Preparation method of a scaffold with a surface microtopography pattern

Abstract

The present invention discloses a preparation method of a scaffold with a surface microtopography pattern. The preparation method includes the following steps: (1) Preparation of polylactic acid-based microspheres: Mix a stabilizer with water to obtain an aqueous phase; mix polylactic acid or poly(lactic acid-glycolic acid) copolymer with an organic solvent to obtain an oil phase; mix the aqueous phase and the oil phase, stir, and dry to obtain polylactic acid-based microspheres; the stabilizer includes at least one of polyvinyl alcohol, gelatin, or polysorbate; (2) Hot pressing: Hot press the polylactic acid-based microspheres to obtain a polylactic acid scaffold preform; the pressure of hot pressing is 0.01-0.5 MPa, and the temperature of hot pressing is 40-50 °C; (3) Heat-treat the polylactic acid scaffold preform to obtain a scaffold with a surface microtopography pattern; the temperature of heat treatment is 70-90 °C, and the time of heat treatment is 5-20 min. The scaffold prepared by this method has significant improvement in promoting the osteogenic differentiation performance of cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical material preparation, and particularly relates to a preparation method of a stent with a surface microtopography pattern. Background Art

[0002] Bone defects caused by traffic accidents, major diseases and other reasons cause harm to patients and a huge socio-economic burden. In severe cases, bone transplantation is required. Artificial repair materials have a wide range of sources and flexible designs, but they lack effective guidance for cell and bone regeneration. At present, composition modification endows scaffold materials with more functions, such as compounding with osteogenic active materials, doping osteogenic active ions, etc. In addition, surface physical modification has a certain stability compared with composition modification. Research shows that the surface topography of a scaffold implanted in the human body can greatly affect cell behavior and can affect cell differentiation by changing cell shape. Optimizing the surface of biomaterials by simulating the natural microenvironment can provide an ideal surface for cell response. Cells sense the changes in the microstructure of the scaffold and play a certain contact guidance role on the cells themselves. The surface micromorphology of a scaffold implanted in the human body can promote the contact between the implanted material and bone, increase the mechanical interlocking force between the material and the surrounding bone tissue, promote cell adhesion and spreading, regulate the migration and growth of bone cells, and promote osteoblast expression; the surface nanomorphology can affect protein adsorption and provide spatial and mechanical cues for cell sensing, regulate cell adhesion, proliferation and gene expression, and promote new bone formation. However, the scaffold materials in the prior art often require template imprinting and chemical etching processes during preparation, the preparation process is cumbersome, and the prepared scaffold materials have poor performance in promoting cell osteogenic differentiation.

[0003] Therefore, it is urgent to provide a new preparation method for scaffold materials. This preparation method not only does not require template imprinting (template-assisted) and chemical etching processes, simplifies the preparation method, but also the prepared scaffold has a significant improvement in promoting cell osteogenic differentiation performance, which is very necessary. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above prior art. For this purpose, the present invention provides a preparation method of a stent with a surface microtopography pattern. The preparation method not only does not require template imprinting (template-assisted) and chemical etching processes, simplifies the preparation method, but also the prepared stent has a surface with a microtopography pattern and has a significant improvement in promoting cell osteogenic differentiation performance.

[0005] Inventive concept of the present invention: The present invention utilizes an oil phase and a water phase, and prepares polylactic acid-based microspheres by the emulsion solvent evaporation method. Then, under specific hot pressing conditions (pressure of 0.01 - 0.5 MPa, temperature of 40 - 50 °C), a scaffold preform is obtained. Finally, under suitable heat treatment conditions (70 - 90 °C, 5 - 20 min), a scaffold with a surface microtopography pattern is obtained. The preparation method of the present invention can flexibly regulate the undulation change of the scaffold surface morphology by regulating the size of the polylactic acid-based microspheres, thereby endowing the scaffold surface with a specific wavy morphology (microtopography pattern), which serves as a physical stimulus factor to enable cell-matrix interaction, ultimately affecting cell behavior, and further affecting gene and protein expression. Moreover, it has high biological safety and stability, avoiding damage to surrounding tissues and organs due to the instability of chemical components and growth factors, simplifying the preparation method, and having significant improvement in enhancing the performance of promoting cell osteogenic differentiation.

[0006] Polylactic acid (PLA) has good biocompatibility and biodegradability. The polylactic acid-based microspheres prepared in the present invention have a large specific surface area and fluidity. By fully exerting the function of the polylactic acid-based microsphere carrier and utilizing the unique three-dimensional structure of the polylactic acid-based microspheres, through a certain treatment method, they are adhered to each other and maintain their own three-dimensional structure to form a scaffold, and a wavy microtopography pattern is constructed on the surface of the scaffold. Moreover, due to the adjustable particle size of the polylactic acid-based microspheres, patterns with different undulating waveforms can be constructed. In addition to constructing a relatively uniform undulating pattern with a single particle size, the preparation method of the present invention can construct a composite wavy pattern by mixing polylactic acid-based microspheres of different sizes. The small-sized polylactic acid-based microspheres fill the gaps between the large-sized polylactic acid-based microspheres and act as a bridge for cells to cross. Different mixing ratios of small-sized and large-sized polylactic acid-based microspheres result in different constructed patterns.

[0007] The present invention provides a preparation method for a scaffold with a surface microtopography pattern.

