Bone immunoregulation osteogenesis promotion and osteogenic ability detection method of PLL-MgBGs / SF frozen gel

Abstract

The invention discloses a bone immunoregulation osteogenesis promoting method of PLL-MgBGs / SF frozen gel and an osteogenesis capability detection method of the PLL-MgBGs / SF frozen gel. The bone immunoregulation osteogenesis promoting method comprises the following steps: preparing a magnesium-doped bioactive substance MgBGs; the preparation method comprises the following steps: preparing PLL-MgBGs nano particles; pLL-MgBGs / SF frozen gel is prepared; a sterile PLL-MgBGs / SF frozen gel leaching solution is prepared; the method comprises the following steps: inoculating macrophages (RAW264.7) into a 48-pore plate, culturing by using a complete culture medium containing LPS (100 ng / mL), and then culturing by using a complete culture medium containing a PLL-MgBGs / SF frozen gel leaching solution. The PLL-MgBGs / SF freezing gel has the advantages that the prepared PLL-MgBGs / SF freezing gel is excellent in water response shape memory ability, capable of releasing various active ions such as Mg < 2 + >, Ca < 2 + > and SiO4 < 4->, remarkable in potential in immunoregulation and bone repair, capable of being implanted into bone defects in an injection mode to fill the bone defects, capable of being rapidly matched with irregular bone defects and capable of improving the bone repair effect. According to the present invention, the problem of insufficient matching of the traditional bone scaffold and the irregular defect site is solved, the excellent mechanical stability and the excellent structure retention can be represented during the degradation process, the good supporting ability is provided in the bone defect region, the polarization of the macrophage can be adjusted to be the M2 type, and the collection, the proliferation and the osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) can be promoted.

Description

Technical Field

[0001] The present invention relates to the field of medical technologies, and particularly to a method for detecting the osteoimmunomodulatory osteogenesis and osteogenic ability of PLL-MgBGs / SF cryogels. 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 the repair of bone defects.

[0004] Clinically, currently commonly used substitutes for bone grafts, 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, so as to 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 failures.

[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. As important innate immune cells, macrophages play a key role in the immune response. Highly plastic macrophages can switch between pro-inflammatory M1 and anti-inflammatory M2 phenotypes. If the implanted material activates the M1 phenotype, macrophages secrete inflammatory factors (such as IL-6, IL-1β, iNOS, and TNF-α), and the long-term action of these inflammatory factors will lead to chronic inflammation, induce the formation of a fibrous capsule, and ultimately result in bone repair failure. If the M2 phenotype is activated, it will play a direct role in bone defect regeneration by secreting anti-inflammatory factors (such as Arg-1 and IL-10), promoting the formation of a bone regeneration environment.

[0007] Therefore, it is of great significance to develop bone repair materials with stable structure, good shape adaptability, excellent shape memory ability and mechanical properties, which can adapt to irregular bone defects, regulate macrophage M2 polarization, and form a bone regeneration environment closely related to local osteogenic differentiation and bone integration, so as to achieve minimally invasive regenerative treatment of bone defects.

[0008] In view of this, the inventors of this patent combined clinical experience, deeply thought about the problems encountered in clinical work, consulted a large number of scientific research materials and literature, and gradually conceived and designed this application through novelty search 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 provide a method for detecting the osteoimmunomodulatory osteogenesis and osteogenic ability of PLL-MgBGs / SF cryogels.

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

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

[0012] The magnesium-doped bioactive substance MgBGs is prepared by using the sol-gel method combined with the microemulsion co-template method;

[0013] S2. Prepare PLL-MgBGs nanoparticles;

[0014] Using polylysine PLL as a modifier, mixing it with the magnesium-doped bioactive substance MgBGs and stirring 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 drying to prepare magnesium-doped bioactive substance PLL-MgBGs nanoparticles loaded with polylysine;

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

[0016] Mix the prepared PLL-MgBGs nanoparticles with silk fibroin (SF) solution, and prepare PLL-MgBGs / SF cryogels through low-temperature freezing and lyophilization.

