Preparation method and physical and chemical property detection method of PLL-MgBGs / SF frozen gel

Abstract

The invention discloses a preparation method of PLL-MgBGs / SF frozen gel and a physical and chemical property detection method of the frozen gel. The preparation method comprises the following steps: preparing a magnesium-doped bioactive substance MgBGs; magnesium-doped bioactive matter PLL-MgBGs nanoparticles loaded with the polylysine are prepared; and mixing the prepared PLL-MgBGs nanoparticles with a mulberry silk protein SF solution, and carrying out low-temperature freezing and freeze-drying, so as to prepare the PLL-MgBGs / SF frozen gel. The PLL-MgBGs / SF frozen gel has the advantages that the prepared PLL-MgBGs / SF frozen gel has excellent shape memory ability and mechanical performance and good anti-swelling performance and mechanical strain performance, so that the water response shape memory ability is excellent, active ions such as Mg < 2 + >, Ca < 2 + > and SiO4 < 4-> can be released, remarkable potential can be shown in immunoregulation and bone repair, the biocompatibility is good, and the frozen gel can be applied to the field of bone repair. According to the present invention, the bone repair material has characteristics of cell proliferation promotion, BMSCs cell migration promotion, good BMSCs collection ability, good medical research value, and good application prospect in the field of minimally invasive bone defect repair.

Description

Technical Field

[0001] The present invention relates to the field of medical technologies, and in particular to a method for preparing PLL-MgBGs / SF cryogel and detecting its physicochemical properties. Background Art

[0002] With the extension of the average human lifespan and the aging of the global population, bone-related diseases have increased significantly. In the United States, more than 600,000 bone grafts are used annually to treat bone defects caused by cancer and trauma, costing more than $2.5 billion. Autologous or allogeneic bone grafting is a common method for treating large long bone defects. Autologous bone grafting is considered the "gold standard", but it has a long operation time and a risk of complications at the donor site. Allogeneic transplantation has risks of immune rejection and pathogen transmission, and the source is limited.

[0003] In recent years, the development of bone implants has provided solutions to this problem. Bone implant materials with excellent mechanical properties and corrosion resistance have been developed to improve bone integration performance and bone defect repair.

[0004] Clinically, currently commonly used bone graft substitutes, such as titanium alloy materials, although having certain mechanical properties, their biological inertness leads to a mismatch in integration with host bone defect tissues, resulting in implant loosening and even ineffective bone repair. In addition, existing bone substitutes lack shape adaptability and are insufficient in matching irregular bone defects. Regularizing irregular bones during surgery to match the shape of the implant will further cause bone tissue defects.

[0005] In the field of bone defect repair, 3D printing technology is widely used, mainly including preoperative planning and surgical simulation, which can reduce the surgical difficulty, reduce surgical trauma, and promote postoperative recovery. Although 3D-printed implants can solve the repair problems of large and complex bone defects that are difficult to solve with some allogeneic or autologous bones, the in-situ implantation of 3D-printed implants for small or irregular bone defects into the bone defect position is much more difficult than that of shape-adaptive materials. The implanted biomaterial contacts the damaged bone tissue, forms a new biological interface, induces biological signals, promotes in-situ bone regeneration and bone integration, and reduces failure.

[0006] However, when the implanted biomaterial is recognized as a foreign substance by the immune system, it will trigger an immune response, leading to chronic inflammation and bone repair failure.

[0007] Therefore, it is of great significance to develop bone repair substances with stable structures, good shape adaptability, excellent shape memory ability and mechanical properties, good anti-swelling performance and mechanical strain performance, good biocompatibility, which can promote cell proliferation, and can also promote the migration of BMSCs cells, etc., to adapt to irregular bone defects and achieve minimally invasive regenerative treatment of bone defects.

[0008] In response to this, the inventor of this patent combined clinical experience, deeply pondered the problems encountered in clinical work, consulted a large number of scientific research materials and literature, and through retrieval and novelty search, gradually conceived and designed this application to solve related technical problems. Summary of the Invention

[0009] The present invention aims to solve at least one of the technical problems in the related art to some extent. For this purpose, the object of the present invention is to propose a preparation method of PLL-MgBGs / SF cryogel and its physicochemical property detection method.

[0010] To achieve one of the above objects, according to an embodiment of the present invention, the preparation method of PLL-MgBGs / SF cryogel includes the following steps:

[0011] S1. Prepare magnesium-doped bioactive substance MgBGs;

[0012] Use the sol-gel method combined with the microemulsion co-template method to prepare magnesium-doped bioactive substance MgBGs;

[0013] S2. Prepare PLL-MgBGs nanoparticles;

[0014] Use polylysine PLL as a modifier, mix it with magnesium-doped bioactive substance MgBGs, and stir evenly, so that the positively charged polylysine PLL and the negatively charged magnesium-doped bioactive substance MgBGs are combined with each other through electrostatic interaction, and then dried to prepare magnesium-doped bioactive substance PLL-MgBGs nanoparticles loaded with polylysine;

[0015] S3. Prepare PLL-MgBGs / SF cryogel;

[0016] Mix the prepared PLL-MgBGs nanoparticles with silk fibroin SF solution, and through low-temperature freezing and freeze-drying, prepare PLL-MgBGs / SF cryogel.