[0008] Specifically, a preparation method for a scaffold with a surface microtopography pattern includes the following steps:

[0009] (1) Preparation of polylactic acid-based microspheres: Mix a stabilizer with water to obtain an aqueous phase; mix polylactic acid or poly(lactic-co-glycolic acid) with an organic solvent to obtain an oil phase; then mix the aqueous phase with the oil phase, stir to volatilize the organic solvent, and dry to obtain the polylactic acid-based microspheres; the stabilizer includes at least one of polyvinyl alcohol, gelatin, or polysorbate.

[0010] (2) Hot pressing: Hot press the polylactic acid-based microspheres to obtain a polylactic acid scaffold preform; the pressure of the hot pressing is 0.01 - 0.5 MPa, and the temperature of the hot pressing is 40 - 50 °C.

[0011] (3) Heat-treat the polylactic acid scaffold preform to obtain the scaffold with surface microtopography patterns; the temperature of the heat treatment is 70-90 °C, and the time of the heat treatment is 5-20 min.

[0012] Preferably, in step (1), the water is deionized water or ultrapure water.

[0013] Preferably, in step (1), the organic solvent is a halogenated alkane; more preferably, the organic solvent is dichloromethane.

[0014] Preferably, in step (1), in the aqueous phase, the mass-volume ratio of the stabilizer to water is 0.5-5 g:(300-800) mL; more preferably, in the aqueous phase, the mass-volume ratio of the stabilizer to water is 1.5-5 g:(500-800) mL.

[0015] Preferably, in step (1), in the oil phase, the mass-volume ratio of polylactic acid or poly(lactic acid-glycolic acid) copolymer to the organic solvent is 2-10 g:(30-80) mL; more preferably, in the oil phase, the mass-volume ratio of polylactic acid or poly(lactic acid-glycolic acid) copolymer to the organic solvent is 4-10 g:(50-80) mL.

[0016] Preferably, in step (1), the volume ratio of the aqueous phase to the oil phase is 8-15:1; more preferably, the volume ratio of the aqueous phase to the oil phase is 10-15:1.

[0017] Preferably, in step (1), during the process of stirring and volatilizing the organic solvent, the stirring speed is 200-5500 revolutions per minute, and the stirring time is 2-5 hours; more preferably, the stirring speed is 250-5000 revolutions per minute, and the stirring time is 2-5 hours. The stirring method can be mechanical stirring and / or high-speed homogeneous shearing.

[0018] Preferably, in step (1), after stirring and volatilizing the organic solvent, let it stand and take the precipitate, and obtain the polylactic acid-based microspheres after centrifugation, washing, and freeze-drying.

[0019] Preferably, in step (1), the particle size of the polylactic acid-based microspheres is 1-160 μm; more preferably, the particle size of the polylactic acid-based microspheres is 2-150 μm, such as 2-30 μm, 40-90 μm, 100-150 μm. When performing the operation of step (2), polylactic acid-based microspheres with different particle sizes can be mixed and hot-pressed to control the surface microtopography patterns of the scaffold. For example, polylactic acid-based microspheres of 100-150 μm and polylactic acid-based microspheres of 2-30 μm are mixed in a mass ratio of 1-10:1.

[0020] Preferably, the polylactic acid is racemic polylactic acid; more preferably, the molecular weight of the polylactic acid is 50,000 - 200,000.

[0021] Preferably, in step (1), after the polylactic acid-based microspheres and nano-hydroxyapatite are mixed, hot pressing is carried out; the mass of the nano-hydroxyapatite is 1% - 15% of the polylactic acid-based microspheres. Thus, a submicron structure is constructed on the surface of the scaffold.

[0022] Preferably, in step (2), the pressure of the hot pressing is 0.1 - 0.5 MPa, and the temperature of the hot pressing is 40 - 50 °C.

[0023] Preferably, in step (2), the time of the hot pressing is 3 - 15 min; more preferably, the time of the hot pressing is 5 - 10 min.

[0024] Preferably, in step (2), the hot pressing is carried out in a hot press.

[0025] Preferably, in step (2), the polylactic acid-based microspheres are poured into the mold of the hot press for hot pressing to obtain a polylactic acid scaffold preform; the pressure of the hot pressing is 0.01 - 0.5 MPa, the temperature of the hot pressing is 40 - 50 °C; the time of the hot pressing is 5 - 10 min.

[0026] Preferably, in step (3), the temperature of the heat treatment is 75 - 85 °C, and the time of the heat treatment is 8 - 15 min.

[0027] A scaffold with a surface microtopography pattern is prepared by the above preparation method.

[0028] Preferably, the surface of the scaffold has a three-dimensional wavy microtopography pattern, the ridge width is 1 - 100 μm, and the depth is 0.5 - 50 μm.

[0029] The application of the above scaffold with a surface microtopography pattern in the field of cell culture. The purpose of the application in the field of cell culture is not for the treatment of diseases, but for the preparation of medical products.

[0030] The application of the above scaffold with a surface microtopography pattern in the preparation of bone defect repair materials.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) The present invention uses an oil phase and a water phase, and prepares polylactic acid-based microspheres by the emulsion solvent evaporation method (i.e., step (1) of the present invention, the water-in-oil emulsion solvent evaporation method), and then obtains a scaffold preform under specific hot pressing conditions (pressure is 0.01 - 0.5 MPa, temperature is 40 - 50 °C), and finally obtains a scaffold with a surface microtopography pattern under suitable heat treatment conditions (70 - 90 °C, 5 - 20 min). The preparation method of the present invention can flexibly regulate the undulation change of the scaffold surface morphology by regulating the size of the polylactic acid-based microspheres, thereby endowing the scaffold surface with a specific wavy morphology (microtopography pattern), which serves as a physical stimulation factor to enable cell-matrix interaction, ultimately affecting cell behavior, and further affecting gene and protein expression. Moreover, it has high biological safety and stability, avoiding damage to surrounding tissues and organs due to the instability of chemical components and growth factors, and has significant improvement in promoting the osteogenic differentiation performance of cells. The preparation method of the present invention not only does not require template imprinting (template-assisted) and chemical etching processes, but also simplifies the preparation method.