[0017] S4. Make a complete medium suspension containing 1 mg / mL of PLL-MgBGs / SF cryogels, incubate it on a shaker at 37 °C 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.

[0018] S5. Seed macrophages (RAW264.7) in a 48-well plate, culture them with medium for 24 h, then change the medium to a complete medium containing LPS (100 ng / mL) and culture for 8 h, and then change it to a complete medium containing PLL-MgBGs / SF cryogel extract and culture for 3 days. Extract total mRNA using a total RNA kit, and detect the expression of pro-inflammatory genes (TNF-α, iNOS) and anti-inflammatory genes (IL-10, Arg-1) by RT-qPCR.

[0019] In addition, according to the method for detecting the osteoimmunomodulatory osteogenesis promotion and osteogenic ability of PLL-MgBGs / SF cryogels in the above embodiments of the present invention, the following additional technical features may also be included:

[0020] According to an embodiment of the present invention, step S1 specifically includes:

[0021] 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.

[0022] S12. Dropwise add tetraethyl orthosilicate (TEOS) and continue stirring for 30 min.

[0023] 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.

[0024] S14. Centrifuge and wash 3 times with absolute ethanol and DIW to obtain a crude product.

[0025] S15. Sinter the crude product in air at 650 °C for 3 h to obtain magnesium-doped bioactive glass MgBGs.

[0026] According to an embodiment of the present invention, step S2 specifically includes:

[0027] S21. Weigh 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;

[0028] S22. Weigh 0.06 g of polylysine PLL and add it to the formed mixed solution, and continue to stir for 24 h;

[0029] 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.

[0030] According to an embodiment of the present invention, step S3 specifically includes:

[0031] S31. Using silk fibroin SF as the main material, remove the sericin protein in silk fibroin SF;

[0032] S32. Dissolve the silk fibroin SF with sericin protein removed in 9.3 M LiBr solution, and dialyze to remove LiBr to obtain a pure SF solution;

[0033] S33. Mix the prepared PLL-MgBGs nanoparticles with the obtained pure SF solution, and through low-temperature freezing and freeze-drying, prepare PLL-MgBGs / SF cryogel.

[0034] To achieve the second above-mentioned object, the osteogenic ability detection method of PLL-MgBGs / SF cryogel according to an embodiment of the present invention includes the following steps:

[0035] S101. Seed bone marrow mesenchymal stem cells BMSCs into a 24-well plate and culture for 24 h, then add complete medium containing the extraction solution of PLL-MgBGs / SF cryogel for culture;

[0036] S102. After 14 days, remove the complete medium, wash 3 times with PBS, fix in 4% paraformaldehyde for 30 min, and then wash with PBS;

[0037] S103. Stain the bone marrow mesenchymal stem cells BMSCs with an alkaline phosphatase (ALP) chromogenic kit for 30 min, and use an inverted fluorescence microscope to obtain pictures and analyze the results.

[0038] To achieve the third above-mentioned object, the osteogenic ability detection method of PLL-MgBGs / SF cryogel according to an embodiment of the present invention includes the following steps:

[0039] S201. Inoculate bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culture for 24 h. Then add complete medium containing the extraction solution of PLL-MgBGs / SF cryogel for culture, and incubate in an incubator at 37 °C with 5% CO2.

[0040] S202. After 14 days, remove the complete medium, wash 3 times with PBS, fix in 4% paraformaldehyde for 30 min, and then wash thoroughly with PBS.

[0041] S203. Add alizarin red staining solution and incubate at 37 °C for 30 min. Wash 3 times with PBS, and use an inverted fluorescence microscope to obtain pictures and analyze the results.

