable membrane has good mechanical properties, and the upper and lower surfaces thereof can construct the same or different ordered micro-nano structures, thereby guiding the repair of the same or different tissues; at the same time, the degradation cycle, mechanical properties, etc. of the corresponding structural layer can be regulated by structural differences and material selection to adapt to the repair cycle and mechanical requirements of different tissue types, thereby improving the repair effect; in addition, the provision of an intermediate disordered fiber layer not only gives the tissue engineering scaffold selective permeability but also ensures that the repair of the first ordered layer and the second ordered layer does not interfere with each other, thereby expanding the scope of application of the tissue engineering scaffold. The preparation method of the present invention adopts a near-field direct writing 3D printing method, and controls the structure of each layer by regulating the printing parameters of different structural layers to give the product the expected performance; the preparation method is simple, easy to operate, and easy to implement.

[0007] A biodegradable membrane with a three-dimensional ordered porous structure, comprising a middle disordered fiber layer formed by disorderly stacking spiral fibers, and a first ordered fiber layer and a second ordered fiber layer arranged on both sides of the middle disordered fiber layer, each independently formed by stacking a single layer or multiple layers of straight fibers and having an ordered structure;

[0008] The material of the first ordered fiber layer, the material of the middle disordered fiber layer, and the material of the second ordered fiber layer are each independently selected from biodegradable materials.

[0009] The first ordered fiber layer and the second ordered fiber layer are each independently formed by stacking a single layer or multiple layers of straight fibers. The first ordered fiber layer and the second ordered fiber layer can have the same structure or different structures as needed. The ordered structure can be a regular grid structure, and the grid can be square, diamond, triangular, etc.

[0010] Preferably, the biodegradable material is a copolymer or mixture of one or more of polylactic acid, polyglycolic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polyethylene glycol, polyurethane, polyhydroxybutyrate, polyhydroxyvalerate, polydioxanone, polytrimethylene carbonate, polybutylene succinate, chitosan and derivatives, carboxymethyl cellulose, gelatin, hyaluronic acid, collagen, alginate, gum, whey protein and derivatives thereof.

[0011] Preferably, the fiber spacing of the first ordered fiber layer and the second ordered fiber layer is independently selected from 1 to 500 μm.

[0012] Preferably, the diameter of the fiber is 0.1 to 200 μm.

[0013] As further preferred, the diameters of the first ordered fibrous layer and the second ordered fibrous layer are each independently selected from 2 to 50 μm.

[0014] As a further preference, the diameter of the middle random fiber layer is 1 to 20 μm.

[0015] A method for preparing a biodegradable membrane having a three-dimensional ordered porous structure as described above, wherein the first ordered fiber layer and the second ordered fiber layer are printed using a near-field direct writing 3D printing process with the following parameters: a nozzle-substrate distance of 0.5 to 5 mm, an extrusion pressure of 0.1 kPa to 1 MPa, and a voltage of 1 to 6 kV;

[0016] The following parameters were used to print the middle disordered fiber layer: nozzle-substrate distance of 1 to 50 mm, extrusion pressure of 0.5 to 50 kPa, and voltage of 1 to 30 kV.

[0017] The preparation method of the present invention utilizes a near-field direct writing 3D printing process to overcome the surface tension of the material by controlling printing parameters (such as nozzle-substrate distance, extrusion pressure, voltage, etc.) to produce a Taylor cone and a "catenary model" effect, thereby forming controllable straight fibers and printing a set ordered fiber structure; at the same time, by controlling the temperature of the printing platform, the crossing, adhesion, and stacking of the straight fibers are achieved, further improving the uniformity and stacking stability of the ordered structure.

[0018] When printing the disordered fiber layer, by controlling the printing parameters (such as nozzle-substrate distance, extrusion pressure, voltage, etc.), a "catenary model" effect is achieved during printing, so that curved spiral fibers are formed during the printing process, cross-stacked, and a dense disordered barrier structure, namely the middle disordered fiber layer; at the same time, by controlling the temperature of the printing platform and attracting the ordered fibers at the bottom, the density of the disordered fiber layer is improved, the size of its micropores and the porosity of the membrane are reduced, and the selective permeability of the middle disordered fiber layer is achieved (it can block the passage of cells while allowing small molecules such as gas, tissue fluid, and cytokines to pass through). Compared with the ordered fiber layer, the middle disordered fiber layer is disorderly stacked with spiral fibers, with smaller pores, finer fiber diameters, and denser arrangements.