[0033] (2) The scaffold of the present invention can be used to regulate cell behavior and guide cell adhesion, growth, proliferation and differentiation as a physical stimulation signal; cells can adhere and grow on the surface of the microspheres, and the pits on the surface pattern of the scaffold can be used to simulate the absorption pits after osteoclasts remove damaged or useless bone, enabling osteoblasts to gather and promoting osteogenic differentiation. Description of the Drawings

[0034] Figure 1 Scanning electron microscope image and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 1 of the present invention;

[0035] Figure 2 Scanning electron microscope image of the surface of the scaffold prepared in Example 1 of the present invention;

[0036] Figure 3 Scanning electron microscope image and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 2 of the present invention;

[0037] Figure 4 Scanning electron microscope image of the surface of the scaffold prepared in Example 2 of the present invention;

[0038] Figure 5 Scanning electron microscope image and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 3 of the present invention;

[0039] Figure 6 Scanning electron microscope image of the surface of the scaffold prepared in Example 3 of the present invention;

[0040] Figure 7 Scanning electron microscope image and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 4 of the present invention;

[0041] Figure 8Scanning electron micrograph of the surface of the scaffold prepared in Example 4 of the present invention;

[0042] Figure 9 Scanning electron micrograph and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 5 of the present invention;

[0043] Figure 10 Scanning electron micrograph of the surface of the scaffold prepared in Example 5 of the present invention;

[0044] Figure 11 Schematic diagram of the structure of the surface of the scaffold constructed by single-size polylactic acid-based microspheres;

[0045] Figure 12 Scanning electron micrograph of the surface of the scaffold prepared in Example 6 of the present invention;

[0046] Figure 13 Scanning electron micrograph of the surface of the scaffold prepared in Example 7 of the present invention;

[0047] Figure 14 Scanning electron micrograph of the surface of the scaffold prepared in Example 8 of the present invention;

[0048] Figure 15 Schematic diagram of the structure of the surface of the scaffold constructed by the composite of small-sized polylactic acid-based microspheres and large-sized polylactic acid-based microspheres;

[0049] Figure 16 Spreading of cells on the surface pattern of the scaffold in Example 9;

[0050] Figure 17 Alizarin red staining in Example 9;

[0051] Figure 18 Spreading of cells on the surface pattern of the scaffold in Example 10;

[0052] Figure 19 Alizarin red staining in Example 10;

[0053] Figure 20 Spreading of cells on the surface pattern of the scaffold in Example 11;

[0054] Figure 21 Alizarin red staining in Example 11;

[0055] Figure 22 Scanning electron micrograph of the surface of the scaffold prepared in Comparative Example 1;

[0056] Figure 23 Spreading of cells on the surface pattern of the scaffold in Comparative Example 1;

[0057] Figure 24 Alizarin red staining in Example 11. Detailed implementation manners

[0058] In order to make the technical solutions described in the present invention clearer and more understandable to those skilled in the art, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0059] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0060] Example 1: Preparation method of scaffold

[0061] A preparation method of a scaffold with a surface microtopography pattern includes the following steps:

[0062] (1) Preparation of polylactic acid-based microspheres: Mix 1.5 g of polyvinyl alcohol with 500 mL of ultrapure water at 80 °C with stirring for 1 hour, then cool to room temperature of 20 °C to obtain an aqueous phase; mix 4 g of polylactic acid with 50 mL of dichloromethane to obtain an oil phase; then mix the aqueous phase and the oil phase at a volume ratio of 10:1 (add the oil phase to the aqueous phase), use a constant-speed electric stirrer, set the rotation speed to 250 revolutions per minute, mechanically stir for 5 hours to volatilize dichloromethane, then let it stand to take the precipitate, and obtain polylactic acid-based microspheres after centrifugation, washing, and freeze-drying; the average particle size of the prepared polylactic acid (PLA)-based microspheres is 135.38 ± 23.94 μm;

[0063] (2) Hot pressing: Take a hot press mold with a diameter of 10 mm, weigh 0.02 g of polylactic acid-based microspheres and pour them between two gaskets of the hot press, set the hot pressing temperature to 40 °C, the hot pressing time to 9 min, and the hot pressing pressure to 0.1 MPa, perform hot pressing molding, and cool and demold to obtain a polylactic acid scaffold preform;

[0064] (3) Place the polylactic acid scaffold preform in an oven for heat treatment and cool it naturally to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature is 90 °C and the heat treatment time is 10 min.

[0065] A scaffold with a surface microtopography pattern is obtained by the above preparation method.

[0066] Figure 1 is the scanning electron microscope image and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 1 of the present invention; Figure 1 in (a) is the scanning electron microscope image of the polylactic acid-based microspheres, Figure 1 in (b) is the different particle size distribution diagram of the polylactic acid-based microspheres (in Figure (b), the abscissa "Diameter" represents the diameter, and the ordinate "Counts" represents the quantity); Figure 2Scanning electron micrograph of the surface of the scaffold prepared in Example 1 of the present invention.

