An engineered spirulina-based composite material, a preparation method and application thereof

Abstract

The application discloses a kind of composite material based on engineering spirulina, its preparation method and application, the composite material includes hydrogel matrix and the engineering spirulina carrier dispersed in the hydrogel matrix;Wherein, the surface of spirulina is loaded with polyethyleneimine-coated Fe3O4 Nanoparticles, obtain the engineering spirulina carrier, and the engineering spirulina high expression arginine.One aspect, the present application is metabolically restructured to spirulina, so that its intracellular L-arginine content is significantly improved, L-arginine can simultaneously improve vascular function through iNOS path, and through Arg-1 path, promote macrophages to repair type M2 polarization and tissue repair, from metabolic root to intervene the core obstacle of bone defect;On the other hand, spirulina is surface modified, can effectively activate osteogenesis-related gene expression, drive new bone formation.By the double modification of L-arginine enrichment and Fe3O4 Nanoparticles loading to spirulina, obtain the composite material of synergistic regulation anti-inflammatory, pro-angiogenic and pro-osteogenic, accurately match the demand of complex pathological microenvironment of bone defect, to effectively treat bone defect, especially drug-related jaw osteonecrosis.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and oral tissue engineering technology, specifically to a composite material based on engineered spirulina, its preparation method, and its application. Background Technology

[0002] Drug-related osteonecrosis of the jaw (MRONJ) is a serious complication caused by long-term use of anti-resorption drugs (such as bisphosphonates and denosumab) or anti-angiogenic drugs. Clinically, it manifests as persistent bone exposure, pain, infection, and impaired bone healing. Its pathological mechanisms are complex, involving persistent inflammatory responses, impaired angiogenesis, and osteogenic dysfunction, forming a unique pathological microenvironment.

[0003] Traditional treatment methods include: (1) Conservative treatment: mainly to control infection and relieve symptoms, including oral irrigation (such as chlorhexidine), antibiotic treatment and analgesia, but it is usually difficult to achieve complete repair of the lesion; (2) Surgical treatment: for patients in the advanced stage, surgical methods such as necrotic bone removal, curettage or partial / segmental jaw resection are often used, but the trauma is large and the risk of recurrence after surgery still exists; (3) Adjunctive treatment methods: such as hyperbaric oxygen therapy (HBOT) and low-energy laser therapy (LLLT), which aim to improve local blood supply or promote healing, but the efficacy varies from person to person and there is no unified standard; (4) Drug adjustment: including pausing or adjusting anti-bone resorption drugs (drug holiday), but its effect on improving the progression of MRONJ is still controversial; (5) Regenerative medicine strategy: in recent years, platelet-rich plasma (PRP), stem cell therapy and biomaterial implantation have been tried, but the overall approach is still in the exploratory stage and lacks large-scale clinical evidence. Overall, the existing treatment methods are mostly to relieve symptoms or delay the course of the disease, and it is difficult to solve the core pathological problems such as inflammatory imbalance, impaired angiogenesis and bone regeneration disorders at the same time. In recent years, tissue engineering technology has provided new ideas for bone defect repair. However, most existing biomaterials have single functions, or only focus on promoting bone growth, or only emphasize anti-inflammation, making it difficult to address the multiple pathological obstacles coexisting in MRONJ lesions at the same time. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies in the lack of effective treatment methods and drugs for drug-related osteonecrosis of the jaw.

[0005] To achieve the above objectives, the present invention provides a composite material based on engineered spirulina, the composite material comprising a hydrogel matrix and an engineered spirulina carrier dispersed in the hydrogel matrix; wherein, the engineered spirulina carrier is obtained by loading Fe3O4 nanoparticles coated with polyethyleneimine onto the surface of spirulina, and the engineered spirulina highly expresses arginine.

[0006] Optionally, the arginine is L-arginine.

[0007] Optionally, the hydrogel matrix comprises any one of the following: methacrylamide gelatin, sodium alginate composite hydrogel, sodium alginate hydrogel, hyaluronic acid hydrogel, methacrylamide hyaluronic acid hydrogel, collagen hydrogel, fibroin hydrogel, chitosan hydrogel, polyethylene glycol hydrogel, polyethylene glycol diacrylate hydrogel, and silk fibroin hydrogel.

[0008] The present invention also provides a method for preparing the composite material as described above, comprising the following steps: Step S1: FeCl2·4H2O solution is mixed with ammonia water, and then polyethyleneimine aqueous solution is added to it. The mixture is reacted under high pressure to obtain Fe3O4 nanoparticles coated with polyethyleneimine. Step S2: Spirulina is cultured in a 50% sulfur-deficient spirulina basal medium to obtain a spirulina solution; Step S3: Disperse the polyethyleneimine-coated Fe3O4 nanoparticles in the spirulina solution, collect the precipitate by centrifugation, and obtain the engineered spirulina carrier modified with polyethyleneimine-coated Fe3O4 nanoparticles. Step S4: Disperse the engineered spirulina carrier in a hydrogel solution to obtain the composite material.