[0017] In addition, according to the preparation method of PLL-MgBGs / SF cryogel and its physicochemical property detection method of the above embodiment of the present invention, it may also have the following additional technical features:

[0018] According to an embodiment of the present invention, the step S1 specifically includes:

[0019] S11. Dissolve 4 g of dodecylamine (DDA) in a mixture of 80 mL of absolute ethanol and 25 mL of deionized water (DIW), and slowly dissolve it at 40 °C;

[0020] S12. Add tetraethyl orthosilicate (TEOS) dropwise and continue stirring for 30 min;

[0021] S13. Add 1.66 mL of TEP, 8.837 g of calcium nitrate tetrahydrate (CN), and 1.547 g of magnesium nitrate (MN) successively every 30 min, and keep the temperature at 40 °C all the time, and stir for 3 h;

[0022] S14. Centrifuge and wash 3 times with absolute ethanol and DIW to obtain the crude product;

[0023] S15. Sinter the crude product in air at 650 °C for 3 h to obtain magnesium-doped bioactive substance MgBGs.

[0024] According to an embodiment of the present invention, the step S2 specifically includes:

[0025] S21. Measure 1 g of magnesium-doped bioactive substance MgBGs and disperse it in 100 mL of deionized water (DIW) to form a mixed solution, and ultrasonicate for 30 min;

[0026] S22. Measure 0.06 g of polylysine PLL and add it to the formed mixed solution, and continue stirring for 24 h;

[0027] S23. Centrifuge the stirred mixed solution for 10 min, wash it with deionized water (DIW) at least 3 times, and dry it at 60 °C to obtain PLL-MgBGs nanoparticles.

[0028] According to an embodiment of the present invention, the step S3 specifically includes:

[0029] S31. Use silk fibroin SF as the main material and remove the sericin protein in the silk fibroin SF;

[0030] S32. Dissolve the silk fibroin SF from which the sericin protein has been removed in 9.3 M LiBr solution, and dialyze to remove LiBr to obtain a pure SF solution;

[0031] S33. Mix the prepared PLL-MgBGs nanoparticles with the obtained pure SF solution, and prepare PLL-MgBGs / SF cryogel through low-temperature freezing and lyophilization.

[0032] To achieve the second above-mentioned object, the physical and chemical property detection method of the PLL-MgBGs / SF cryogel according to the embodiment of the present invention includes the following steps:

[0033] S101. Make the above-prepared PLL-MgBGs / SF cryogel into several cylindrical scaffolds with exactly the same height and diameter, and weigh their masses respectively;

[0034] S102. At room temperature, immerse several prepared PLL-MgBGs / SF cryogel cylinder scaffolds in multiple groups of PBS solutions at different time points (0.5 days, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, and 28 days). When the immersion time is reached, wipe and weigh them respectively with a wet tissue paper.

[0035] S103. Calculate the equilibrium swelling ratio of several prepared PLL-MgBGs / SF cryogel cylinder scaffolds according to the following formula: SD = Ws / Wd;

[0036] Where SD is the equilibrium swelling ratio, Ws is the weight of the swollen PLL-MgBGs / SF cryogel cylinder scaffold, and Wd is the weight of the dry PLL-MgBGs / SF cryogel cylinder scaffold; the finally calculated equilibrium swelling degree is the average value of the equilibrium swelling ratios of the corresponding several PLL-MgBGs / SF cryogel cylinder scaffolds.

[0037] S104. Measure the water absorption rate of the PLL-MgBGs / SF cryogel cylinder scaffold in PBS solution. The calculation formula is: water absorption rate = (We - Wd) / Wd;

[0038] Where We refers to the weight of the PLL-MgBGs / SF cryogel cylinder scaffold equilibrated in PBS solution, and Wd refers to the weight of the dry PLL-MgBGs / SF cryogel cylinder scaffold; the finally calculated water absorption rate is the average value of the water absorption rates of the corresponding several PLL-MgBGs / SF cryogel cylinder scaffolds.

[0039] To achieve the third above-mentioned purpose, the physicochemical property detection method of PLL-MgBGs / SF cryogel according to the embodiment of the present invention includes the following steps:

[0040] S201. Make the above-prepared PLL-MgBGs / SF cryogel into a cylinder scaffold.

[0041] S202. In the compression test, set the compression strain rate to 0.5 N / min, and increase the force from 0 to 18 N to make the strain degree of the prepared PLL-MgBGs / SF cryogel cylinder scaffold reach 80%.

[0042] S203. In the cyclic compression test, drop a drop of water on the surface of the prepared PLL-MgBGs / SF cryogel cylinder scaffold and let it strain cycle 50 times with a compression degree of 30%.

[0043] To achieve the fourth above-mentioned object, a method for detecting the physicochemical properties of PLL-MgBGs / SF cryogels according to an embodiment of the present invention includes the following steps:

[0044] S301. Fabricate several cylindrical scaffolds with exactly the same height and diameter from the PLL-MgBGs / SF cryogel prepared above.

[0045] S302. Compress the front, side, and top views of several prepared PLL-MgBGs / SF cryogel cylindrical scaffolds respectively, drop PBS solution onto their top views, observe their recovery, take pictures and record for analysis.

[0046] S303. Place several 30-mg PLL-MgBGs / SF cryogel cylindrical scaffolds into multiple groups of 15 mL of simulated body fluid (SBF), and incubate them at 37 °C on a shaker at a speed of 120 rpm for 0 min, 30 min, 12 h, 1 day, 3 days, 5 days, and 7 days respectively.

[0047] S304. Collect the supernatant at the above different incubation time points, and detect the concentrations of Mg2+, Ca 2+ and SiO 4 4- in the SBF solution collected at each time point by ICP-AES.