[0067] Example 2: Preparation method of the scaffold

[0068] A preparation method of a scaffold with a surface microtopography pattern, comprising the following steps:

[0069] (1) Preparation of polylactic acid-based microspheres: 2.5 g of polyvinyl alcohol and 500 mL of ultrapure water were stirred and mixed at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain an aqueous phase; 2.5 g of polylactic acid was mixed with 50 mL of dichloromethane to obtain an oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a constant-speed electric stirrer was used with a rotation speed set at 250 revolutions per minute, and mechanically stirred for 5 hours to volatilize dichloromethane, and then allowed to stand to take the precipitate, which was centrifuged, washed, and freeze-dried to obtain polylactic acid-based microspheres; the average particle diameter of the prepared polylactic acid (PLA)-based microspheres was 92.94 ± 15.22 μm;

[0070] (2) Hot pressing: A hot press mold with a diameter of 10 mm was taken, 0.02 g of polylactic acid-based microspheres was weighed and poured between two gaskets of the hot press, the hot pressing temperature was set at 40 °C, the hot pressing time was 9 min, the hot pressing pressure was 0.1 MPa, hot pressing was carried out, and after cooling and demolding, a polylactic acid scaffold preform was obtained;

[0071] (3) The polylactic acid scaffold preform was placed in an oven for heat treatment and naturally cooled to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature was 90 °C and the heat treatment time was 10 min.

[0072] A scaffold with a surface microtopography pattern, prepared by the above preparation method.

[0073] Figure 3 Scanning electron micrograph and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 2 of the present invention; Figure 3 (a) in is the scanning electron micrograph of the polylactic acid-based microspheres, Figure 3 (b) in is the different particle size distribution diagram of the polylactic acid-based microspheres (in Figure (b), the abscissa "Diameter" represents the diameter, and the ordinate "Counts" represents the quantity); Figure 4 Scanning electron micrograph of the surface of the scaffold prepared in Example 2 of the present invention.

[0074] Example 3: Preparation method of the scaffold

[0075] A preparation method of a scaffold with a surface microtopography pattern, comprising the following steps:

[0076] (1) Preparation of polylactic acid-based microspheres: 2.5 g of polyvinyl alcohol and 500 mL of ultrapure water were stirred and mixed at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 2.5 g of polylactic acid was mixed with 50 mL of dichloromethane to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a constant-speed electric stirrer was used with the rotation speed set at 1000 revolutions per minute. The mixture was mechanically stirred for 5 hours to volatilize dichloromethane, and then allowed to stand to collect the precipitate. After centrifugation, washing, and freeze-drying, polylactic acid-based microspheres were obtained; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 51.29 ± 6.52 μm;

[0077] (2) Hot pressing: A hot press mold with a diameter of 10 mm was taken, and 0.02 g of polylactic acid-based microspheres was weighed and poured between two gaskets of the hot press. The hot pressing temperature was set at 40 °C, the hot pressing time was 9 min, and the hot pressing pressure was 0.1 MPa. After hot pressing and cooling to demold, a polylactic acid scaffold preform was obtained;

[0078] (3) The polylactic acid scaffold preform was placed in an oven for heat treatment and then naturally cooled to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature was 90 °C and the heat treatment time was 10 min.

[0079] A scaffold with a surface microtopography pattern was prepared by the above preparation method.

[0080] Figure 5 This is the scanning electron microscope image and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 3 of the present invention; Figure 5 In (a) is the scanning electron microscope image of the polylactic acid-based microspheres, Figure 5 In (b) is the particle size distribution diagram of different polylactic acid-based microspheres (in Figure (b), the abscissa "Diameter" represents the diameter and the ordinate "Counts" represents the quantity); Figure 6 This is the scanning electron microscope image of the surface of the scaffold prepared in Example 3 of the present invention.

[0081] Example 4: Preparation method of the scaffold

[0082] A preparation method of a scaffold with a surface microtopography pattern, comprising the following steps:

[0083] (1) Preparation of polylactic acid-based microspheres: 2.5 g of polyvinyl alcohol and 500 mL of ultrapure water were stirred and mixed at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 2.5 g of polylactic acid was mixed with 50 mL of dichloromethane to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a high-speed homogenizing shear machine was used with the rotation speed set at 1500 revolutions per minute, and high-shear stirring was carried out for 3 hours to volatilize dichloromethane, and then the precipitate was taken by standing, and the polylactic acid-based microspheres were obtained after centrifugation, washing, and freeze-drying; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 13.46 ± 2.48 μm;

[0084] (2) Hot pressing: A hot press mold with a diameter of 10 mm was taken, and 0.02 g of polylactic acid-based microspheres was weighed and poured between two gaskets of the hot press. The hot pressing temperature was set at 40 °C, the hot pressing time was 9 min, and the hot pressing pressure was 0.1 MPa. Hot pressing was carried out for molding, and after cooling, demolding was carried out to obtain a polylactic acid scaffold preform;

[0085] (3) The polylactic acid scaffold preform was placed in an oven for heat treatment and cooled naturally to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature was 80 °C and the heat treatment time was 10 min.

[0086] A scaffold with a surface microtopography pattern was prepared by the above preparation method.

[0087] Figure 7 This is the scanning electron microscope image and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 4 of the present invention; Figure 7 In (a) is the scanning electron microscope image of the polylactic acid-based microspheres, Figure 7 In (b) is the different particle size distribution diagram of the polylactic acid-based microspheres (in Figure (b), the abscissa "Diameter" represents the diameter, and the ordinate "Counts" represents the quantity); Figure 8 This is the scanning electron microscope image of the surface of the scaffold prepared in Example 4 of the present invention.