[0042] To achieve the fourth above-mentioned object, the method for detecting the osteogenic ability of PLL-MgBGs / SF cryogel according to the embodiment of the present invention includes the following steps:

[0043] S301. Select 60 male SD rats at 8 weeks old with a body weight of 200 - 250 g, and anesthetize the rats with 3% pentobarbital solution (150 μL / 100 g).

[0044] S302. Make a 1-cm incision on the head of the rat, and use a trephine to drill a defect with a diameter of 0.5 cm on the skull within the incision on the rat's head, and ensure that the dura mater is not damaged.

[0045] S303. Thaw the prepared PLL-MgBGs / SF cryogel, and implant it into the defect by injection to fill it, and suture the incision.

[0046] S304. After the operation, put the rats back into the breeding room for free activities, and continuously inject penicillin for 3 days to prevent infection.

[0047] S305. After 12 weeks, sacrifice the rats by CO2 inhalation method, remove the excess muscle tissue, take out the rat skull specimens, and use a Micro-CT scanner to examine the rat skull specimens and analyze the results.

[0048] The beneficial effects of the present invention are:

[0049] In the method for promoting osteogenesis by bone immunomodulation of PLL-MgBGs / SF cryogels provided in this application, the prepared PLL-MgBGs / SF cryogel as a whole presents a regular spherical structure. It has 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, and it is a typical type I crystal. This crystal can endow the PLL-MgBGs / SF cryogel with excellent shape memory ability and mechanical properties. Moreover, it has a macroporous connected structure and can return to its original state after mechanical strain, resulting in excellent water-responsive shape memory ability.

[0050] At the same time, it can release various active ions such as Mg2+, Ca 2+ and SiO 4 4- etc., showing significant potential in immune regulation and bone repair. Furthermore, by injecting the prepared PLL-MgBGs / SF cryogel into the bone defect site to fill it, it can quickly match irregular bone defects, tightly combine with the irregular bone defect site, solve the problem of insufficient matching between traditional bone scaffolds and irregular defect sites, and show excellent mechanical stability and structural retention during the degradation process, having good supporting ability in the bone defect area, and being able to regulate macrophage polarization into the M2 type, promoting the recruitment, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Therefore, the PLL-MgBGs / SF cryogel prepared in this application has good medical research value and has good application prospects in the field of minimally invasive bone defect repair.

[0051] 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 understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or 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.

[0053] Figure 1 is a flowchart of the steps of the method for promoting osteogenesis by bone immunomodulation of PLL-MgBGs / SF cryogel of the present invention;

[0054] Figure 2 is a flowchart of the steps of step S1 in the embodiment of the present invention;

[0055] Figure 3It is the flowchart of step S2 in the embodiments of the present invention;

[0056] Figure 4 It is the flowchart of step S3 in the embodiments of the present invention;

[0057] Figure 5 It is the flowchart of the steps for detecting the osteogenic differentiation ability of the prepared PLL-MgBGs / SF cryogel in the embodiments of the present invention Figure 1 ;

[0058] Figure 6 It is the flowchart of the steps for detecting the osteogenic differentiation ability of the prepared PLL-MgBGs / SF cryogel in the embodiments of the present invention Figure 2 ;

[0059] Figure 7 It is the flowchart of the steps for detecting the bone regeneration and repair promoting ability of the prepared PLL-MgBGs / SF cryogel in the embodiments of the present invention;

[0060] Figure 8 It is the topological diagram of the preparation process of the PLL-MgBGs / SF cryogel in the embodiments of the present invention Figure 1 ;

[0061] Figure 9 It is the topological diagram of the process for the method of detecting the osteogenic and osteogenic ability of the bone immune regulation of the PLL-MgBGs / SF cryogel of the present invention;

[0062] Figure 10 It is the diagram showing the analysis of the properties and potentials of the substances formed in each step in the embodiments of the present invention;

[0063] Figure 11 It is the diagram showing the characteristics of the substances formed in each step in the embodiments of the present invention;