[0048] To achieve the fifth above-mentioned object, a method for detecting the physicochemical properties of PLL-MgBGs / SF cryogels according to an embodiment of the present invention includes the following steps:

[0049] S401. Prepare a complete medium suspension containing 1 mg / mL of PLL-MgBGs / SF cryogel, incubate it at 37 °C on a shaker at a speed of 100 rpm for 24 h, and filter it through a 0.22-μm sterile filter head to prepare a sterile PLL-MgBGs / SF cryogel extract.

[0050] S402. Seed bone marrow mesenchymal stem cells BMSCs (CRL-12424, ATCC, America) into a 48-well plate at an initial concentration of 10,000 cells / well, and incubate them in a culture medium in an incubator at 37 °C with 5% CO2 for 24 h.

[0051] S403. Remove the culture medium, replace it with the PLL-MgBGs / SF cryogel extract, continue culturing for 1 day, 3 days, and 7 days, wash 3 times with PBS, add 500 μL / well of CCK8 working solution, incubate at 37 °C for 30 min in the dark, then aliquot into a 96-well plate, and detect the absorbance using an enzyme-linked immunosorbent assay (ELISA) reader.

[0052] S404. Use Live / Dead cell staining. Remove the culture medium from the cultured cells and wash them 3 times with PBS. Add Calcein AM diluted 1:1000 and propidium iodide (PI) diluted 2:1000 respectively, incubate at 37 °C for 30 min, and wash 3 times with PBS. Obtain pictures and analyze them using an inverted fluorescence microscope.

[0053] To achieve the sixth above-mentioned object, the physical and chemical property detection method of PLL-MgBGs / SF cryogel according to the embodiment of the present invention includes the following steps:

[0054] S501. Prepare a complete medium suspension containing 1 mg / mL of PLL-MgBGs / SF cryogel, incubate at 100 rpm on a shaker at 37 °C for 24 h, and filter with a 0.22 μm sterile filter head to prepare a sterile PLL-MgBGs / SF cryogel extract.

[0055] S502. Seed bone marrow mesenchymal stem cells BMSCs (CRL-12424, ATCC, America) in a 48-well plate at an initial concentration of 10,000 cells / well, and incubate with the medium in an incubator at 37 °C and containing 5% CO2 for 24 h.

[0056] S503. Add 600 μL of the PLL-MgBGs / SF cryogel extract to the lower chamber of the Transwell, continue to culture for 12 h, remove the medium, wash 3 times with PBS, and add 4% paraformaldehyde to fix for 30 min.

[0057] S504. Add 0.1% crystal violet staining solution, incubate at room temperature for 30 min, wash 3 times with PBS, obtain pictures using a microscope, and randomly select 6 regions and analyze them using Image-J software.

[0058] The beneficial effects of the present invention are:

[0059] The PLL-MgBGs / SF cryogel prepared in this application has excellent shape memory ability and mechanical properties, good anti-swelling performance and mechanical strain performance, resulting in excellent water-responsive shape memory ability, and can release Mg2+, Ca 2+ and SiO 4 4- and other active ions, and can show significant potential in immunomodulation and bone repair. It has good biocompatibility, the ability to promote cell proliferation, can also promote the migration of BMSCs cells, has a good ability to recruit BMSCs, making it have good medical research value and good application prospects in the field of minimally invasive bone defect repair.

[0060] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned by practice of the present invention. Brief Description of the Drawings

[0061] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0062] Figure 1 is a flowchart of the steps of the preparation method of the PLL-MgBGs / SF cryogel of the present invention;

[0063] Figure 2 is a flowchart of the steps of step S1 in the embodiment of the present invention;

[0064] Figure 3 is a flowchart of the steps of step S2 in the embodiment of the present invention;

[0065] Figure 4 is a flowchart of the steps of step S3 in the embodiment of the present invention;

[0066] Figure 5 is a flowchart of the steps for detecting the swelling characteristics of the prepared PLL-MgBGs / SF cryogel in the embodiment of the present invention;

[0067] Figure 6 is a flowchart of the steps for detecting the mechanical properties of the prepared PLL-MgBGs / SF cryogel in the embodiment of the present invention;

[0068] Figure 7 is a flowchart of the steps for detecting the active ions contained in the prepared PLL-MgBGs / SF cryogel in the embodiment of the present invention;

[0069] Figure 8 is a flowchart of the steps for detecting the biocompatibility of the prepared PLL-MgBGs / SF cryogel in the embodiment of the present invention;

[0070] Figure 9 is a flowchart of the steps for detecting the ability of the prepared PLL-MgBGs / SF cryogel to recruit BMSCs in the embodiment of the present invention;

[0071] Figure 10 is the preparation process topology of the PLL-MgBGs / SF cryogel of the present invention Figure 1 ;

[0072] Figure 11 is the topological diagram of the preparation process of the PLL-MgBGs / SF cryogel of the present invention Figure 2 ;

[0073] Figure 12 is the diagram showing the properties and potential analysis of the substances formed in each step in the embodiment of the present invention;

[0074] Figure 13 is the diagram showing the characteristics and anti-swelling properties of the substances formed in each step in the embodiment of the present invention;

[0075] Figure 14 is the diagram showing the water-responsive shape memory test results of the prepared PLL-MgBGs / SF cryogel in the embodiment of the present invention;

[0076] Figure 15 is the diagram showing the biocompatibility and the ability to recruit BMSCs of the prepared PLL-MgBGs / SF cryogel in the embodiment of the present invention;

[0077] The realization, functional features and advantages of the object of the present invention will be further described with reference to the embodiments and the accompanying drawings. Detailed Embodiments

[0078] The following describes in detail the embodiments of the present invention. The examples of the embodiments are shown in the accompanying drawings of the specification, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0079] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "circumferential", "radial", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings of the specification. These are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present invention.