[0019] Preferably, when printing the first ordered fiber layer and the second ordered fiber layer, the nozzle-substrate distance is 1-3 mm, the extrusion pressure is 0.1-50 KPa, and the voltage is 2-5 KV.

[0020] Preferably, when printing the middle disordered fiber layer, the nozzle-substrate distance is 5 to 25 mm, the extrusion pressure is 1 to 20 KPa, and the voltage value is 8 to 20 KV.

[0021] As a preference, a continuous printing method is used for printing:

[0022] The first ordered fiber layer, the middle disordered fiber layer and the second ordered fiber layer are printed in sequence on the substrate to obtain the biodegradable membrane with a three-dimensional ordered porous structure.

[0023] Specifically, a near-field direct writing 3D printing process is used to print an ordered structure on a smooth substrate. By overcoming the "catenary model" effect (adjusting the printing process parameters), controllable straight fibers are formed, and the spatial arrangement of the fibers is regulated to form a straight ordered structure (first ordered fiber layer); using the first ordered fiber layer as the substrate, utilizing the "catenary model" effect (achieved by adjusting the printing process parameters), the fibers are changed into curved spirals during the printing process, and are disorderly cross-stacked on the first ordered fiber layer to form a dense barrier layer, i.e., the middle disordered fiber layer; and then the ordered-disordered (first ordered fiber layer + middle disordered fiber layer) obtained above is used as the base to print an ordered structure. By overcoming the "catenary model" effect (adjusting the printing process parameters), the spatial arrangement of the fibers is regulated to form a straight ordered structure, and a second ordered fiber layer is obtained on the middle disordered fiber layer, thereby obtaining the biodegradable membrane with a three-dimensional ordered porous structure.

[0024] As a preferred method, thermal printing is performed, and the specific steps are as follows:

[0025] printing a disordered spiral fiber layer and a first ordered fiber layer in sequence on a substrate to obtain a first composite layer;

[0026] printing an intermediate disordered fiber layer and a second ordered fiber layer on another substrate in sequence to obtain a second composite layer;

[0027] Heat-sealing the disordered spiral fiber layer of the first composite layer and the middle disordered fiber layer of the second composite layer to obtain the biodegradable film having a three-dimensional ordered porous structure;

[0028] When printing the disordered spiral fiber layer, the nozzle-substrate distance is controlled to be 1 to 50 mm, the extrusion pressure is 0.5 to 50 KPa, and the voltage value is 1 to 30 KV.

[0029] As a further preference, when printing the disordered spiral fiber layer, the nozzle-substrate distance is 5 to 25 mm, the extrusion pressure is 1 to 20 KPa, and the voltage value is 8 to 20 KV.

[0030] Preferably, the heat-sealing temperature is 0.2 to 0.9 times the melting point of the material with the lower melting point in the middle random fiber layer material and the random spiral fiber layer material, more preferably 0.4 to 0.9 times.

[0031] Preferably, during the heat-sealing process, a certain weight may be placed on top of the first composite layer and the second composite layer to ensure tight bonding.

[0032] Preferably, the fiber diameter of the disordered spiral fiber layer is 0.01 to 1 times the fiber diameter of the first ordered fiber layer, and the fiber path is the same as that of the first ordered fiber layer.

[0033] The disordered helical fiber layer is obtained by disorderly stacking one or more layers of helical fibers.

[0034] Preferably, the roughness R of the substrate is a ≤1.6μm. For the continuous printing method, when the bottom surface of the ordered layer contacts the substrate, it is easier to adhere to the smooth substrate. The roughness R a The substrate thickness of ≤1.6μm further ensures the flatness and stacking of the ordered layer at the bottom. For thermal bonding, printing the disordered layer onto the substrate first also makes the bottom surface of the disordered layer smooth. The spiral fibers will spread out, increasing the bottom area and facilitating thermal bonding.

[0035] Preferably, the substrate is selected from one of glass plate, single crystal silicon wafer, polytetrafluoroethylene, nylon, polyurethane and the like.

[0036] Preferably, during printing, the substrate temperature is controlled to be 0.3 to 0.9 times the melting point of the current printing material. Controlling the substrate temperature close to the melting point of the material slows down the cooling speed of the fiber, allowing the fiber to be fixed on the substrate.

[0037] The preparation method of the present invention can customize scaffolds with different three-dimensional ordered structural surfaces according to the requirements of different tissues for the scaffold, thereby better promoting the repair of different tissues.