[0064] Figure 12 It is the diagram showing the detection results of the water-responsive shape memory of the prepared PLL-MgBGs / SF cryogel in the embodiments of the present invention;

[0065] Figure 13 It is the diagram showing the ability of the prepared PLL-MgBGs / SF cryogel to regulate the polarization of macrophages from M1 type to M2 type in the embodiments of the present invention;

[0066] Figure 14 It is the diagram showing the ability of the prepared PLL-MgBGs / SF cryogel to promote the osteogenic differentiation of bone marrow mesenchymal stem cells BMSCs in the embodiments of the present invention;

[0067] Figure 15This is a demonstration diagram showing that the prepared PLL-MgBGs / SF cryogel in the embodiments of the present invention has excellent bone regeneration and repair capabilities;

[0068] The realization, functional characteristics, and advantages of the objectives of the present invention will be further described in conjunction with the embodiments and with reference to the accompanying drawings. Detailed implementation manners

[0069] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings of the specification, where the same or similar reference numerals represent the same or similar elements or elements with 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.

[0070] 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.

[0071] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the 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" means two or more unless otherwise specifically defined.

[0072] In the present invention, unless otherwise clearly defined and limited, the terms "installed", "connected", "connected to", "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 internal communication of two elements. 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.

[0073] In the present invention, unless otherwise clearly defined and limited, the first feature being "on" or "under" the second feature may include direct contact between the first and second features, or may include indirect contact between the first and second features through additional features therebetween. Moreover, the first feature being "above", "over" and "on top of" the second feature includes the first feature being directly above and obliquely above the second feature, or merely indicating that the horizontal height of the first feature is higher than that of the second feature. The first feature being "under", "below" and "beneath" the second feature includes the first feature being directly below and obliquely below the second feature, or merely indicating that the horizontal height of the first feature is lower than that of the second feature.

[0074] The following is a detailed description of the bone immunomodulatory osteogenesis promotion and osteogenic ability detection method of PLL-MgBGs / SF cryogel according to an embodiment of the present invention with reference to the accompanying drawings.

[0075] Example 1

[0076] Refer to Figure 1 、 Figure 8 and Figure 9 As shown, according to the bone immunomodulatory osteogenesis promotion method of PLL-MgBGs / SF cryogel provided by an embodiment of the present invention, the method includes the following steps:

[0077] S1. Prepare magnesium-doped bioactive glass MgBGs;

[0078] Prepare magnesium-doped bioactive substance MgBGs using the sol-gel method combined with the microemulsion co-template method;

[0079] 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, commercially available magnesium-doped bioactive glass (MgBGs) can release various active ions such as magnesium ions (Mg 2+ ), calcium ions (Ca 2+ ) and silicate ions (SiO 4 4- ), and shows significant potential in immunomodulation and bone repair. Magnesium plays an important role in bone health, can regulate cell functions such as proliferation, adhesion and migration, and promote neovascularization.

[0080] Therefore, compared with pure magnesium ions, magnesium-doped bioactive substance MgBGs is more suitable as a bone tissue repair material due to its slow and continuous release of magnesium ions, avoiding the problems of rapid degradation and hydrogen release. In addition, magnesium-doped bioactive substance MgBGs is a material that can form chemical bonds with biological tissues. By introducing magnesium elements, magnesium-doped bioactive substance MgBGs can not only enhance the biological activity of the material, but also improve its mechanical properties.

[0081] S2. Prepare PLL-MgBGs nanoparticles;

[0082] 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;

[0083] S3. Prepare PLL-MgBGs / SF cryogel;

[0084] Mix the prepared PLL-MgBGs nanoparticles with silk fibroin SF solution, and through low-temperature freezing and lyophilization, prepare PLL-MgBGs / SF cryogel.