[0088] Example 5: Preparation method of the scaffold

[0089] A preparation method of a scaffold with a surface microtopography pattern, comprising the following steps:

[0090] (1) Preparation of polylactic acid-based microspheres: 2.5 g of polyvinyl alcohol was stirred and mixed with 500 mL of ultrapure water at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 2.5 g of polylactic acid was mixed with 50 mL of dichloromethane to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a high-speed homogenizing shear machine was used with the rotation speed set at 4000 revolutions per minute, and high-shear stirring was carried out for 2 hours to volatilize dichloromethane, and then the precipitate was taken by standing, and the polylactic acid-based microspheres were obtained after centrifugation, washing, and freeze-drying; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 4.21 ± 0.96 μm;

[0091] (2) Hot pressing: A hot press mold with a diameter of 10 mm was taken, and 0.02 g of polylactic acid-based microspheres was weighed and poured between two gaskets of the hot press. The hot pressing temperature was set at 40 °C, the hot pressing time was 9 min, and the hot pressing pressure was 0.1 MPa. After hot pressing and forming and cooling and demolding, a polylactic acid scaffold preform was obtained;

[0092] (3) The polylactic acid scaffold preform was placed in an oven for heat treatment and naturally cooled to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature was 70 °C and the heat treatment time was 10 min.

[0093] A scaffold with a surface microtopography pattern was prepared by the above preparation method.

[0094] Figure 9 This is the scanning electron microscope image and particle size distribution diagram of the polylactic acid-based microspheres prepared in Example 5 of the present invention; Figure 9 In (a) is the scanning electron microscope image of the polylactic acid-based microspheres, Figure 9 In (b) is the particle size distribution diagram of different polylactic acid-based microspheres (in Figure (b), the abscissa "Diameter" represents the diameter, and the ordinate "Counts" represents the quantity); Figure 10 This is the scanning electron microscope image of the surface of the scaffold prepared in Example 5 of the present invention.

[0095] Example 6: Preparation method of the scaffold

[0096] A preparation method of a scaffold with a surface microtopography pattern, comprising the following steps:

[0097] (1) Preparation of polylactic acid-based microspheres: 2.5 g of polyvinyl alcohol was stirred and mixed with 500 mL of ultrapure water at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 2.5 g of polylactic acid was mixed with 50 mL of dichloromethane, and then 0.15 g of nano-hydroxyapatite was added and ultrasonically dispersed for 20 min to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a constant-speed electric stirrer was used with the rotation speed set at 250 revolutions per minute, and mechanically stirred for 5 hours to volatilize dichloromethane, and then the precipitate was taken by standing, and the polylactic acid-based microspheres were obtained after centrifugation, washing, and freeze-drying; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 92.94 ± 15.22 μm;

[0098] (2) Hot pressing: A hot press mold with a diameter of 10 mm was taken, 0.02 g of polylactic acid-based microspheres was weighed and poured between two gaskets of the hot press, the hot pressing temperature was set at 40 °C, the hot pressing time was 10 min, and the hot pressing pressure was 0.1 MPa. After hot pressing and forming and cooling and demolding, a polylactic acid scaffold preform was obtained;

[0099] (3) The polylactic acid scaffold preform was placed in an oven for heat treatment and naturally cooled to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature was 90 °C and the heat treatment time was 10 min.

[0100] A scaffold with a surface microtopography pattern was prepared by the above preparation method.

[0101] Figure 11 Schematic diagram of the structure of the surface of the scaffold constructed by single-size polylactic acid-based microspheres ( Figure 11 in which "microtopography length" represents the microtopography length and "microtopography height" represents the microtopography height); that is, the surface of the scaffold is wavy.

[0102] Figure 12 This is the scanning electron micrograph of the surface of the scaffold prepared in Example 6 of the present invention; Figure 12 Figures (a), (b), and (c) in it are the scanning electron micrographs of the surface of the scaffold at different magnifications.

[0103] Example 7: Preparation method of the scaffold

[0104] A preparation method of a scaffold with a surface microtopography pattern, comprising the following steps:

[0105] (1) Preparation of polylactic acid-based microspheres: 2.5 g of polyvinyl alcohol was stirred and mixed with 500 mL of ultrapure water at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 2.5 g of polylactic acid was mixed with 50 mL of dichloromethane to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a high-speed homogenizing shear machine was used with the rotation speed set at 4000 revolutions per minute, and high-shear stirring was carried out for 2 hours to volatilize dichloromethane, and then the precipitate was taken by standing, and after centrifugation, washing, and freeze-drying, polylactic acid-based microspheres were obtained; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 4.21 ± 0.96 μm;

[0106] 1.5 g of polyvinyl alcohol was stirred and mixed with 500 mL of ultrapure water at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 4 g of polylactic acid was mixed with 50 mL of dichloromethane to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a constant-speed electric stirrer was used with the rotation speed set at 250 revolutions per minute, and mechanical stirring was carried out for 5 hours to volatilize dichloromethane, and then the precipitate was taken by standing, and after centrifugation, washing, and freeze-drying, polylactic acid-based microspheres were obtained; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 135.38 ± 23.94 μm;

[0107] (2) Hot pressing: The polylactic acid (PLA)-based microspheres with an average particle size of 4.21 ± 0.96 μm and the polylactic acid (PLA)-based microspheres with an average particle size of 135.38 ± 23.94 μm were mixed in a mass ratio of 1:1 to obtain mixed polylactic acid (PLA)-based microspheres; a hot press mold with a diameter of 10 mm was taken, and 0.02 g of the mixed polylactic acid (PLA)-based microspheres was weighed and poured between two gaskets of the hot press. The hot pressing temperature was set at 40 °C, the hot pressing time was 10 min, and the hot pressing pressure was 0.1 MPa. After hot pressing and forming and cooling and demolding, a polylactic acid scaffold preform was obtained;

[0108] (3) The polylactic acid scaffold preform was placed in an oven for heat treatment and then cooled naturally to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature was 80 °C and the heat treatment time was 15 min.