[0080] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "a plurality of" means two or more unless otherwise specifically defined.

[0081] In the present invention, unless otherwise clearly defined and limited, the terms such as "installed", "connected", "connected", "fixed", etc. should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0082] In the present invention, unless otherwise clearly defined and limited, the first feature being "on" or "under" the second feature may include the direct contact between the first and second features, or may include the situation where the first and second features are not in direct contact but in contact through other features therebetween. Moreover, the first feature being "above", "over" and "on" the second feature includes that the first feature is directly above and obliquely above the second feature, or merely indicates that the horizontal height of the first feature is higher than that of the second feature. The first feature being "under", "beneath" and "under" the second feature includes that the first feature is directly below and obliquely below the second feature, or merely indicates that the horizontal height of the first feature is lower than that of the second feature.

[0083] The preparation of PLL-MgBGs / SF cryogel and the detection method of its physical and chemical properties according to the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0084] Example 1

[0085] Referring to Figure 1 、 Figure 10 and Figure 11 as shown, according to the preparation method of PLL-MgBGs / SF cryogel provided by the embodiments of the present invention, the following steps are included:

[0086] S1. Prepare magnesium-doped bioactive glass MgBGs;

[0087] Prepare magnesium-doped bioactive substance MgBGs by using the sol-gel method combined with the microemulsion co-template method;

[0088] It should be noted here that bioactive ions with strong immunomodulatory properties have been applied to various inflammatory and degenerative diseases as well as the field of bone immunomodulation. Currently, the commercially available magnesium-doped bioactive glass (MgBGs), because it can release magnesium ions (Mg2+ ) Calcium ions (Ca 2+ ) and silicate ions (SiO 4 4- ) and other active ions show significant potential in immunomodulation and bone repair. Therefore, magnesium plays an important role in bone health, regulating cell functions such as proliferation, adhesion, and migration, and promoting angiogenesis.

[0089] Thus, compared with pure magnesium ions, magnesium-doped bioactive substances MgBGs are more suitable as bone tissue repair materials due to their slow and continuous magnesium ion release, avoiding the problems of rapid degradation and hydrogen release. In addition, magnesium-doped bioactive substances MgBGs are materials that can form chemical bonds with biological tissues. By introducing magnesium elements, magnesium-doped bioactive substances MgBGs can not only enhance the bioactivity of the materials but also improve their mechanical properties.

[0090] S2. Prepare PLL-MgBGs nanoparticles;

[0091] Using polylysine PLL as a modifier, mix it with magnesium-doped bioactive substance MgBGs and stir evenly, so that the positively charged polylysine PLL and the negatively charged magnesium-doped bioactive substance MgBGs are combined with each other through electrostatic interaction, and then dried to prepare magnesium-doped bioactive substance PLL-MgBGs nanoparticles loaded with polylysine;

[0092] S3. Prepare PLL-MgBGs / SF cryogel;

[0093] Mix the prepared PLL-MgBGs nanoparticles with silk fibroin SF solution, and through low-temperature freezing and freeze-drying, prepare PLL-MgBGs / SF cryogel.

[0094] It should be noted here that silk fibroin (SF) is a natural protein biomaterial extracted from silkworm cocoons, with good biocompatibility, controllable biodegradability, and excellent mechanical properties, and is widely used in the fields of tissue regeneration, drug delivery, and medical implants.

[0095] However, due to the lack of shape memory effect in traditional SF materials, their structures are easily damaged under external forces, resulting in poor integration with bone defects after implantation. Shape memory SF materials need to be tough enough, fatigue-resistant, and have sufficient resilience to expand in a limited space. In addition, bone repair is a complex process involving various cells, signaling molecules, and growth factors. These biochemical signals can promote bone formation by triggering cell proliferation and differentiation, accelerating bone healing, and improving the repair quality. To improve the bioactivity of SF materials, they are usually combined with other bioactive factors. However, due to biosafety and ethical issues, materials with added cells or factors are restricted in clinical applications.

[0096] Therefore, in this application, magnesium-doped bioactive substance MgBGs, polylysine PLL, and silk fibroin (SF) are combined to prepare PLL-MgBGs / SF cryogel by the above method.

[0097] Preferably, in this technical solution, the control Figure 2 As shown, according to an embodiment of the present invention, the step S1 specifically includes:

[0098] S11. Dissolve 4 g of dodecylamine (DDA) in a mixture of 80 mL of absolute ethanol and 25 mL of deionized water (DIW), and slowly dissolve it at 40 °C.

[0099] S12. Dropwise add tetraethyl orthosilicate (TEOS) and continue stirring for 30 min.

[0100] S13. Add 1.66 mL of TEP, 8.837 g of calcium nitrate tetrahydrate (CN), and 1.547 g of magnesium nitrate (MN) every 30 min in sequence, and keep the temperature at 40 °C all the time, and stir for 3 h.

[0101] S14. Centrifuge and wash with absolute ethanol and DIW three times to obtain a crude product.

[0102] S15. Sinter the crude product in air at 650 °C for 3 h to obtain magnesium-doped bioactive substance MgBGs.

[0103] Preferably, in this technical solution, the control Figure 3 As shown, according to an embodiment of the present invention, the step S2 specifically includes:

[0104] S21. Measure 1 g of magnesium-doped bioactive substance MgBGs and disperse it in 100 mL of deionized water (DIW) to form a mixed solution, and sonicate for 30 min.

[0105] S22. Weigh 0.06 g of polylysine PLL and add it to the formed mixed solution, then continue stirring for 24 h;

[0106] S23. Centrifuge the stirred mixed solution for 10 min, wash it with deionized water (DIW) at least 3 times, and dry it at 60 °C to obtain PLL-MgBGs nanoparticles with a PLL content of 6%.