[0038] The present invention also discloses the use of any of the above-mentioned biodegradable membranes with a three-dimensional ordered porous structure as a tissue engineering scaffold.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The tissue engineering scaffold constructed in this invention possesses a finely ordered three-dimensional surface structure. With adjustable specific surface area and porosity, it effectively promotes cell adhesion, growth, and migration, while also facilitating the exchange of nutrients and metabolic products around the tissue. Furthermore, the upper and lower surfaces (the first and second ordered fiber layers) can be designed with different structures depending on the type of tissue they contact and the needs of clinical application.

[0041] 2. The tissue engineering scaffold constructed in the present invention has a multilayer structure, and its in vivo degradation time can be adjusted by changing the composition of the polymer material. At the same time, different three-dimensional porous structures can change the specific surface area and also affect the degradation time of the material. Therefore, the degradation time of the scaffold can be regulated according to different application scenarios.

[0042] 3. The present invention adopts a near-field direct writing process based on 3D printing, which realizes the printing of ordered straight fiber layers and disordered spiral fiber layers by adjusting the printing process parameters of different layers; and this printing method is an efficient micro-nanoscale additive manufacturing method with zero additives in the preparation process, and does not introduce organic solvents and chemical cross-linking agents.

[0043] 4. The printing process adopted by the present invention is simple and does not require complicated post-processing processes, which can greatly improve production efficiency and reduce production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG1 is a photo of the biodegradable film obtained in Example 1;

[0045] FIG2 is a scanning electron microscope image of the biodegradable membrane obtained in Example 1 taken from the first ordered fiber layer;

[0046] FIG3 is a scanning electron microscope image of the biodegradable membrane obtained in Example 1 taken from the second ordered fiber layer;

[0047] FIG4 shows: (a) a photograph of the second composite layer prepared in Example 2; (b) a photograph of the first composite layer prepared in Example 2; and (c) a photograph of the biodegradable film prepared in Example 2.

[0048] 5 : (A) is a micrograph of the second composite layer prepared in Example 2; (B) is a micrograph of the first composite layer prepared in Example 2; (C) is a micrograph of the biodegradable film prepared in Example 2;

[0049] Figure 6 shows the experimental results of cells cultured on disordered membranes;

[0050] FIG. 7 shows the experimental results of culturing cells using the biodegradable membrane prepared in Example 1. DETAILED DESCRIPTION

[0051] Taking the preparation of a 10*10 mm diaphragm as an example, the design concept and technical solution of the present invention are further illustrated.

[0052] Implementation 1: Direct stacking printing (continuous printing method)

[0053] Specific methods:

[0054] (1) The material used for this printing is polycaprolactone (PCL). The required patterns of the first ordered fiber layer and the second ordered fiber layer are produced using CAD, which is then exported and converted into Gcode files. The generated Gcode files are then imported into the 3D printing software to generate the printing path.

[0055] (2) According to the printing path generated in step (1), a smooth glass plate is used as the substrate, and a linear grid structure with a spacing of 100 μm and a fiber diameter of 5-10 μm is first printed on the substrate to obtain the first ordered fiber layer; the printing process parameters for printing this layer are: substrate temperature of 33°C, substrate-nozzle distance of 2.5 mm, nozzle diameter of 350 μm, voltage of 4.7 kV, barrel heating of 90°C, needle heating temperature of 120°C, printing speed of 1200 mm / min, air pressure of 1 KPa, and distance compensation of 5 μm per layer.

[0056] (3) On the basis of the first ordered fiber layer, a spiral fiber disordered layer with a pore size of less than 18 μm and a diameter of 5-9 μm was printed to obtain an intermediate disordered fiber layer on the first ordered fiber layer; the printing process parameters for printing this layer were: substrate-nozzle distance 10 mm, nozzle diameter 350 μm, voltage 12 kV, barrel heating temperature 90 °C, needle heating temperature 120 °C, printing speed 550 mm / min, and air pressure 2 kPa.

[0057] (4) Finally, a second ordered fiber layer is printed on the middle disordered fiber layer to obtain a biodegradable membrane with a three-dimensional ordered porous structure; the second ordered fiber layer is a straight square grid structure with a fiber spacing of 200 μm and a fiber diameter of 15-20 μm; the printing process parameters for printing this layer are: substrate-nozzle distance 2.6 mm, nozzle diameter 350 μm, voltage 4.7 kV, barrel heating temperature 90 ° C, needle heating temperature 120 ° C, printing speed 1200 mm / min, air pressure 5 kPa, and distance compensation of 10 μm per layer.