[0109] A scaffold with a surface microtopography pattern was prepared by the above preparation method.

[0110] Figure 13 This is the scanning electron micrograph of the surface of the scaffold prepared in Example 7 of the present invention.

[0111] Example 8: Preparation method of the scaffold

[0112] A preparation method of a scaffold with a surface microtopography pattern, comprising the following steps:

[0113] (1) Preparation of polylactic acid-based microspheres: 2.5 g of polyvinyl alcohol was stirred and mixed with 500 mL of ultrapure water at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 2.5 g of polylactic acid was mixed with 50 mL of dichloromethane to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a high-speed homogenizing shear machine was used with the rotation speed set at 4000 revolutions per minute, and high-shear stirring was carried out for 2 hours to volatilize dichloromethane, and then the precipitate was taken by standing, and after centrifugation, washing, and freeze-drying, polylactic acid-based microspheres were obtained; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 4.21 ± 0.96 μm;

[0114] 1.5 g of polyvinyl alcohol was stirred and mixed with 500 mL of ultrapure water at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 4 g of polylactic acid was mixed with 50 mL of dichloromethane to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a constant-speed electric stirrer was used with the rotation speed set at 250 revolutions per minute, and mechanical stirring was carried out for 5 hours to volatilize dichloromethane, and then the precipitate was taken by standing, and after centrifugation, washing, and freeze-drying, polylactic acid-based microspheres were obtained; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 135.38 ± 23.94 μm;

[0115] (2) Hot pressing: The polylactic acid (PLA)-based microspheres with an average particle size of 4.21 ± 0.96 μm and the polylactic acid (PLA)-based microspheres with an average particle size of 135.38 ± 23.94 μm were mixed at a mass ratio of 1:8 to obtain mixed polylactic acid (PLA)-based microspheres; a hot press mold with a diameter of 10 mm was taken, and 0.02 g of the mixed polylactic acid (PLA)-based microspheres was weighed and poured between two gaskets of the hot press. The hot pressing temperature was set at 40 °C, the hot pressing time was 10 min, and the hot pressing pressure was 0.1 MPa. After hot pressing and cooling and demolding, a polylactic acid scaffold preform was obtained;

[0116] (3) The polylactic acid scaffold preform was placed in an oven for heat treatment and then cooled naturally to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature was 80 °C and the heat treatment time was 15 min.

[0117] A scaffold with a surface microtopography pattern was prepared by the above preparation method.

[0118] Figure 14 This is the scanning electron microscope image of the surface of the scaffold prepared in Example 8 of the present invention.

[0119] Figure 15 This is the structural schematic diagram of the surface of the scaffold constructed by the composite of small-particle-size polylactic acid-based microspheres and large-particle-size polylactic acid-based microspheres. Figure 15100 in it represents polylactic acid-based microspheres with small particle size, and 200 represents polylactic acid-based microspheres with large particle size.

[0120] Example 9

[0121] After sterilizing the surface of the scaffold prepared in Example 2, it was placed in a 24-well plate. Each well was soaked overnight with PBS buffer (phosphate buffered saline). The next day, the PBS buffer in the well plate was aspirated. Then, 50 μL of cell suspension with a cell concentration of 2×10 5 cells / mL (MC3T3-E1 cells, which can be provided by the manufacturer Qingqi (Shanghai) Biotechnology Development Co., Ltd.) was added dropwise to the well plate containing the scaffold. After culturing for 2 h, when the cells adhered to the wall, another 950 μL of culture medium (the culture medium is DMEM medium, product model: AF29498406; manufacturer: Hyclone) was added dropwise to the well plate. The cell culture environment was 37 °C and 5% volume fraction of CO2. The time interval for changing the culture medium was 2 days. The cells on the scaffold were observed by confocal microscopy photography to analyze the spreading morphology and growth status of the cells on the scaffolds composed of microspheres with different particle sizes. Among them, the red staining was actin (i.e., Actin, labeled with Alexa Fluor568), and the blue staining was the cell nucleus (i.e., Nucleus, labeled with DAPI 4',6-diamidino-2-phenylindole). It was found through actin labeling that the cells tended to spread in all directions with a larger area, presenting a polygon shape, and the pseudopodia were more obvious. Figure 16 It shows the spreading situation of the cells on the surface pattern of the scaffold in Example 9. Figure 16 In (a) “Merge” represents the merged state, (b) represents actin, and (c) represents the cell nucleus.

[0122] On the 21st day, the surface cells were stained with alizarin red solution (a 0.1% mass concentration ARS alkylresorcinol Tris-HCl buffer solution, pH = 8.0) to evaluate the osteogenic differentiation of MC3T3-E1 cells. The specific operation was as follows: The scaffold rinsed with PBS buffer was fixed with 4% mass concentration paraformaldehyde for 30 min. After washing, alizarin red solution was added dropwise until the surface of the scaffold was immersed, and it was incubated at 37 °C for 1 h. Then, it was washed with deionized water, and images were taken through a microscope.

[0123] Figure 17 It shows the alizarin red staining situation in Example 9. It can be found from Figure 17 that the red area is the calcium ion deposition and mineralization area. The darker the color of the area, the higher the mineralization.