[0107] Preferably, in this technical solution, the control Figure 4 As shown, according to an embodiment of the present invention, the step S3 specifically includes:

[0108] S31. Using silk fibroin SF as the main material, remove the sericin protein in the silk fibroin SF;

[0109] S32. Dissolve the silk fibroin SF from which the sericin protein has been removed in a 9.3 M LiBr solution and dialyze to remove LiBr to obtain a pure SF solution;

[0110] S33. Mix the prepared PLL-MgBGs nanoparticles with a PLL content of 6% with the obtained pure SF solution, and after low-temperature freezing and lyophilization, prepare a PLL-MgBGs / SF cryogel with a PLL content of 6%.

[0111] The control Figure 12 As shown in (a), the SEM results show that the prepared magnesium-doped bioactive agent MgBGs, the PLL-MgBGs / SF cryogel with a PLL content of 3%, and the PLL-MgBGs / SF cryogel with a PLL content of 6% all exhibit a regular spherical structure.

[0112] The control Figure 12 As shown in (b), the XRD results show that the prepared magnesium-doped bioactive agent MgBGs, the PLL-MgBGs / SF cryogel with a PLL content of 3%, and the PLL-MgBGs / SF cryogel with a PLL content of 6% all have characteristic peaks of bioactive glass in the range of 20° - 30°, indicating that the surface PLL modification does not affect the microstructure and crystal configuration of MgBGs.

[0113] In addition, the potential analysis results show that PLL-MgBGs modified with 0.06 g of PLL has a positive potential and has the potential to bind to silk fibroin (SF) through electrostatic interaction.

[0114] Regarding the above operation steps, it should be noted here that the control Figure 12As shown in (c), in the above step S2, 0.06 g of polylysine PLL is measured as the modifier. The reason is that through experiments, it is found that when the measured polylysine PLL is less than 0.06 g, such as 0.03 g or 0.00 g, when its potential is detected using a nanoparticle size analyzer, it shows a negative potential, which is not the result required by this application. Only when the measured polylysine PLL is 0.06 g or more, it shows a positive potential, so that an appropriate amount of polylysine PLL carrying a positive potential and an appropriate amount of magnesium-doped bioactive substance MgBGs carrying a negative potential can bind to each other through electrostatic interaction, and finally magnesium-doped bioactive substance nanoparticles PLL-MgBGs loaded with polylysine can be prepared.

[0115] Furthermore, for the prepared PLL-MgBGs / SF cryogel, the preparation schematic diagram is referred to Figure 13 as shown in (a); the crystalline configuration of the PLL-MgBGs / SF cryogel is characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The results show that it is a typical type I crystal. Specifically, it can be referred to Figure 13 as shown in (b) and Figure 13 as shown in (c). This crystal can endow the PLL-MgBGs / SF cryogel with excellent shape memory ability and mechanical properties; the SEM results show that the prepared magnesium-doped bioactive substance MgBGs, the PLL-MgBGs / SF cryogel with 3% PLL content, and the PLL-MgBGs / SF cryogel with 6% PLL content all have a macroporous connected structure. Specifically, it can be referred to Figure 13 as shown in (d).

[0116] Example Two

[0117] Control Figure 5 As shown, according to the method for detecting the physicochemical properties of the PLL-MgBGs / SF cryogel according to the embodiments of the present invention, its swelling characteristics are detected, including the following steps:

[0118] S101. The prepared PLL-MgBGs / SF cryogel is made into several cylindrical scaffolds with exactly the same height and diameter (height is 0.5 cm, diameter is 0.5 cm), and their masses are weighed respectively.

[0119] S102. At room temperature, the prepared several PLL-MgBGs / SF cryogel cylindrical scaffolds are correspondingly immersed in multiple groups of PBS solutions at different time points (0.5 days, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, and 28 days). When the immersion time is reached, they are wiped and weighed with a wet tissue paper respectively.

[0120] S103. Calculate the equilibrium swelling ratio of several prepared PLL-MgBGs / SF cryogel cylinder scaffolds according to the following formula: SD = Ws / Wd;

[0121] Where SD is the equilibrium swelling ratio, Ws is the weight of the swollen PLL-MgBGs / SF cryogel cylinder scaffold, and Wd is the weight of the dried PLL-MgBGs / SF cryogel cylinder scaffold;

[0122] The calculated final equilibrium swelling degree is the average value of the equilibrium swelling ratios of the corresponding several PLL-MgBGs / SF cryogel cylinder scaffolds;

[0123] S104. Measure the water absorption rate of the PLL-MgBGs / SF cryogel cylinder scaffold in PBS solution. The calculation formula is: water absorption rate = (We - Wd) / Wd;

[0124] Where We refers to the weight of the PLL-MgBGs / SF cryogel cylinder scaffold equilibrated in PBS solution, and Wd refers to the weight of the dried PLL-MgBGs / SF cryogel cylinder scaffold;

[0125] The calculated final water absorption rate is the average value of the water absorption rates of the corresponding several PLL-MgBGs / SF cryogel cylinder scaffolds.

[0126] Control Figure 13 As shown in (e), the results show that the PLL-MgBGs / SF cryogel prepared in this application has good anti-swelling performance.