[0009] Optionally, the Fe3O4 nanoparticles have a nanoparticle size of 10 nm to 200 nm.

[0010] Optionally, in step S1, the reaction under high pressure specifically refers to a high-pressure reaction at 120℃~160℃ for 2h~5h in a high-pressure reactor.

[0011] Optionally, in step S2, the cultivation conditions are a temperature of 20℃~35℃ and a light intensity of 3000~6000 1x.

[0012] Optionally, the method for preparing the 50% sulfur-deficient spirulina basal culture medium includes: reducing K2SO4 and MgSO4·7H2O in the spirulina basal culture medium by 50% respectively, and supplementing KCl and MgCl2·6H2O in equal molar amounts to achieve a sulfur concentration of 106 mg S / L.

[0013] The present invention also provides an application of the composite material described above, wherein the composite material is used to prepare a drug for treating bone defects.

[0014] Optionally, the bone defect includes at least one of the following: traumatic bone defect, infectious bone defect, post-tumor resection bone defect, radiation-induced osteonecrosis-related defect, alveolar bone resorption or bone defect, drug-related bone defect, congenital or developmental bone defect.

[0015] Compared to the prior art, the beneficial effects of the present invention include at least the following: (1) The composite material of the present invention comprises an engineered spirulina carrier that has undergone dual modification of spirulina. On the one hand, the spirulina is metabolically reconstructed to highly express L-arginine. L-arginine, as a key metabolic hub connecting immunity and repair, can simultaneously improve vascular function through the iNOS pathway and promote macrophage polarization towards repair-type M2 and tissue repair through the Arg-1 pathway. By improving the vascular and immune microenvironment, it intervenes in the core barrier of MRONJ from the metabolic root. On the other hand, Fe3O4 nanoparticles coated with polyethyleneimine are loaded onto the surface of spirulina, which can controllably release Fe3O4 in an inflammatory environment. 2+ It can be taken up by bone marrow mesenchymal stem cells, effectively activating the expression of osteogenic-related genes and driving new bone formation. Spirulina itself and engineered spirulina carriers can effectively induce macrophages to polarize from the pro-inflammatory M1 phenotype to the anti-inflammatory and repair-oriented M2 phenotype, significantly inhibiting the secretion of pro-inflammatory factors such as TNF-α and increasing the levels of anti-inflammatory factors such as IL-10, fundamentally improving the persistent inflammatory microenvironment of MRONJ. The composite material of this invention can synergistically regulate anti-inflammation, angiogenesis, and osteogenic effects, effectively treating bone defects.

[0016] (2) Combining 3D printing technology, a personalized scaffold that perfectly matches the shape of the bone defect can be customized according to the patient's CBCT data, so as to achieve precise implantation and close fit, maximize the repair effect and reduce foreign body reaction. Attached Figure Description

[0017] Figure 1 This is a diagram showing the single-cell analysis results of the present invention.

[0018] Figure 2 This is a diagram showing the experimental results of the sulfur deficiency gradient of the present invention.

[0019] Figure 3 This is a graph showing the flow cytometry analysis results of the present invention.

[0020] Figure 4 This is a graph showing the statistical analysis results of the ELISA detection in this invention.

[0021] Figure 5 The figure shows the results of the cell migration scratch experiment and statistical quantitative analysis of the present invention.

[0022] Figure 6 The image shows the results of the ALP and ARS staining experiments of this invention.

[0023] Figure 7 This is a graph showing the results of multicolor immunofluorescence detection and statistical quantitative analysis of the present invention. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. All experimental materials not otherwise mentioned in this invention are commercially available, and all experimental methods not otherwise mentioned in this invention are conventional experimental methods in the art.

[0025] The spirulina used in this invention was purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0026] Current research has explored the microenvironment of MRONJ to some extent, but overall it remains largely focused on single-level studies, lacking systematic and high-resolution analysis. For example, previous studies have found abnormal enrichment of macrophages and their pro-inflammatory M1 polarization in MRONJ lesions, as well as suppressed osteogenic differentiation capacity of mesenchymal stem cells, but these findings mostly rely on immunohistochemistry or in vitro experiments, resulting in relatively limited levels of evidence and a lack of in-depth analysis of cellular heterogeneity and interactions.