[0124] Example 10

[0125] The scaffold prepared in Example 4 was subjected to surface sterilization and then placed in a 24-well plate. Each well was soaked overnight with PBS buffer. The next day, the PBS buffer in the well plate was aspirated. Then, 50 μL of cell suspension with a cell (MC3T3-E1 cells) concentration of 2*10 5 cells / ml was added dropwise to the well plate containing the scaffold. After culturing for 2 h, when the cells adhered to the wall, another 950 μL of culture medium (the culture medium was DMEM medium) was added dropwise to the well plate. The cell culture environment was 37 °C and 5% by volume of CO2. The time interval for changing the culture medium was 2 days. The cells on the scaffold were photographed and observed by confocal microscopy to analyze the spreading morphology and growth status of the cells on the scaffolds composed of microspheres with different particle sizes. Among them, the red staining was actin (i.e., Actin, labeled with Alexa Fluor 568), and the blue staining was the cell nucleus (i.e., Nucleus, labeled with DAPI 4',6-diamidino-2-phenylindole).

[0126] Figure 18 For the spreading of cells on the surface pattern of the scaffold in Example 10, Figure 18 in (a) "Merge" represents the merged state, (b) represents actin, and (c) represents the cell nucleus.

[0127] From Figure 18 it can be seen that when the scaffold of Example 4 was used for the above experiment, the cell density was the highest, almost covering the entire surface of the scaffold. The cells showed obvious arrangement with the microspheres on the surface of the scaffold, indicating that the scaffold with this morphology has a certain contact guidance effect.

[0128] On the 21st day, alizarin red staining solution (0.1% mass concentration of ARS alkylresorcinol in Tris-HCl buffer solution, pH = 8.0) was used to stain the surface cells to evaluate the osteogenic differentiation of MC3T3-E1. The specific operation was as follows: The scaffold rinsed with PBS buffer was fixed with 4% mass concentration of paraformaldehyde for 30 min. After washing, alizarin red staining solution was added dropwise until the surface of the scaffold was immersed, and incubated at 37 °C for 1 hour. Then, it was washed with deionized water, and images were taken by a microscope.

[0129] Figure 19 For the alizarin red staining in Example 10. Figure 19 It can be found that the area of the mineralized region is large and the color is darker, indicating higher osteogenic differentiation performance.

[0130] Example 11

[0131] After surface sterilization of the scaffold prepared in Example 5, it was placed in a 24-well plate. Each well was soaked overnight with PBS buffer. The next day, the PBS buffer in the well was aspirated. Then, 50 μL of cell suspension with a cell (MC3T3-E1 cell) concentration of 2*10 5 cells / ml was added dropwise to the well plate containing the scaffold. After culturing for 2 h, when the cells adhered to the wall, another 950 μL of culture medium (the culture medium was DMEM medium) was added dropwise to the well plate. The cell culture environment was 37 °C and 5% volume fraction of CO2. The time interval for changing the culture medium was 2 days. The cells on the scaffold were photographed and observed by confocal microscopy to analyze the spreading morphology and growth status of the cells on the microsphere scaffolds with different particle sizes. Among them, the red staining was for actin (labeled with Alexa Fluor568), and the blue staining was for cell nuclei (labeled with DAPI).

[0132] Figure 20 For the spreading of cells on the surface pattern of the scaffold in Example 11, Figure 20 in (a) “Merge” represents the merged state, (b) represents actin, and (c) represents cell nuclei. As can be seen from Figure 20 it, the undulating surface has relatively small protrusions, and the adherent cells are elongated on its surface. The elongated extension and polarization of the cytoskeleton are beneficial to osteogenic differentiation.

[0133] On the 21st day, the surface cells were stained with alizarin red staining solution (0.1% ARS Tris-HCl buffer solution, pH = 8.0) to evaluate the osteogenic differentiation of MC3T3-E1 cells. The specific operation was as follows: The scaffold after being rinsed with PBS was fixed with 4% mass concentration paraformaldehyde for 30 min. After washing, alizarin red staining solution was added dropwise until the surface of the scaffold was immersed, and it was incubated at 37 °C for 1 hour. Then, it was washed with deionized water, and images were taken by a microscope.

[0134] Figure 21 For the alizarin red staining in Example 11; As can be found from Figure 21 it, there are dark red mineralized areas, indicating that the protrusion morphology promotes the osteogenic differentiation performance of cells.

[0135] Comparative Example 1 (The hot pressing and heat treatment conditions in the preparation process of the Comparative Example 1 scaffold are different from those in Example 2)

[0136] A method for preparing a scaffold, comprising the following steps:

[0137] (1) Preparation of polylactic acid-based microspheres: 2.5 g of polyvinyl alcohol was stirred and mixed with 500 mL of ultrapure water at 80 °C for 1 hour, and then cooled to room temperature of 20 °C to obtain the aqueous phase; 2.5 g of polylactic acid was mixed with 50 mL of dichloromethane, and then 0.15 g of nano-hydroxyapatite was added and ultrasonically dispersed for 20 min to obtain the oil phase; then the aqueous phase and the oil phase were mixed at a volume ratio of 10:1 (the oil phase was added to the aqueous phase), and a constant-speed electric stirrer was used with the rotation speed set at 250 revolutions per minute, and mechanically stirred for 5 hours to volatilize dichloromethane, and then the precipitate was taken by standing, and the polylactic acid-based microspheres were obtained after centrifugation, washing, and freeze-drying; the average particle size of the prepared polylactic acid (PLA)-based microspheres was 92.94 ± 15.22 μm;

[0138] (2) Hot pressing: A hot press mold with a diameter of 10 mm was taken, and 0.02 g of polylactic acid-based microspheres was weighed and poured between two gaskets of the hot press. The hot pressing temperature was set at 40 °C, the hot pressing time was 10 min, and the hot pressing pressure was 5 MPa. After hot pressing and forming, it was cooled and demolded to obtain a polylactic acid scaffold preform;

[0139] (3) The polylactic acid scaffold preform was placed in an oven for heat treatment and naturally cooled to obtain a scaffold with a surface microtopography pattern; the heat treatment temperature was 110 °C and the heat treatment time was 20 min.