[0085] It should be noted here that silk fibroin (SF) is a natural protein biomaterial extracted from silkworm cocoons, which has good biocompatibility, controllable biodegradability and excellent mechanical properties, and is widely used in tissue regeneration, drug delivery and medical implants and other fields. However, traditional SF materials lack shape memory effect, and 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 enough resilience to expand in a limited space. In addition, bone repair is a complex process involving multiple cells, signaling molecules and growth factors, and 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. Due to biosafety and ethical issues, materials added with cells or factors are restricted in clinical applications.

[0086] Therefore, this application combines magnesium-doped bioactive substance MgBGs, polylysine PLL and silk fibroin (SF), and prepares PLL-MgBGs / SF cryogel by the above method;

[0087] Control Figure 12 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.

[0088] S4. Make 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 with a 0.22 μm sterile filter head to prepare a sterile PLL-MgBGs / SF cryogel extract;

[0089] S5. Seed macrophages (RAW264.7) in a 48-well plate at a density of 1.0×10 5 cells / well, culture them in medium for 24 h, then replace the medium with complete medium containing LPS (100 ng / mL) and culture for 8 h, and then replace it with complete medium containing the extract of PLL-MgBGs / SF cryogel and culture for 3 days. Extract total mRNA using a total RNA kit, and detect the expression of pro-inflammatory genes (TNF-α, iNOS) and anti-inflammatory genes (IL-10, Arg-1) by RT-qPCR.

[0090] As shown in the following table, the primer sequences of the relevant genes for detecting pro-inflammatory genes (TNF-α, iNOS) and anti-inflammatory genes (IL-10, Arg-1) by RT-qPCR are as follows;

[0091] The primers of RAW264.7 cells-related genes for RT-qPCR

[0092]

[0093] Control Figure 13 As shown, the results show that after the above step S5, the PLL-MgBGs / SF cryogel prepared in this application can promote the expression of alkaline phosphatase (ALP). Detect the expression of M2 marker genes (Arg-1, IL-10) and M1 marker genes (TNF-α, iNOS) in macrophages by RT-qPCR. The results show that the PLL-MgBGs / SF cryogel down-regulates M1 marker genes and up-regulates M2 marker genes, indicating that the PLL-MgBGs / SF cryogel prepared in this application has the ability to regulate the polarization of macrophages from M1 type to M2 type.

[0094] Preferably, in this technical solution, as shown in the control Figure 2 According to an embodiment of the present invention, the step S1 specifically includes:

[0095] 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;

[0096] S12. Dropwise add tetraethyl orthosilicate (TEOS) and continue stirring for 30 min;

[0097] 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 minutes, and keep the temperature at 40 °C all the time, and stir for 3 hours continuously.

[0098] S14. Centrifuge and wash the obtained crude product 3 times with absolute ethanol and DIW.

[0099] S15. Sinter the crude product in air at 650 °C for 3 hours to obtain magnesium-doped bioactive substance MgBGs.

[0100] Preferably, in this technical solution, as shown in the control Figure 3 According to an embodiment of the present invention, the step S2 specifically includes:

[0101] 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 minutes.

[0102] S22. Measure 0.06 g of polylysine PLL and add it to the formed mixed solution, and continue to stir for 24 hours.

[0103] S23. Centrifuge the stirred mixed solution for 10 minutes, 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%.

[0104] Preferably, in this technical solution, as shown in the control Figure 4 According to an embodiment of the present invention, the step S3 specifically includes:

[0105] S31. Use silk fibroin SF as the main material and remove the sericin in silk fibroin SF.

[0106] S32. Dissolve the silk fibroin SF with sericin removed in 9.3 M LiBr solution, and dialyze to remove LiBr to obtain a pure SF solution.

[0107] S33. Mix the prepared PLL-MgBGs nanoparticles with a PLL content of 6% with the obtained pure SF solution, and through low-temperature freezing and lyophilization, prepare PLL-MgBGs / SF cryogel with a PLL content of 6%.

