Porous scaffold as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a porous scaffold as well as a preparation method and application thereof. The porous scaffold comprises a scaffold substrate and polydopamine deposited on the surface of the scaffold substrate, the stent base body comprises a base body material and mesoporous silicon dispersed in the base body material. The polydopamine coating is beneficial to cell adhesion and proliferation, promotes stem cell osteogenic differentiation and induces macrophages to differentiate in a direction beneficial to tissue regeneration and repair, so that the porous scaffold has osteogenesis promoting and immune regulation functions. Mesoporous silicon has the characteristics of good biocompatibility, large specific surface area, high safety and the like, so that the porous scaffold not only has good biocompatibility and degradability, but also has a three-dimensional communicated porous structure, the pore structure is uniform, the strength is high, the porosity is high, and enough space can be provided. Under the combined action of the mesoporous silicon and the polydopamine layer, the porous scaffold has the functions of promoting bone regeneration and immune regulation and control, and is good in mechanical property.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a porous scaffold and a preparation method and application thereof. Background Art

[0002] Exploring new artificial bone repair materials is an urgent issue for treating large-scale clinical bone defects. In recent years, the continued advancement of bone tissue engineering and other related research on bone defect treatment has brought hope for bone defect repair. Ideal bone repair materials should possess excellent biocompatibility and biodegradability, as well as osteoconductivity, high porosity, mechanical strength, an extracellular matrix-like structure, and the ability to effectively induce new bone formation. Sustained and dynamic osteogenesis and angiogenesis are key factors in the bone repair process.

[0003] Currently, biomaterials suitable for bone regeneration remain a hot topic of debate. Poly(lactic-co-glycolic acid) (PLGA) has been used in bone tissue engineering due to its good biocompatibility and tunable biodegradability. However, PLGA still suffers from insufficient mechanical strength and lack of bioactivity, which limits its application.

[0004] Numerous studies are currently underway on bone repair materials, including combining PLGA and hydroxyapatite to create PLGA-hydroxyapatite composite porous microspheres; combining mechanical and biochemical stimulation to enhance the osteoinductive properties of bone repair scaffolds, creating bone repair scaffolds with excellent osteogenic properties; 3D printing of polylactic acid bone repair scaffolds and studying their osteoinductive properties; and 3D printing of bioactive ceramic / PLGA core-shell fiber scaffolds for bone repair research. However, the bone repair hydrogels produced by these studies generally suffer from the following issues: 1) difficulty in simultaneously promoting bone regeneration and immune regulation; 2) insufficient mechanical strength of the materials; and 3) complex preparation processes, making them difficult to commercialize.

[0005] Therefore, it is of great significance to provide a porous scaffold that can promote bone regeneration and immune regulation and has good mechanical properties. Summary of the Invention

[0006] The present invention aims to solve one or more technical problems existing in the above-mentioned prior art and at least provide a beneficial alternative. Specifically, the present invention provides a porous scaffold that promotes bone regeneration and immune regulation, has good mechanical properties, and is simple to prepare.

[0007] The inventive concept of the present invention is as follows: The porous scaffold comprises a scaffold matrix and polydopamine deposited on the surface of the scaffold matrix; the scaffold matrix comprises a base material and mesoporous silica dispersed within the base material. The polydopamine coating on the surface of the porous scaffold facilitates cell adhesion and proliferation, promotes the osteogenic differentiation of stem cells, and induces macrophage differentiation in a direction conducive to tissue regeneration and repair, thus endowing the porous scaffold with osteogenesis and immunomodulatory functions. Furthermore, the base material and the deposited polydopamine layer render the porous scaffold degradable. Mesoporous silica possesses excellent biocompatibility, a large specific surface area, and high safety. This porous scaffold not only exhibits excellent biocompatibility but also possesses a three-dimensional interconnected porous structure with uniform pore structure, high strength, and high porosity, providing space for the regeneration of new bone and vascular tissue. The scaffold gradually degrades, creating more space. However, if the pore space of the porous scaffold is too small, macrophages can be polarized in a pro-inflammatory direction, leading to inflammation and fibrous encapsulation, which is detrimental to bone regeneration. Through the combined action of mesoporous silica and polydopamine layers, the porous scaffold has the functions of promoting bone regeneration and immune regulation, and has good mechanical properties, and can be well used for the repair and reconstruction of bone tissue defects.