[0127] Example 3

[0128] Control Figure 6 As shown, according to the method for detecting the physicochemical properties of the PLL-MgBGs / SF cryogel according to the embodiment of the present invention, its mechanical properties are detected, including the following steps:

[0129] S201. Fabricate the above-prepared PLL-MgBGs / SF cryogel into a cylinder scaffold (height 0.5 cm, diameter 0.5 cm);

[0130] S202. In the compression test, the compression strain rate is set to 0.5 N / min. Under the condition that the force continuously increases from 0 to 18 N, the strain degree of the prepared PLL-MgBGs / SF cryogel cylinder scaffold reaches 80%;

[0131] S203. In the cyclic compression test, drop a drop of water on the surface of the prepared PLL-MgBGs / SF cryogel cylinder scaffold and let it strain cyclically 50 times at a compression degree of 30%.

[0132] Control Figure 13 (f) and Figure 13 as shown in (g), Figure 13 (f) shows the stress-strain curve in the compression test, Figure 13 (g) shows the curve of cyclic compression results;

[0133] Control Figure 14 As shown, the results show that the PLL-MgBGs / SF cryogel prepared in this application has good mechanical strain performance and can return to its original state after mechanical strain, resulting in excellent water-responsive shape memory ability.

[0134] Example 4

[0135] Control Figure 7 As shown, according to the method for detecting the physicochemical properties of the PLL-MgBGs / SF cryogel according to the embodiment of the present invention, the active ions contained therein are detected, including the following steps:

[0136] S301. Fabricate several cylindrical scaffolds with exactly the same height and diameter (height: 0.5 cm, diameter: 0.5 cm) from the above-prepared PLL-MgBGs / SF cryogel;

[0137] S302. Compress the front, side, and top views of several prepared PLL-MgBGs / SF cryogel cylindrical scaffolds respectively, drop PBS solution onto their top views, observe their recovery, take pictures and record for analysis;

[0138] S303. Place several 30-mg PLL-MgBGs / SF cryogel cylindrical scaffolds into multiple groups of 15 mL of simulated body fluid (SBF) and incubate them on a shaker at 37 °C at a speed of 120 rpm for 0 min, 30 min, 12 h, 1 day, 3 days, 5 days, and 7 days respectively;

[0139] S304. Collect the supernatant at the above different incubation time points and detect the concentrations of Mg2+, Ca 2+ and SiO 4 4- in the SBF solution collected at each time point by ICP-AES.

[0140] The results show that the PLL-MgBGs / SF cryogel prepared in this application can release various active ions such as Mg2+, Ca 2+ and SiO 4 4- etc., showing significant potential in immunomodulation and bone repair.

[0141] Example 5

[0142] ControlFigure 8 As shown, for the method of detecting the physical and chemical properties of PLL-MgBGs / SF cryogels according to an embodiment of the present invention, the biocompatibility thereof is detected, including the following steps:

[0143] S401. Prepare a complete medium suspension containing 1 mg / mL of PLL-MgBGs / SF cryogel, incubate it at 100 rpm on a shaker at 37 °C for 24 h, and filter it through a 0.22 μm sterile filter head to prepare a sterile PLL-MgBGs / SF cryogel extract;

[0144] S402. Seed bone marrow mesenchymal stem cells BMSCs (CRL-12424, ATCC, America) in a 48-well plate at an initial concentration of 10,000 cells / well, and incubate them with the medium in an incubator at 37 °C and containing 5% CO2 for 24 h;

[0145] S403. Remove the medium, replace it with the PLL-MgBGs / SF cryogel extract, continue culturing for 1 day, 3 days, and 7 days, wash 3 times with PBS, add 500 μL / well of CCK8 working solution, incubate at 37 °C for 30 min in the dark, then aliquot into a 96-well plate, and detect the absorbance using an enzyme-linked immunosorbent assay (ELISA) reader;

[0146] S404. Perform Live / Dead cell staining. For the cultured cells, remove the medium and wash 3 times with PBS; add Calcein AM diluted 1:1000 and propidium iodide (PI) diluted 2:1000 respectively, incubate at 37 °C for 30 min, and wash 3 times with PBS; obtain pictures and analyze them using an inverted fluorescence microscope.

[0147] For step S403 therein, it is the PLL-MgBGs / SF group with a PLL content of 6%. Correspondingly, a control group (Control group, without filling material) and an SF group (filled with SF cryogel) are also set;

[0148] The control group is: Remove the medium, continue culturing for 1 day, 3 days, and 7 days, wash 3 times with PBS, add 500 μL / well of CCK8 working solution, incubate at 37 °C for 30 min in the dark, then aliquot into a 96-well plate, and detect the absorbance using an ELISA reader;

[0149] The SF group is: Remove the medium, replace it with the SF cryogel extract, continue culturing for 1 day, 3 days, and 7 days, wash 3 times with PBS, add 500 μL / well of CCK8 working solution, incubate at 37 °C for 30 min in the dark, then aliquot into a 96-well plate, and detect the absorbance using an ELISA reader;

[0150] Control Figure 15(a) and Figure 15 As shown in (b), the results show that the PLL-MgBGs / SF cryogel prepared in this application, corresponding to the detection by CCK-8 and live / dead cell staining experiments, shows that the PLL-MgBGs / SF cryogel with 6% PLL content has good biocompatibility, is non-toxic, and has the ability to promote cell proliferation.

[0151] Example Six

[0152] Control Figure 9 As shown, according to the method for detecting the physicochemical properties of the PLL-MgBGs / SF cryogel according to the embodiments of the present invention, its ability to recruit bone marrow mesenchymal stem cells BMSCs is detected, including the following steps:

[0153] S501. Prepare a complete medium suspension containing 1 mg / mL of PLL-MgBGs / SF cryogel, incubate it at 100 rpm on a shaker at 37 °C for 24 h, and filter it through a 0.22 μm sterile filter head to prepare a sterile PLL-MgBGs / SF cryogel extract.