[0027] To elucidate the mechanism of impaired bone regeneration in MRONJ mice, this invention performed single-cell RNA sequencing (scRNA-seq) analysis on the bone marrow microenvironment of the mandible of MRONJ mice. The results showed that bone immune homeostasis was significantly disrupted in MRONJ mice, manifested as enhanced macrophage-driven inflammatory responses, impaired angiogenesis signaling, and a marked inhibition of osteogenic differentiation. Compared with controls, the cellular composition in MRONJ mice was significantly altered, with a significantly increased proportion of macrophages exhibiting the most significant pathological reprogramming; conversely, endothelial cells were significantly reduced, suggesting impaired angiogenesis maintenance. Figure 1 A, Figure 1 Differential expression analysis showed that inflammation-related pathways were significantly upregulated in MRONJ macrophages, exhibiting a pro-inflammatory phenotype; while endothelial cells showed widespread transcriptional repression, with significantly downregulated angiogenesis, migration, and proliferation-related pathways. Figure 1 C, Figure 1 Further metabolic analysis revealed impaired amino acid metabolism in endothelial cells, with the arginine biosynthesis pathway being most significantly inhibited and positively correlated with cell proliferation capacity. Figure 1 E, Figure 1 (F). Furthermore, the persistent activation of pro-inflammatory macrophages coexisted with impaired endothelial cell metabolism, suggesting a pathological coupling between inflammation and vascular dysfunction. Overall, this invention reveals that the dysregulation of bone-immune-angiogenesis synergy in MRONJ may be an important mechanism in its pathogenesis.

[0028] As can be seen, this invention is the first to systematically and accurately verify the above phenomena at the level of the whole bone marrow microenvironment through single-cell transcriptome sequencing, and further reveals the key mechanism of significantly impaired arginine metabolism in endothelial cells, which has not been reported before, elevating the vascular dysfunction of MRONJ from the traditional structural or signaling abnormalities to the level of metabolic regulation.

[0029] Existing biomaterials often focus only on one aspect of function, such as osteogenic or anti-inflammatory properties, making them difficult to adapt to the complex microenvironment of MRONJ. To address these technical problems, this invention provides a composite material based on engineered spirulina. The composite material comprises a hydrogel matrix and an engineered spirulina carrier dispersed within the hydrogel matrix. The engineered spirulina carrier is obtained by loading polyethyleneimine-coated Fe3O4 nanoparticles onto the surface of the spirulina, which highly expresses arginine. Through dual biological and chemical modification of the spirulina—namely, conditioned cultivation to achieve high arginine expression and loading Fe3O4 nanoparticles onto its surface—the composite material possesses triple functions: anti-inflammatory, angiogenic, and osteogenic.

[0030] The present invention provides a method for preparing a composite material based on engineered spirulina, comprising the following steps: Step S1: FeCl2·4H2O solution is mixed with ammonia water, and then polyethyleneimine aqueous solution is added to it. The mixture is reacted under high pressure to obtain Fe3O4 nanoparticles coated with polyethyleneimine. Step S2: Spirulina is cultured in a 50% sulfur-deficient spirulina basal medium to obtain a spirulina solution; Step S3: Disperse the polyethyleneimine-coated Fe3O4 nanoparticles in the spirulina solution, collect the precipitate by centrifugation, and obtain the engineered spirulina carrier modified with polyethyleneimine-coated Fe3O4 nanoparticles. Step S4: Disperse the engineered spirulina carrier in a hydrogel solution to obtain the composite material.

[0031] In some embodiments, in step S1, a freshly prepared FeCl2·4H2O solution is mixed with ammonia (6.5 mL) and continuously stirred in air for 10 minutes. The reaction mixture is then transferred to a 50 mL autoclave, and 5 mL of a polyethyleneimine (PEI) (25 kDa) aqueous solution is added. The mass ratio of PEI to Fe is determined to be 2:1. After thorough stirring, the reaction mixture is autoclaved at 140 °C for 3 hours. After cooling to room temperature, the black precipitate is collected by magnetic separation and washed five times with pure water to remove excess reactants and byproducts, yielding Fe3O4-PEI nanoparticles with a nanoparticle size of 10 nm to 200 nm (i.e., polyethyleneimine-coated Fe3O4 nanoparticles). The Fe3O4-PEI nanoparticles are redispersed in water for later use. PEI coating of Fe3O4 nanoparticles significantly improves their dispersion stability in aqueous solution and enhances cellular uptake efficiency by utilizing the positive surface charge. Meanwhile, PEI coating imparts a positive charge to the Fe3O4 nanoparticles, allowing them to firmly bind to the negatively charged spirulina via electrostatic interactions, thus endowing the spirulina with superparamagnetism. This magnetic spirulina can achieve directional movement, rapid enrichment, and easy separation using an external magnetic field, facilitating precise operations in targeted delivery, magnetically controlled bioreactors, or environmental remediation.