[0008] Thus, a first aspect of the present invention provides a porous scaffold.

[0009] Specifically, the porous scaffold includes a scaffold matrix and polydopamine deposited on the surface of the scaffold matrix;

[0010] The support matrix includes a matrix material and mesoporous silicon dispersed in the matrix material.

[0011] Specifically, the surface of the stent matrix includes the outer surface of the stent matrix and the surface of the pore wall of the porous structure of the stent matrix.

[0012] Preferably, the matrix material comprises poly(lactic-co-glycolic acid).

[0013] Preferably, the molecular weight of the polylactic acid-glycolic acid copolymer is 20,000-60,000 Daltons; more preferably, the molecular weight of the polylactic acid-glycolic acid copolymer is 25,000-45,000 Daltons.

[0014] Preferably, the particle size of the mesoporous silicon is 270-850 nm; further preferably, the particle size of the mesoporous silicon is 300-800 nm; even more preferably, the particle size of the mesoporous silicon is 500-700 nm or 300-600 nm or 600-800 nm.

[0015] Preferably, the specific surface area of ​​the mesoporous silicon is 450-1600m 2 / g; Further preferably, the specific surface area of ​​the mesoporous silicon is 500-1500m 2 / g.

[0016] Preferably, the average pore size of the mesoporous silicon is 2-11 nm; further preferably, the average pore size of the mesoporous silicon is 2-10 nm.

[0017] Preferably, the pore volume of the mesoporous silica is 0.9-1.6 cm 3 / g; Further preferably, the pore volume of the mesoporous silica is 1.0-1.5cm 3 / g.

[0018] The second aspect of the present invention provides a method for preparing the porous scaffold described in the first aspect of the present invention.

[0019] Specifically, the method for preparing the porous scaffold comprises the following steps:

[0020] (1) mixing a matrix material solution with mesoporous silica to obtain a mixed solution; then mixing the mixed solution with a surfactant solution, and freeze-drying the mixture to obtain composite microspheres;

[0021] (2) Filling the composite microspheres obtained in step (1) into a mold, keeping the mold warm and shaping it to obtain a mesoporous silicon / matrix material porous scaffold, and soaking it in a dopamine solution to obtain the porous scaffold.

[0022] Preferably, in step (1), the concentration of the matrix material solution is 0.045-0.32 g / mL.

[0023] Preferably, in step (1), the mass ratio of the matrix material to the mesoporous silicon in the matrix material solution is 10:(0.45-2.8); further preferably, the mass ratio of the matrix material to the mesoporous silicon in the matrix material solution is 10:(0.5-2.5).

[0024] Preferably, in step (1), the matrix material solution and mesoporous silicon are mixed and stirred, the stirring speed is 450-11000 rpm, and the stirring time is 5 min-2.2 h; further preferably, the stirring speed is 500-10000 rpm, and the stirring time is 5 min-2 h.

[0025] Preferably, the matrix material in the matrix material solution includes polylactic acid-glycolic acid copolymer.

[0026] Preferably, in step (1), the volume ratio of the mixed solution to the surfactant solution is 1:(18-110); further preferably, the volume ratio of the mixed solution to the surfactant solution is 1:(20-100).

[0027] Preferably, in step (1), the concentration of the surfactant solution is 2.5-55 mg / mL.

[0028] Preferably, in step (1), the solute in the surfactant solution includes at least one of polyvinyl alcohol, gelatin, and carboxymethyl cellulose.

[0029] Preferably, in step (1), the surfactant solution is an aqueous solution containing a surfactant.