[0058] The photos and scanning electron microscope images of the biodegradable film prepared in this example are shown in Figures 1 to 3.

[0059] Example 2: Thermal Legal Printing

[0060] Specific methods:

[0061] (1) The material used for this printing is polycaprolactone. The patterns required for the first ordered fiber layer and the second ordered fiber layer are made using CAD, which is then exported and converted into Gcode files. The generated Gcode files are then imported into the 3D printing software to generate the printing path.

[0062] (2) First, a spiral disordered layer with pores less than 18 μm and a diameter of 5-9 μm was printed on a smooth glass plate to obtain a middle disordered fiber layer; the printing process parameters for printing this layer were: substrate temperature of 33 °C, substrate-nozzle distance of 10 mm, nozzle diameter of 350 μm, voltage of 12 kV, barrel heating of 90 °C, needle heating temperature of 120 °C, printing speed of 550 mm / min, and air pressure of 2 kPa.

[0063] (3) Printing a straight square grid structure with a gap of 100 μm and a fiber diameter of 5-10 μm (second ordered fiber layer) on the intermediate disordered fiber layer obtained in step (2) to obtain a second composite layer; the printing process parameters for printing the intermediate disordered fiber layer are: substrate-nozzle distance 2.5 mm, nozzle diameter 350 μm, voltage 4.7 kV, barrel heating temperature 90°C, needle heating temperature 120°C, printing speed 1200 mm / min, air pressure 1 KPa, and distance compensation 5 μm per layer.

[0064] (4) Print a spiral disordered layer with a pore size of about 150 μm and a diameter of 2-6 μm on another smooth glass plate to obtain a disordered spiral fiber layer; the printing process parameters are: substrate temperature of 33 °C, substrate-nozzle distance of 10 mm, nozzle diameter of 350 μm, voltage of 12 kV, barrel heating of 90 °C, needle heating temperature of 120 °C, printing speed of 1000 mm / min, and air pressure of 1 kPa.

[0065] (5) Printing a linear square grid structure with a fiber spacing of 200 μm and a fiber diameter of 15-20 μm (i.e., the first ordered fiber layer) on the disordered spiral fiber layer obtained in step (4) to obtain a first composite layer; the printing process parameters are: substrate-nozzle distance 2.6 mm, nozzle diameter 350 μm, voltage 4.7 kV, barrel heating temperature 90 ° C, needle heating temperature 120 ° C, printing speed 1200 mm / min, air pressure 5 kPa, and distance compensation of 10 μm per layer.

[0066] (6) The first composite layer and the second composite layer printed in steps (3) and (5) are removed and placed in a vacuum drying oven with the disordered spiral fiber layer and the middle disordered fiber layer facing each other. The layers are heated at 52° C. for 15 minutes and heat-sealed to obtain a biodegradable film having a three-dimensional ordered porous structure. During the heat-sealing process, a glass slide is pressed against the first composite layer and the second composite layer.

[0067] The photographs and micrographs of the first composite layer, the second composite layer and the biodegradable film prepared in this example are shown in Figures 4 and 5 .

[0068] Product performance testing

[0069] To evaluate the adhesion and oriented growth of gingival fibroblasts (HGF-1) on disordered and ordered PCL membrane surfaces.

[0070] Preparation of disordered films:

[0071] A disordered layer of spiral fibers with pores less than 18 μm and a diameter of 5-9 μm was printed on a smooth glass plate to prepare a disordered film. The printing process parameters were: substrate-nozzle distance 10 mm, nozzle diameter 350 μm, voltage 12 kV, barrel heating temperature 90°C, needle heating temperature 120°C, printing speed 550 mm / min, and air pressure 2 kPa.

[0072] An HGF-1 cell suspension was prepared and inoculated onto the surface of the disordered membrane prepared above and the biodegradable membrane obtained in Example 1. After 24 hours of incubation, the cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.5% Triton X-100. The cells were then stained for cytoskeleton using a working solution of Phalloidin (Cytoshelle Inc., USA) for 40 minutes in the dark, followed by three washes with PBS. The nuclei were then stained with a working solution of DAPI (Beyotime, China) for 5 minutes, followed by three washes with PBS.