[0154] S502. Seed bone marrow mesenchymal stem cells BMSCs (CRL-12424, ATCC, America) in a 48-well plate at an initial concentration of 10,000 cells / well, and incubate them with the medium in an incubator at 37 °C and containing 5% CO2 for 24 h.

[0155] S503. Add 600 μL of the PLL-MgBGs / SF cryogel extract to the lower chamber of the Transwell, continue to culture for 12 h, remove the medium, wash it 3 times with PBS, and add 4% paraformaldehyde to fix it for 30 min.

[0156] S504. Add 0.1% crystal violet staining solution, incubate it at room temperature for 30 min, wash it 3 times with PBS, obtain pictures with a microscope, and randomly select 6 regions and analyze them using Image-J software.

[0157] For step S503 therein, it is the PLL-MgBGs / SF group with 6% PLL content. Correspondingly, a control group (Control group, without filling material) and an SF group (filling SF cryogel) will also be set;

[0158] The control group is: Do not add other materials to the lower chamber of the Transwell, continue to culture for 12 h, remove the medium, wash it 3 times with PBS, and add 4% paraformaldehyde to fix it for 30 min.

[0159] The SF group was as follows: 600 μL of SF cryogel extract was added to the lower chamber of Transwell, and the cells were cultured for another 12 h. The culture medium was removed, and the cells were washed 3 times with PBS and then fixed with 4% paraformaldehyde for 30 min.

[0160] Control Figure 15 (c) As shown, the results showed that the PLL-MgBGs / SF cryogel with 6% PLL prepared in this application could promote the migration of bone marrow mesenchymal stem cells (BMSCs) and had good ability to recruit BMSCs when detected by Transwell assay.

[0161] For other embodiments, etc., no examples will be given here.

[0162] Therefore, in summary, the PLL-MgBGs / SF cryogel prepared in this application generally presented a regular spherical structure. It had characteristic peaks of bioactive glass in the range of 20°-30°, indicating that the surface PLL modification did not affect the microstructure and crystal configuration of MgBGs. And it was a typical type I crystal, which could endow the PLL-MgBGs / SF cryogel with excellent shape memory ability and mechanical properties. Moreover, it had a macroporous connected structure, good anti-swelling performance, and good mechanical strain performance, and could return to its original state after mechanical strain, resulting in excellent water-responsive shape memory ability.

[0163] At the same time, it could release multiple active ions such as Mg2+ and Ca 2+ and SiO 4 4- etc., showing significant potential in immunomodulation and bone repair. When detected by CCK-8 and live / dead cell staining assays, it showed good biocompatibility, was non-toxic, and had the ability to promote cell proliferation. When detected by Transwell assay, it could promote the migration of BMSCs and had good ability to recruit BMSCs.

[0164] Therefore, the PLL-MgBGs / SF cryogel prepared in this application had good medical research value and had good application prospects in the field of minimally invasive bone defect repair.

[0165] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0166] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structural transformation made by using the content of the specification and drawings of the present invention under the inventive concept of the present invention, or direct / indirect application in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A method for preparing PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S1, preparing magnesium-doped bioactive substances MgBGs; The magnesium-doped bioactive material MgBGs was prepared by using the sol-gel method combined with the microemulsion co-template method. S2, preparing PLL-MgBGs nanoparticles; Polylysine PLL is used as a modifier, and it is mixed with magnesium-doped bioactive material MgBGs, and stirred evenly, so that the polylysine PLL carrying a positive potential and the magnesium-doped bioactive material MgBGs carrying a negative potential are combined with each other through electrostatic interaction, and then dried to prepare polylysine-loaded magnesium-doped bioactive material PLL-MgBGs nanoparticles; S3, preparing PLL-MgBGs / SF cryogel; The prepared PLL-MgBGs nanoparticles were mixed with mulberry silk protein SF solution, and then cryogenically frozen and freeze-dried to prepare PLL-MgBGs / SF cryogel.

2. The method for preparing PLL-MgBGs / SF cryogel according to claim 1, characterized in that: The step S1 specifically includes: S11, dissolving 4 g of dodecylamine (DDA) in a mixture of 80 mL of anhydrous ethanol and 25 mL of deionized water (DIW), and slowly dissolving at 40° C.; S12, add tetraethyl orthosilicate (TEOS) dropwise and continue stirring for 30 min; S13, adding 1.66 mL of TEP, 8.837 g of calcium nitrate tetrahydrate (CN) and 1.547 g of magnesium nitrate (MN) in sequence every 30 min, and keeping the temperature at 40° C. and stirring for 3 h; S14, washing by centrifugation with anhydrous ethanol and DIW three times to obtain a crude product; S15. Sinter the crude product at 650° C. in air for 3 h to obtain magnesium-doped bioactive material MgBGs.

3. The method for preparing PLL-MgBGs / SF cryogel according to claim 2, characterized in that: The step S2 specifically includes: S21, 1 g of magnesium-doped bioactive material MgBGs was dispersed in 100 mL of deionized water (DIW) to form a mixed solution, and ultrasonicated for 30 min; S22, taking 0.06 g of polylysine PLL, adding it to the formed mixed solution, and continuing stirring for 24 h; S23, centrifuging the stirred mixture for 10 minutes, washing with deionized water (DIW) for at least 3 times, and drying at 60° C. to obtain PLL-MgBGs nanoparticles.