[0030] Preferably, in step (1), after the mixed solution and the surfactant solution are mixed, they are first stirred at room temperature and then freeze-dried. The rotation speed of the room temperature stirring is 180-1100 rpm, and the time of the room temperature stirring is 3.5-13 h; further preferably, the rotation speed of the room temperature stirring is 200-1000 rpm, and the time of the room temperature stirring is 4-12 h.

[0031] Preferably, the freeze-drying time is 22-80 hours; further preferably, the freeze-drying time is 24-72 hours.

[0032] Preferably, the freeze-drying temperature is -85 to -18°C; more preferably, the freeze-drying temperature is -80 to -20°C.

[0033] Preferably, in step (2), the temperature of the heat preservation and shaping is 55-85°C, and the heat preservation and shaping time is 3.5-8.5h; further preferably, in step (2), the temperature of the heat preservation and shaping is 60-80°C, and the heat preservation and shaping time is 4-8h.

[0034] Preferably, in step (2), the concentration of the dopamine solution is 1.4-3.3 mg / mL; further preferably, in step (2), the concentration of the dopamine solution is 1.5-3 mg / mL.

[0035] Preferably, in step (2), the pH of the dopamine solution is 8.0-9.0.

[0036] Specifically, the dopamine solution is a weakly alkaline aqueous solution containing dopamine.

[0037] Preferably, in step (2), the soaking time is 1.5-8.5 hours; further preferably, the soaking time is 2-8 hours.

[0038] The third aspect of the present invention provides a use of the porous scaffold described in the first aspect of the present invention in the field of bone repair.

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

[0040] (1) The porous scaffold of the present invention has high strength, high porosity, and a three-dimensional open-pore structure, which can provide space for the regeneration of new bone / vascular tissue.

[0041] (2) The surface of the porous scaffold is provided with a polydopamine coating, which is conducive to cell adhesion and proliferation, promotes the osteogenic differentiation of stem cells, and induces macrophage differentiation in a direction that is conducive to tissue regeneration and repair. In addition, it works together with mesoporous silica, so that the porous scaffold has the functions of promoting bone regeneration and immune regulation, and has good mechanical properties, which can be well used for the repair and reconstruction of bone tissue defects.

[0042] (3) The preparation method of the present invention has a simple process, low requirements on equipment, and the raw materials are cheap and easily available, which is conducive to industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The scanning electron microscope images of the porous scaffolds of Example 1 and Comparative Example 1 of the present invention are shown;

[0044] Figure 2 The Fourier transform infrared spectrum test graphs of the porous stents of Example 1 and Comparative Example 1 of the present invention;

[0045] Figure 3 The mechanical properties of the porous scaffolds of Examples 1-3 and Comparative Example 1 of the present invention are shown;

[0046] Figure 4 The water contact angle test results of the porous scaffolds of Example 1 and Comparative Example 1 of the present invention are shown;

[0047] Figure 5 The porosity test results of the porous scaffolds of Examples 1-3 and Comparative Example 1 of the present invention are shown;

[0048] Figure 6 Graph showing the cell proliferation promotion results of the porous scaffolds of Example 1 and Comparative Example 1 of the present invention;

[0049] Figure 7 This is an alkaline phosphatase staining image of Example 1 and Comparative Example 1 of the present invention to promote the osteogenic differentiation of rat bone marrow mesenchymal stem cells;

[0050] Figure 8 This is a quantitative analysis result of Example 1 and Comparative Example 1 of the present invention on the effect of porous scaffolds on the osteogenic differentiation of rat bone marrow mesenchymal stem cells;

[0051] Figure 9 This is a graph showing the test results of the porous scaffold promoting macrophage polarization in Example 1 and Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0052] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0053] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0054] Example 1

[0055] A method for preparing a porous scaffold comprises the following steps:

[0056] (1) 3 g of PLGA (molecular weight 30,000 Daltons) was dissolved in 15 mL of dichloromethane to obtain a PLGA solution. The PLGA solution and 0.5 g of mesoporous silica (particle size range 500-700 nm, specific surface area 1457 m 2 / g, the average pore diameter is 2.32nm, and the pore volume is 1.45cm 3 / g) and stirred at a speed of 2000 rpm for 30 min to mix evenly to obtain a mesoporous silica / PLGA mixed solution;

[0057] (2) adding the mesoporous silica / PLGA mixture obtained in step (1) to 600 mL of a 15 mg / mL aqueous solution of polyvinyl alcohol 1799, stirring continuously at 400 rpm for 10 h at room temperature, washing with deionized water, and freeze-drying for 48 h to obtain mesoporous silica / PLGA composite microspheres;

[0058] (3) Filling the composite microspheres obtained in step (2) into a cylindrical mold, keeping the temperature at 80°C for 4 hours, then cooling to room temperature, and demolding to obtain a mesoporous silica / PLGA porous scaffold; placing the mesoporous silica / PLGA porous scaffold in a weakly alkaline aqueous solution of dopamine with a concentration of 2.5 mg / mL (pH of 8.4), soaking for 6 hours, washing, and drying to obtain a mesoporous silica / PLGA porous scaffold containing a polydopamine coating.

[0059] Example 2

[0060] A method for preparing a porous scaffold comprises the following steps:

[0061] 0.5 g of PLGA (molecular weight of 20,000 Daltons) was dissolved in 10 mL of dichloromethane to obtain a PLGA solution. The PLGA solution and 0.125 g of mesoporous silica (particle size range of 300-600 nm, specific surface area of ​​864 m 2 / g, the average pore diameter is 5.85nm, and the pore volume is 1.18cm 3 / g) and stirred at a speed of 500 rpm for 120 min to mix evenly to obtain a mesoporous silica / PLGA mixed solution;

[0062] The mesoporous silica / PLGA mixture obtained in step (1) was added to 1000 mL of a 2.5 mg / mL carboxymethyl cellulose aqueous solution, stirred at 1000 rpm for 4 h at room temperature, washed with deionized water, and freeze-dried for 24 h to obtain mesoporous silica / PLGA composite microspheres;

[0063] The composite microspheres obtained in step (2) were filled into a cylindrical mold, kept warm at 60°C for 8 hours, then cooled to room temperature and demolded to obtain a mesoporous silica / PLGA porous scaffold; the mesoporous silica / PLGA porous scaffold was placed in a weakly alkaline aqueous solution of dopamine with a concentration of 3 mg / mL (pH value of 8.0), soaked for 2 hours, washed and dried to obtain a mesoporous silica / PLGA porous scaffold containing a polydopamine coating.

[0064] Example 3

[0065] A method for preparing a porous scaffold comprises the following steps:

[0066] 2.5 g of PLGA (molecular weight of 60,000 Daltons) was dissolved in 10 mL of dichloromethane to obtain a PLGA solution. The PLGA solution and 0.125 g of mesoporous silica (particle size range of 600-800 nm, specific surface area of ​​521 m 2 / g, the average pore diameter is 9.87nm, and the pore volume is 1.03cm 3 / g) and stirred at a speed of 10000 rpm for 5 min to obtain a mesoporous silica / PLGA mixed solution;

[0067] The mesoporous silica / PLGA mixture obtained in step (1) was added to 200 mL of a 50 mg / mL gelatin aqueous solution, stirred at 200 rpm for 12 h at room temperature, washed with deionized water, and freeze-dried for 72 h to obtain mesoporous silica / PLGA composite microspheres;

[0068] The composite microspheres obtained in step (2) were filled into a cylindrical mold, kept warm at 70°C for 6 hours, then cooled to room temperature and demolded to obtain a mesoporous silica / PLGA porous scaffold; the mesoporous silica / PLGA porous scaffold was placed in a weakly alkaline aqueous solution of dopamine (pH 9.0) with a concentration of 1.5 mg / mL, soaked for 8 hours, washed and dried to obtain a mesoporous silica / PLGA porous scaffold containing a polydopamine coating.