[0108] As shown in the control Figure 10 As shown in (a), the SEM results show that the prepared magnesium-doped bioactive substance MgBGs, PLL-MgBGs / SF cryogel with a PLL content of 3%, and PLL-MgBGs / SF cryogel with a PLL content of 6% all show regular spherical structures.

[0109] Control Figure 10 (b) shows that the XRD results indicate that for the prepared magnesium-doped bioactive material MgBGs, PLL-MgBGs / SF cryogels with 3% PLL content, and PLL-MgBGs / SF cryogels with 6% PLL content, they 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.

[0110] In addition, the potential analysis results show that PLL-MgBGs modified with 0.06 g of PLL is positively charged and has the potential to bind to silk fibroin (SF) through electrostatic interaction.

[0111] Regarding the above operation steps, it should be noted here that the control Figure 10 (c) shows that in the above step S2, the reason for measuring 0.06 g of polylysine PLL as the modifier 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 using a nanoparticle size analyzer to detect its potential, 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 positively charged polylysine PLL and an appropriate amount of negatively charged magnesium-doped bioactive material MgBGs can bind to each other through electrostatic interaction, and finally, magnesium-doped bioactive material PLL-MgBGs nanoparticles loaded with polylysine can be prepared.

[0112] Furthermore, for the prepared PLL-MgBGs / SF cryogel, the preparation schematic diagram is referred to Figure 11 (a). The crystallization configuration of PLL-MgBGs / SF cryogel is characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), and the results show that it is a typical type I crystallization. Specifically, it can be referred to Figure 11 (b) and Figure 11 (c). This crystallization can endow PLL-MgBGs / SF cryogel with excellent shape memory ability and mechanical properties. The SEM results show that the prepared magnesium-doped bioactive material MgBGs, PLL-MgBGs / SF cryogels with 3% PLL content, and PLL-MgBGs / SF cryogels with 6% PLL content all have a macroporous connected structure. Specifically, it can be referred to Figure 11 (d).

[0113] Example 2

[0114] Control Figure 5As shown, the osteogenic ability detection method of PLL-MgBGs / SF cryogel according to the embodiment of the present invention detects its osteogenic differentiation ability, including the following steps:

[0115] S101. Inoculate bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culture for 24 h, then add complete medium containing the extract of PLL-MgBGs / SF cryogel for culture;

[0116] S102. After 14 days, remove the complete medium, wash 3 times with PBS, fix in 4% paraformaldehyde for 30 min, and then wash with PBS;

[0117] S103. Stain the bone marrow mesenchymal stem cells (BMSCs) with an alkaline phosphatase (ALP) chromogenic kit for 30 min, and use an inverted fluorescence microscope to obtain pictures and analyze the results.

[0118] For step S101 therein, it is the PLL-MgBGs / SF group. Correspondingly, a control group (Control group, without filling material) and an SF group (filling SF cryogel) will also be set;

[0119] The control group is: inoculate bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culture for 24 h, without adding the extract of PLL-MgBGs / SF cryogel;

[0120] The SF group is: inoculate bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culture for 24 h, then add complete medium containing the extract of SF cryogel for culture;

[0121] Control Figure 14 (a) As shown, the experimental results of the three groups show that the PLL-MgBGs / SF cryogel prepared in this application can promote the expression of alkaline phosphatase (ALP).

[0122] And control Figure 14 (b) As shown, the experimental results of the three groups show that for the PLL-MgBGs / SF cryogel prepared in this application, the alkaline phosphatase (ALP) staining effect is more obvious.

[0123] Example 3

[0124] Control Figure 6 As shown, the osteogenic ability detection method of PLL-MgBGs / SF cryogel according to the embodiment of the present invention detects its osteogenic differentiation ability, including the following steps:

[0125] S201. Seed bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culture for 24 h. Then add complete medium containing the extraction solution of PLL-MgBGs / SF cryogel for culture, and incubate in an incubator at 37 °C with 5% CO₂.