[0140] A scaffold was prepared by the above preparation method.

[0141] Figure 22 It is the scanning electron micrograph of the surface of the scaffold prepared in Comparative Example 1; from Figure 22 It can be seen that the surface of the scaffold prepared in Comparative Example 1 is similar to a plane.

[0142] After the surface of the scaffold prepared in Comparative Example 1 was sterilized, it was placed in a 24-well plate, and each well was soaked overnight with PBS buffer. The next day, the PBS buffer in the well plate was aspirated. Then, 50 μL of a cell suspension with a cell (MC3T3-E1 cell) concentration of 2*10 5 cells / ml was added dropwise to the well plate containing the scaffold. After culturing for 2 h, after the cells adhered, 950 μL of culture medium (the culture medium was DMEM medium) was added dropwise to the well plate again. The cell culture environment was 37 °C and 5% by volume of CO2. The medium replacement time interval was 2 days. The cells on the scaffold were photographed and observed by confocal microscopy to analyze the spreading morphology and growth status of the cells on the microsphere scaffolds with different particle sizes. Among them, the red staining was actin (labeled with Alexa Fluor568), and the blue staining was the cell nucleus (labeled with DAPI).

[0143] Figure 23 It is the spreading situation of the cells on the surface pattern in Comparative Example 1, Figure 20In (a), "Merge" represents the fusion state, (b) represents actin, and (c) represents the cell nucleus. From Figure 23 It can be seen that the cells on the scaffold spread in a near-circular shape, while a circular shape is not conducive to cell differentiation.

[0144] On the 21st day, alizarin red staining solution (Tris-HCl buffer solution with 0.1% ARS, pH = 8.0) was used to stain the surface cells to evaluate the osteogenic differentiation of MC3T3-E1 cells. The specific operation is as follows: The scaffold rinsed with PBS was fixed with 4% paraformaldehyde by mass concentration for 30 min. After washing, alizarin red staining solution was dropped until the surface of the scaffold was immersed, and incubated at 37 °C for 1 hour. Then, it was washed with deionized water, and images were taken through a microscope.

[0145] Figure 24 This is the alizarin red staining situation in Comparative Example 1; from Figure 24 it can be found that the red mineralized areas are sparse and the area of the region is small, and the osteogenic differentiation effect of the cells is poor.

Claims

1. A method for preparing a bracket, characterized in that, It includes the following steps: (1) Preparation of polylactic acid-based microspheres: Mix a stabilizer with water to obtain an aqueous phase; mix polylactic acid or poly(lactic-co-glycolic acid) with an organic solvent to obtain an oil phase; then mix the aqueous phase with the oil phase, stir to volatilize the organic solvent, and dry to obtain the polylactic acid-based microspheres; the stabilizer includes at least one of polyvinyl alcohol, gelatin, or polysorbate; (2) Hot pressing: Hot press the polylactic acid-based microspheres to obtain a polylactic acid scaffold preform; the pressure of the hot pressing is 0.01 - 0.5 MPa, and the temperature of the hot pressing is 40 - 50 °C; (3) Heat-treat the polylactic acid scaffold preform to obtain the scaffold with a surface microtopography pattern; the temperature of the heat treatment is 70 - 90 °C, and the time of the heat treatment is 5 - 20 min.

2. The preparation method according to claim 1, characterized in that, In step (1), the organic solvent is a halogenated alkane.

3. The preparation method according to claim 1, characterized in that, In step (1), in the aqueous phase, the mass-volume ratio of the stabilizer to water is 0.5 - 5 g : (300 - 800) mL.

4. The preparation method according to claim 1, characterized in that, In step (1), in the oil phase, the mass-volume ratio of polylactic acid or poly(lactic-co-glycolic acid) to the organic solvent is 2 - 10 g : (30 - 80) mL.

5. The preparation method according to claim 1, characterized in that, In step (1), the volume ratio of the aqueous phase to the oil phase is 8 - 15 :

1.

6. The preparation method according to claim 1, characterized in that In step (1), the particle size of the polylactic acid-based microspheres is 1 - 160 μm.

7. The preparation method according to claim 1, characterized in that In step (1), mix the polylactic acid-based microspheres with nano-hydroxyapatite and then prepare the oil phase; the mass of the nano-hydroxyapatite is 1 - 15% of the polylactic acid.

8. The preparation method according to claim 1, wherein, In step (2), the pressure of the hot pressing is 0.1 - 0.5 MPa, the temperature of the hot pressing is 40 - 50 °C; the time of the hot pressing is 3 - 15 min.

9. A bracket, characterized in that, Prepared by the preparation method according to any one of claims 1 - 8; the surface of the scaffold has a three-dimensional wavy microtopography pattern, the ridge width is 1 - 100 μm, and the depth is 0.5 - 50 μm.

10. Use of the scaffold according to claim 9 in the field of cell culture.

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