[0069] Comparative Example 1

[0070] The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain a polydopamine layer, that is, in step (3) of Comparative Example 1, the mesoporous silicon / PLGA porous scaffold is not immersed in a weakly alkaline aqueous solution (pH 8.4) of dopamine at a concentration of 2.5 mg / mL. The rest is the same as Example 1.

[0071] Performance Testing

[0072] 1. Scanning electron microscopy observation

[0073] The surfaces of the porous scaffolds of Example 1 and Comparative Example 1 were observed using a scanning electron microscope. The samples were freeze-dried and gold-sprayed before observation, and then the surface morphologies of Example 1 and Comparative Example were observed using a scanning electron microscope.

[0074] The scanning electron microscope images of the porous scaffold of Example 1 and Comparative Example 1 are as follows: Figure 1 As shown. Among them, Figure 1 Figures (a) and (c) are scanning electron micrographs of the porous scaffold of Comparative Example 1 at different magnifications; Figure 1 Figures (b) and (d) are scanning electron micrographs of the porous scaffold of Example 1 at different magnifications; Figure 1 The scale of Figure (a) is the same as Figure 1 In Figure (b), Figure 1 The scale of Figure (c) is the same as Figure 1 Figure (d) in .

[0075] Depend on Figure 1 It can be seen that the mesoporous silica / PLGA porous scaffold in Comparative Example 1 without a polydopamine layer has a pore structure and good pore connectivity, and the surface is relatively rough; while the surface of the porous scaffold in Example 1 becomes smoother after the polydopamine coating is deposited.

[0076] 2. Fourier transform infrared spectroscopy detection

[0077] The Fourier transform infrared spectra of the porous scaffolds of Example 1 and Comparative Example 1 were tested. The testing method was as follows: the porous scaffolds of Example 1 and Comparative Example 1 were mixed and ground in a mortar to obtain a sample powder. The sample powder was evenly mixed with KBr and pressed into a sheet. The sample powder was then preheated for 15-30 minutes using a Fourier transform infrared spectrometer (FTIR). The sample spectrum was then detected by FTIR in the spectral range of 400-4000 cm -1 .

[0078] The Fourier transform infrared spectroscopy test results of the porous scaffold of Example 1 and Comparative Example 1 are as follows: Figure 2 shown.

[0079] Depend on Figure 2 It can be seen that after the polydopamine coating is deposited in Example 1, the -1New characteristic absorption peaks appeared on the left and right, which may be caused by the stretching vibration of the -NH- group in polydopamine, indicating that polydopamine was successfully deposited on the surface of the porous scaffold.

[0080] 3. Mechanical properties test

[0081] The porous scaffolds of Examples 1-3 and Comparative Example 1 were subjected to compression tests at room temperature at a speed of 1 mm / min using a universal testing machine (INSTRON 5967). Five samples were prepared for each group, each with a diameter of 10 mm and a height of 8 mm. On the stress-strain curve, the compressive strength of the scaffold was measured as the pressure at which the compression deformation reached 60%.

[0082] The mechanical properties of the porous scaffolds of Examples 1-3 and Comparative Example 1 are as follows: Figure 3 As shown. Among them, Figure 3 Figure (a) is the compressive strength diagram. Figure 3 Figure (b) is the compressive modulus diagram.

[0083] Depend on Figure 3 It can be seen that the compressive strength and Young's modulus of the porous scaffolds of Examples 1-3 are better than those of Comparative Example 1, and have good mechanical properties.

[0084] 4. Water contact angle test

[0085] The porous supports of Example 1 and Comparative Example 1 were placed on a lifting platform respectively, and the height of the lifting platform was adjusted. Liquid droplets were dropped on the surface of the supports using a contact angle meter. The amount of liquid dropped each time was 2 μL. The position where the sample and the droplet contacted was adjusted to coincide with the measurement baseline. A static image of the droplet was obtained by taking a picture, and the contact angle (θ) of each group of supports was calculated using the contact angle measurement software.