[0126] S202. After 14 days, remove the complete medium, wash 3 times with PBS, fix in 4% paraformaldehyde for 30 min, and then wash thoroughly with PBS.

[0127] S203. Add alizarin red for staining and incubate at 37 °C for 30 min. Wash 3 times with PBS, and obtain pictures and analyze the results using an inverted fluorescence microscope.

[0128] Similarly to Example 2 above, for step S201, it is the PLL-MgBGs / SF group. Correspondingly, a control group (Control group, without filling material) and an SF group (filling SF cryogel) will also be set up.

[0129] The control group is as follows: Seed bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culture for 24 h without adding the extraction solution of PLL-MgBGs / SF cryogel.

[0130] The SF group is as follows: Seed bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culture for 24 h, and then add complete medium containing the extraction solution of SF cryogel for culture.

[0131] Control Figure 14 As shown in (c), the experimental results of the three groups show that for the PLL-MgBGs / SF cryogel prepared in this application, the alizarin red staining effect is more obvious, indicating that it can better promote the expression of calcium nodules.

[0132] Therefore, based on the above, from Figure 14 (a), (b), and (c), it can be concluded that the PLL-MgBGs / SF cryogel prepared in this application has excellent ability to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

[0133] Example 4

[0134] Control Figure 7 As shown, according to the osteogenic ability detection method of the PLL-MgBGs / SF cryogel according to the embodiments of the present invention, its bone regeneration and repair ability is detected, including the following steps:

[0135] S301. Select 60 male Sprague-Dawley (SD) rats at 8 weeks old with a body weight of 200 - 250 g, and anesthetize the rats with 3% pentobarbital solution (150 μL / 100 g).

[0136] S302. Make an incision of 1 cm on the head of the rat, and use a trephine to drill a defect with a diameter of 0.5 cm on the skull within the incision on the rat's head, ensuring that the dura mater is not damaged;

[0137] S303. Thaw the prepared PLL-MgBGs / SF cryogel, and implant it into the defect by injection to fill it, and suture the incision;

[0138] S304. After the operation, return the rat to the breeding room for free movement, and continuously inject penicillin for 3 days to prevent infection;

[0139] S305. After 12 weeks, euthanize the rat by the CO2 inhalation method, remove the excess muscle tissue, take out the rat skull specimen, and use a Micro-CT scanner to examine the rat skull specimen and analyze the results.

[0140] Similarly to the above-mentioned Example 2 and the above-mentioned Example 3, for step S303 therein, it is the PLL-MgBGs / SF group. Correspondingly, a control group (Control group, without filling materials) and an SF group (filling SF cryogel) will also be set;

[0141] The control group is: Do not fill any materials into the defect;

[0142] The SF group is: Thaw the prepared SF cryogel, and implant it into the defect by injection to fill it, and suture the incision;

[0143] Control Figure 15 As shown in (a), the experimental results of the three groups by the Micro-CT scanner show that the PLL-MgBGs / SF cryogel prepared in this application has excellent bone regeneration and repair effects.

[0144] All of the above results indicate that the PLL-MgBGs / SF cryogel prepared in this application has excellent repair ability for promoting osteogenesis.

[0145] For other embodiments, etc., they will not be exemplified here.

[0146] Therefore, in summary, in the method for promoting osteogenesis by bone immunomodulation of the PLL-MgBGs / SF cryogel provided in this application, the prepared PLL-MgBGs / SF cryogel as a whole presents a regular spherical structure, and it has 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, and it is a typical type I crystal, which can endow the PLL-MgBGs / SF cryogel with excellent shape memory ability and mechanical properties. Moreover, it has a macroporous connected structure and can return to its original state after mechanical strain, resulting in excellent water-responsive shape memory ability.