4. The method for preparing PLL-MgBGs / SF cryogel according to claim 3, characterized in that: The step S3 specifically includes: S31, using mulberry silk protein SF as the main material, removing sericin from the mulberry silk protein SF; S32, dissolving the mulberry silk protein SF from which sericin has been removed in a 9.3 M LiBr solution, and dialyzing to remove LiBr to obtain a pure SF solution; S33, mixing the prepared PLL-MgBGs nanoparticles with the obtained pure SF solution, and preparing PLL-MgBGs / SF cryogel through low-temperature freezing and freeze-drying.

5. A method for detecting the physical and chemical properties of PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S101, making the PLL-MgBGs / SF cryogel prepared according to any one of claims 1 to 4 into several cylindrical stents with exactly the same height and diameter, and weighing the masses thereof respectively; S102, immersing the prepared PLL-MgBGs / SF cryogel cylindrical scaffolds in multiple groups of PBS solutions at room temperature for different time points (0.5 days, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 21 days and 28 days), and wiping and weighing them with wet paper towels when the immersion time is reached; S103, calculating the equilibrium swelling ratio of the prepared PLL-MgBGs / SF cryogel cylindrical scaffolds according to the following formula: SD = Ws / Wd; Wherein, SD is the equilibrium swelling ratio, Ws is the weight of the swollen PLL-MgBGs / SF cryogel cylindrical scaffold, and Wd is the weight of the dry PLL-MgBGs / SF cryogel cylindrical scaffold; S104. The water absorption rate of the PLL-MgBGs / SF cryogel cylindrical scaffold was measured in a PBS solution, and the calculation formula was: water absorption rate = (We-Wd) / Wd; Wherein, We refers to the weight of the PLL-MgBGs / SF cryogel cylindrical scaffold equilibrated in PBS solution, and Wd refers to the weight of the dried PLL-MgBGs / SF cryogel cylindrical scaffold.

6. A method for detecting the physical and chemical properties of PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S201, preparing a cylindrical stent by using the PLL-MgBGs / SF cryogel prepared according to any one of claims 1 to 4; S202, in the compression test, the compression strain rate is set to 0.5N / min, and the strain degree of the prepared PLL-MgBGs / SF cryogel cylindrical scaffold reaches 80% when the force is continuously increased from 0 to 18N; S203. In a cyclic compression test, a drop of water is dropped on the surface of the prepared PLL-MgBGs / SF cryogel cylindrical scaffold, and the scaffold is strained and cycled 50 times at a compression degree of 30%.

7. A method for detecting the physical and chemical properties of PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S301, making the PLL-MgBGs / SF cryogel prepared as claimed in any one of claims 1 to 4 into a plurality of cylindrical stents with exactly the same height and diameter; S302, compressing the front, side and top surfaces of several prepared PLL-MgBGs / SF cryogel cylindrical scaffolds respectively, dripping PBS solution onto the top surfaces thereof, observing their recovery conditions, taking photos and recording and analyzing; S303, placing several 30 mg PLL-MgBGs / SF cryogel cylindrical scaffolds into multiple groups of 15 mL simulated body fluid (SBF), and incubating them on a shaker at 37°C at 120 rpm for 0 min, 30 min, 12 h, 1 day, 3 days, 5 days and 7 days respectively; S304, collecting the supernatants at the above different incubation time points, and detecting the Mg2+, Ca2+ and Mg2+ in the SBF solution collected at each time point by ICP-AES. 2+ and SiO4 4- concentration.

8. A method for detecting the physical and chemical properties of PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S401, preparing a complete culture medium suspension containing 1 mg / mL of PLL-MgBGs / SF cryogel, incubating at 100 rpm on a shaker at 37°C for 24 h, and filtering with a 0.22 μm sterile filter to prepare a sterile PLL-MgBGs / SF cryogel extract; S402, seeding bone marrow mesenchymal stem cells (BMSCs) (CRL-12424, ATCC, America) in a 48-well plate at an initial concentration of 10,000 cells / well, and incubating with culture medium in an incubator at 37° C. and 5% CO 2 for 24 h; S403, remove the culture medium and replace it with PLL-MgBGs / SF cryogel extract, continue culturing for 1 day, 3 days and 7 days, wash 3 times with PBS, add 500 μL / well of CCK8 working solution, incubate at 37°C for 30 min in the dark, then dispense into 96-well plates, and detect absorbance with an enzyme reader; S404. Live / Dead cell staining was used. The cultured cells were removed from the culture medium and washed three times with PBS. Calcein AM diluted 1:1000 and propidium iodide (PI) diluted 2:1000 were added respectively, incubated at 37°C for 30 min, and washed three times with PBS. Images were acquired and analyzed using an inverted fluorescence microscope.

9. A method for detecting the physical and chemical properties of PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S501, preparing a complete culture medium suspension containing 1 mg / mL of PLL-MgBGs / SF cryogel, incubating on a shaker at 37°C at 100 rpm for 24 h, and filtering with a 0.22 μm sterile filter to prepare a sterile PLL-MgBGs / SF cryogel extract; S502, seeding bone marrow mesenchymal stem cells (BMSCs) (CRL-12424, ATCC, America) in a 48-well plate at an initial concentration of 10,000 cells / well, and incubating with culture medium in an incubator at 37° C. and 5% CO 2 for 24 h; S503, add 600 μL of PLL-MgBGs / SF cryogel extract to the lower chamber of Transwell, continue culturing for 12 h, remove the culture medium, wash with PBS three times, and add 4% paraformaldehyde to fix for 30 min; S504, add 0.1% crystal violet staining solution, incubate at room temperature for 30 minutes, wash 3 times with PBS, obtain pictures under a microscope, and use Image-J software to randomly select 6 areas for analysis.

Images