[0086] The water contact angle test results of the porous scaffolds in Example 1 and Comparative Example 1 are as follows: Figure 4 As shown. Figure 4 Figures (a) and (b) are static images of droplets during water contact angle testing of the porous scaffolds in Comparative Example 1 and Example 1, respectively. Figure 4 Figure (c) shows the water contact angles of the porous scaffolds of Example 1 and Comparative Example 1, **p<0.01.

[0087] Depend on Figure 4 It can be seen that the water contact angle of the porous scaffold in Example 1 is significantly smaller than that in Comparative Example 1, indicating that the hydrophilicity of the porous scaffold in Example 1 is significantly better than that in Comparative Example 1. The hydrophilicity of the porous scaffold of the present invention is significantly improved, which is beneficial to cell adhesion and proliferation.

[0088] 5. Scaffold Porosity Test

[0089] The porosity of the porous scaffolds of Examples 1-3 and Comparative Example 1 was tested using the following method:

[0090] The mass of the dry scaffold sample was measured and recorded as M0. The pycnometer was filled with anhydrous ethanol at room temperature, and the total mass was recorded as M1. The sample was placed in the pycnometer and ultrasonicated for 5 minutes to remove all bubbles. The total mass was measured and recorded as M2. The sample was taken out and the remaining mass was recorded as M3. The porosity calculation formula of the scaffold was: P = (M2-M3-M0) / (M1-M3).

[0091] The porosity test results of the porous scaffolds of Examples 1-3 and Comparative Example 1 are as follows: Figure 5 shown.

[0092] Depend on Figure 5 It can be seen that the porous scaffolds of Examples 1-3 of the present invention have good porosity, indicating that after the polydopamine coating is deposited, although the surface of the scaffold becomes smoother, it can still maintain the pore structure and good pore connectivity.

[0093] 6. Cell proliferation promoting properties

[0094] The cell proliferation promoting performance of the porous scaffolds of Example 1 and Comparative Example 1 was tested, and the specific testing method is as follows:

[0095] After recovery, rat bone marrow mesenchymal stem cells (BMSCs) were cultured in a constant temperature cell culture incubator at 37°C and a carbon dioxide volume concentration of 5%. BMSCs were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 μg / mL penicillin, and 100 μg / mL streptomycin. Cells were passaged when the confluence reached 70%-80%. The cell generations used in this experiment were 3-5. Before the experiment, each group of scaffolds was sterilized by cobalt-60 irradiation. The control group consisted of only BMSCs cells without any added materials.

[0096] BMSCs were selected as evaluation cells, and the cells on the sample surface were stained with a live-dead cell staining kit. The morphology and distribution of the cells on the sample surface were observed using an inverted fluorescence microscope, and photos were taken to detect the cell growth viability of BMSCs. The selected cells were tested after 72 hours of adhesion and proliferation on the sample surface. In short, the sterilized scaffold samples were placed in a 48-well plate, and 1×10 cells were seeded into each sample. 4 The culture medium was removed after 1, 4, and 7 days of culture, and the cell proliferation activity on different scaffolds was evaluated using a CCK-8 kit, and the OD value at 450 nm was measured by a microplate reader.

[0097] The results of promoting BMSCs proliferation by porous scaffolds in Example 1 and Comparative Example 1 are as follows: Figure 6 As shown. Among them, Figure 6Figures (a) and (b) show the BMSCs cell growth activity on the porous scaffold surface of Comparative Example 1 and Example 1 on the 4th day, respectively. Figure 6 The scale in (a) is the same as that in (b); Figure 6 Figure (c) shows the results of promoting cell proliferation activity of the porous scaffolds of Example 1 and Comparative Example 1, *p<0.05, **p<0.01, ***p<0.001.