[0147] Meanwhile, it can release various active ions such as Mg2+, Ca 2+ and SiO 4 4- etc., showing significant potential in immunomodulation and bone repair. Moreover, the prepared PLL-MgBGs / SF cryogel is implanted into the bone defect site by injection to fill it, enabling rapid matching of irregular bone defects, being able to closely adhere to the irregular bone defect site, solving the problem of insufficient matching between traditional bone scaffolds and irregular defect sites, and showing excellent mechanical stability and structural retention during the degradation process, having good supporting ability in the bone defect area, being able to regulate macrophage polarization into the M2 type, and promoting the recruitment, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

[0148] Therefore, the PLL-MgBGs / SF cryogel prepared in this application has good medical research value and has good application prospects in the field of minimally invasive bone defect repair.

[0149] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations 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.

[0150] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. All equivalent structural transformations made under the inventive concept of the present invention by using the content of the specification and drawings of the present invention, or directly / indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A method for osteoimmunomodulation and osteogenesis using 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; S4, prepare a complete culture medium suspension containing 1 mg / mL PLL-MgBGs / SF cryogel, incubate at 100 rpm on a shaker at 37°C for 24 h, and filter using a 0.22 μm sterile filter to prepare a sterile PLL-MgBGs / SF cryogel extract; S5. Macrophages (RAW264.7) were inoculated in a 48-well plate and cultured with culture medium for 24 h. The culture medium was then replaced with complete culture medium containing LPS (100 ng / mL) for 8 h, and then replaced with complete culture medium containing PLL-MgBGs / SF cryogel extract for 3 days. Total mRNA was extracted using a total RNA kit, and the expression of pro-inflammatory genes (TNF-ɑ, iNOS) and anti-inflammatory genes (IL-10, Arg-1) was detected by RT-qPCR.

2. The osteoimmunomodulatory and osteogenesis promoting method of 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 osteoimmunomodulatory and osteogenesis promoting method of 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 osteoimmunomodulation and osteogenesis of 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 osteogenic ability of PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S101, inoculating bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culturing for 24 hours, adding complete culture medium containing PLL-MgBGs / SF cryogel extract for culturing; S102, 14 days later, the complete medium was removed, the cells were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min, and then washed with PBS; S103. Use an alkaline phosphatase (ALP) colorimetric kit to stain bone marrow mesenchymal stem cells (BMSCs) for 30 minutes, and use an inverted fluorescence microscope to obtain images and analyze the results.

6. A method for detecting the osteogenic ability of PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S201, inoculating bone marrow mesenchymal stem cells (BMSCs) into a 24-well plate and culturing for 24 hours, adding complete medium containing PLL-MgBGs / SF cryogel extract for culturing, and incubating in an incubator at 37° C. and containing 5% CO 2 ; S202, 14 days later, the complete medium was removed, the cells were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min, and then washed with PBS; S203. Add Alizarin Red dye and incubate at 37°C for 30 min. Wash three times with PBS and use an inverted fluorescence microscope to obtain images and analyze the results.

7. A method for detecting the osteogenic ability of PLL-MgBGs / SF cryogel, characterized in that: The following steps are involved: S301. Select 60 8-week-old male SD rats weighing 200-250 g, and anesthetize the rats with 3% pentobarbital solution (150 μL / 100 g); S302, making a 1 cm incision on the rat's head, and drilling a 0.5 cm diameter defect on the skull within the incision on the rat's head with a circular drill, and ensuring that the dura mater is not damaged; S303, thawing the prepared PLL-MgBGs / SF cryogel, and injecting it into the defect to fill it, and suturing the incision; S304. After surgery, the rats were returned to the feeding room to move freely and were injected with penicillin for 3 consecutive days to prevent infection. S305. After 12 weeks, the rats were killed by CO2 inhalation, the excess muscle tissue was removed, and the rat skull specimens were taken out. The rat skull specimens were examined and the results were analyzed using a Micro-CT scanner.

Images