[0073] Confocal laser scanning microscopy (CLSM, Leica Microsystems, Germany) was used to observe cell growth on the disordered and biodegradable membrane surfaces, and the results are shown in Figures 6 and 7, respectively. As shown in Figures 6 and 7, HGF-1 cells grew in a scattered manner on the disordered PCL membrane (disordered membrane) surface, but were able to grow in a directional manner along the fibers on the ordered layer (biodegradable membrane) surface. Furthermore, the number of cells adhering to the membrane surface was greater than that on the disordered PCL membrane surface within the same field of view, indicating that the ordered PCL surface facilitates the early adhesion and directional growth of HGF-1 cells compared to the disordered surface structure.

Claims

1. A biodegradable film having a three-dimensional ordered porous structure, characterized in that: It includes a middle disordered fiber layer formed by disorderly stacking spiral fibers, and a first ordered fiber layer and a second ordered fiber layer arranged on both sides of the middle disordered fiber layer, each independently formed by stacking a single layer or multiple layers of straight fibers and having an ordered structure; The material of the first ordered fiber layer, the material of the middle disordered fiber layer, and the material of the second ordered fiber layer are each independently selected from biodegradable materials.

2. The biodegradable film having a three-dimensional ordered porous structure according to claim 1, characterized in that: The biodegradable material is a copolymer or mixture of one or more of polylactic acid, polyglycolic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polyethylene glycol, polyurethane, polyhydroxybutyrate, polyhydroxyvalerate, polydioxanone, polytrimethylene carbonate, polybutylene succinate, chitosan and derivatives, carboxymethyl cellulose, gelatin, hyaluronic acid, collagen, alginate, gum, whey protein and derivatives thereof.

3. The biodegradable film having a three-dimensional ordered porous structure according to claim 1, characterized in that: The fiber spacings of the first ordered fiber layer and the second ordered fiber layer are each independently selected from 1 to 500 μm.

4. A method for preparing a biodegradable film having a three-dimensional ordered porous structure as claimed in any one of claims 1 to 3, characterized in that: The first ordered fiber layer and the second ordered fiber layer were printed using the near-field direct writing 3D printing process with the following parameters: nozzle-substrate distance of 0.5-5 mm, extrusion pressure of 0.1 KPa-1 MPa, and voltage of 1-6 KV; The following parameters were used to print the middle disordered fiber layer: nozzle-substrate distance was 1 to 50 mm, extrusion pressure was 0.5 to 50 KPa, and voltage value was 1 to 30 KV.

5. The method for preparing a biodegradable film having a three-dimensional ordered porous structure according to claim 4, characterized in that: Printing using the continuous printing method: The first ordered fiber layer, the middle disordered fiber layer and the second ordered fiber layer are printed in sequence on the substrate to obtain the biodegradable film with a three-dimensional ordered porous structure.

6. The method for preparing a biodegradable film having a three-dimensional ordered porous structure according to claim 4, characterized in that: Use thermal printing, the specific steps are as follows: Printing a disordered spiral fiber layer and a first ordered fiber layer in sequence on a substrate to obtain a first composite layer; printing an intermediate disordered fiber layer and a second ordered fiber layer on another substrate in sequence to obtain a second composite layer; The disordered spiral fiber layer of the first composite layer and the middle disordered fiber layer of the second composite layer are heat-sealed to obtain the biodegradable film having a three-dimensional ordered porous structure; When printing the disordered spiral fiber layer, the nozzle-substrate distance is controlled to be 1 to 50 mm, the extrusion pressure is 0.5 to 50 KPa, and the voltage value is 1 to 30 KV.

7. The method for preparing a biodegradable film having a three-dimensional ordered porous structure according to claim 6, characterized in that: The heat-sealing temperature is 0.2 to 0.9 times the melting point of the middle random fiber layer material.

8. The method for preparing a biodegradable film having a three-dimensional ordered porous structure according to claim 6, characterized in that: The fiber diameter of the disordered spiral fiber layer is 0.01 to 1 times of the fiber diameter of the first ordered fiber layer, and its fiber path is the same as that of the first ordered fiber layer.

9. The method for preparing a biodegradable film having a three-dimensional ordered porous structure according to claim 4, characterized in that: The roughness R of the substrate a ≤1.6μm.

10. Use of the biodegradable membrane with a three-dimensional ordered porous structure according to any one of claims 1 to 3 as a tissue engineering scaffold.

Images