[0098] Depend on Figure 6 It can be seen that compared with comparative example 1 and the control group, Example 1 can significantly promote the proliferation of BMSCs, which also shows that the porous scaffold prepared by the present invention has good biocompatibility.

[0099] 7. Evaluation of Osteogenesis in Vitro

[0100] The in vitro osteogenesis performance of the porous scaffolds of Example 1 and Comparative Example 1 was evaluated, and the specific process was as follows:

[0101] 1×10 5 Individual BMSCs were added to a 48-well plate containing a porous scaffold sample and a blank well. After culturing for 24 hours, the supernatant was discarded and osteogenic induction culture medium (100 mL complete culture medium, 0.39 mg dexamethasone, 1.76 mg vitamin C and 306.11 mg sodium β-glycerophosphate, where 100 mL complete culture medium consists of 90 mL basal culture medium (DMEM) and 10 mL fetal bovine serum) was added respectively. The medium was changed regularly. On the 7th and 14th days after osteogenic induction, the scaffold was stained for alkaline phosphatase (ALP staining) using an ALP staining kit, and the distribution of ALP on the scaffold was observed under an inverted fluorescence microscope and photographed. ALP quantitative analysis was performed using an ALP detection kit and a BCA detection kit.

[0102] Example 1, Comparative Example 1 Porous scaffold promotes BMSCs osteogenic differentiation ALP staining Figure 7 As shown, Figure 7 Figures (a) and (b) are ALP staining images of the porous scaffold in comparative example 1 promoting osteogenic differentiation of BMSCs on the 7th and 14th days of osteogenic induction, respectively; Figure 7 Figures (c) and (d) are ALP staining images of the porous scaffold in Example 1 promoting osteogenic differentiation of BMSCs after 7 days and 14 days of osteogenic induction, respectively; Figure 7 The scales in Figures (a), (b), and (c) are the same as in Figure (d).

[0103] The quantitative analysis results of the in vitro osteogenic performance of the porous scaffolds in Example 1 and Comparative Example 1 are shown in the figure. Figure 8 As shown, ***p<0.001.

[0104] Depend on Figure 7and 8 It can be seen that the in vitro osteogenic performance of the porous scaffold in Example 1 is better than that in Comparative Example 1, indicating that depositing polydopamine on the surface of the porous scaffold can enhance its in vitro osteogenic performance.

[0105] 8. Macrophage Polarization Test

[0106] The porous scaffolds of Example 1 and Comparative Example 1 were tested for promoting macrophage polarization. The specific process is as follows:

[0107] RAW264.7 cells were plated at 5 × 10 4 The cells were seeded at a density of 100 cells / mL on a porous scaffold sample in a 48-well plate, and a control group was set up (the control group consisted of only RAW264.7 cells without any added materials). On days 1 and 3, the culture medium was removed and total RNA was extracted from each sample using the "column extraction method". After successful RNA extraction, cDNA was obtained by reverse transcription using a PCR top heater. Real-time fluorescence quantitative PCR was used to detect the expression of the M1 immune marker gene tumor necrosis factor α (TNF-α) and the M2 immune marker gene arginase (ARG) of macrophages, with 3-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference gene.

[0108] The primer sequences used for real-time quantification of selected genes are shown in Table 1.

[0109] Table 1: Primer sequences used for real-time quantification of selected genes

[0110]

[0111] Example 1, Comparative Example 1 The test results of porous scaffold promoting macrophage polarization are as follows Figure 9 As shown, *p<0.05, **p<0.01, ***p<0.001.

[0112] The polarization state of macrophages affects the process of bone regeneration. Figure 9 It can be seen that in the process of inducing polarization of RAW264.7 cells, the porous scaffold groups of Example 1 and Comparative Example 1 were significantly better than the control group. The polydopamine layer gave the porous scaffold of Example 1 a stronger ability to regulate the polarization of macrophages to the M2 phenotype. Compared with Comparative Example 1, the performance of Example 1 in upregulating the expression of macrophage M2 marker genes (ARG) and inhibiting the expression of M1 marker genes (TNF-α) was more significant. That is, the polydopamine layer can induce macrophages to differentiate in a direction that is conducive to tissue regeneration and repair, so that the porous scaffold of Example 1 has the functions of promoting bone and immune regulation.

[0113] In summary, the surface of the porous scaffold of the present invention is provided with a polydopamine coating, which is conducive to cell adhesion and proliferation, promotes the osteogenic differentiation of stem cells and induces macrophages to differentiate in a direction that is conducive to tissue regeneration and repair, so that the porous scaffold has the functions of promoting bone regeneration and immune regulation. Mesoporous silica has the characteristics of good biocompatibility, large specific surface area, and high safety, so that the porous scaffold not only has good biocompatibility and degradability, but also has a three-dimensional interconnected porous structure, and the pore structure is uniform, high strength, high porosity, and can provide sufficient space. Through the combined action of mesoporous silica and the polydopamine layer, the porous scaffold has the functions of promoting bone regeneration and immune regulation, and has good mechanical properties, and can be well used for the repair and reconstruction of bone tissue defects.

[0114] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A porous scaffold, characterized in that: The porous scaffold comprises a scaffold matrix and polydopamine deposited on the surface of the scaffold matrix; The support matrix includes a matrix material and mesoporous silicon dispersed in the matrix material.

2. The porous scaffold according to claim 1, wherein The matrix material includes poly(lactic acid-co-glycolic acid).

3. The porous scaffold according to claim 2, characterized in that The molecular weight of the polylactic acid-glycolic acid copolymer is 20,000-60,000 Daltons.

4. The porous scaffold according to claim 1, wherein The particle size of the mesoporous silicon is 270-850 nm; and / or the specific surface area of ​​the mesoporous silicon is 450-1600 m 2 / g; and / or, the average pore size of the mesoporous silica is 2-11 nm; and / or, the pore volume of the mesoporous silica is 0.9-1.6 cm 3 / g.

5. The method for preparing the porous scaffold according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) mixing a matrix material solution with mesoporous silica to obtain a mixed solution; then mixing the mixed solution with a surfactant solution, and freeze-drying the mixture to obtain composite microspheres; (2) placing the composite microspheres obtained in step (1) in a mold, keeping them warm and shaping them to obtain a mesoporous silicon / matrix material porous scaffold, and soaking them in a dopamine solution to obtain the porous scaffold.

6. The preparation method according to claim 5, characterized in that In step (1), the concentration of the matrix material solution is 0.045-0.32 g / mL; and / or the mass ratio of the matrix material to the mesoporous silicon in the matrix material solution is 10:(0.45-2.8).

7. The preparation method according to claim 5, characterized in that In step (1), the volume ratio of the mixed solution to the surfactant solution is 1:(18-110); and / or the concentration of the surfactant solution is 2.5-55 mg / mL; and / or the solute in the surfactant solution includes at least one of polyvinyl alcohol, gelatin, and carboxymethyl cellulose.

8. The preparation method according to claim 5, characterized in that In step (1), after the mixed solution and the surfactant solution are mixed, they are first stirred at room temperature and then freeze-dried; the rotation speed of the room temperature stirring is 180-1100 rpm, and the time of the room temperature stirring is 3.5-13 hours; and / or the time of the freeze-drying is 22-80 hours.

9. The preparation method according to claim 5, characterized in that In step (2), the temperature of the heat preservation and setting is 55-85° C., and the heat preservation and setting time is 3.5-8.5 hours; and / or the pH of the dopamine solution is 8.0-9.0; and / or the concentration of the dopamine solution is 1.4-3.3 mg / mL; and / or the immersion time is 1.5-8.5 hours.

10. Use of the porous scaffold according to any one of claims 1 to 4 in the field of bone repair.

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