[0032] Arginine is a key metabolic hub amino acid connecting immune regulation and tissue repair, playing a dual biological role in inflammation and bone repair. On one hand, arginine can be metabolized into nitric oxide (NO) via the nitric oxide synthase (iNOS) pathway, regulating endothelial cell function and vasodilation, thereby improving impaired angiogenesis and the ischemic microenvironment commonly found in MRONJ. On the other hand, arginine can also be metabolized into polyamines and proline via the arginase-1 (Arg-1) pathway, promoting cell proliferation and collagen synthesis, thus supporting tissue repair and bone matrix reconstruction. Simultaneously, arginine metabolism is considered a crucial determinant of macrophage polarization; a shift towards the Arg-1 pathway can promote macrophage transformation into the anti-inflammatory and repair-promoting M2 phenotype, which is critical in MRONJ for suppressing persistent inflammatory responses, promoting angiogenesis, and bone remodeling.

[0033] Based on the above mechanism, this invention induces metabolic remodeling in Spirulina through sulfur restriction, thereby significantly enhancing its arginine enrichment capacity. This transforms it not only as a nutritional metabolic supplement but also into a bioactive carrier with potential for immune-vascular regulation. This "sulfur regulation – arginine enrichment – ​​immune microenvironment remodeling" strategy can improve the endothelial dysfunction and bone immune imbalance characteristic of MRONJ at the metabolic level, thus providing a new bioengineering intervention approach for disease treatment.

[0034] In some embodiments, to create sulfur-deficient conditions, the K₂SO₄ and MgSO₄·7H₂O in standard Zarrouk medium (i.e., the basal medium for Spirulina) were reduced by 50% (to 0.5 g / L and 0.1 g / L, respectively). To maintain equal concentrations of potassium and magnesium ions, KCl (0.428 g / L) and MgCl₂·6H₂O (0.0825 g / L) were supplemented accordingly. This formulation resulted in a final sulfur concentration of approximately 10⁶ mg S / L. Spirulina (SP) was cultured in sulfur-deficient Zarrouk medium to obtain SP solutions at 25 °C and a light intensity of 3000–6000 lx to promote intracellular arginine biosynthesis and metabolism. By reconstructing the arginine metabolic pathway, the engineered Spirulina carrier was able to continuously secrete L-arginine, enhancing local NO production and NOS3 signaling pathway activity, and significantly improving the angiogenesis capacity of vascular endothelial cells. This invention achieves effective regulation of arginine metabolism in Spirulina cells by precisely adjusting the sulfur content in the culture medium, reducing the sulfur content by 50%, while maintaining stable potassium and magnesium ion concentrations. The intracellular arginine concentration was effectively increased by nearly 2.5 times. It should be noted that this invention designed a sulfur deficiency gradient experiment, including conditions of 100% sulfur (control group), 75% sulfur, 50% sulfur, 25% sulfur, and 0% sulfur. All experimental and control groups used a sulfur content of 1×10⁻⁶. 7 SP cells were seeded at a density of [number] cells / mL and cultured in six-well plates, with three replicates per group. Cells were cultured at 25°C under continuous light (3000–6000 lx). After 3 days of culture, SP cells were collected, washed with PBS, lysed according to the kit instructions, and intracellular L-arginine concentration was detected using a competitive ELISA kit. Figure 2 As shown, the increase in intracellular L-arginine concentration was most significant when sulfur content decreased by 50%.

[0035] In some embodiments, the Fe3O4-PEI nanoparticles (500 μL, 1 mg / mL) were dispersed in a sulfur-deficient culture SP solution (5 mL, 1 × 10⁻⁶). 8 The cells were added to a solution and gently stirred at room temperature for 2 hours. The precipitate was collected by centrifugation (4000 rpm, 5 min) and washed twice with phosphate-buffered saline (PBS). A polyvinylimide-coated Fe3O4 nanoparticle-modified engineered spirulina carrier (hereinafter referred to as Fe / SP) was obtained. The engineered spirulina carrier was redispersed in 5 mL of PBS for later use.

[0036] Spirulina and Fe3O4 have synergistic effects. Spirulina is rich in arginine, which can promote angiogenesis and immune regulation through the nitric oxide (NO) pathway, while Fe3O4 can continuously release Fe. 2+Arginine participates in the regulation of cellular metabolism and the maintenance of iron homeostasis. Under their combined action, arginine promotes NO production, enhances angiogenesis and tissue repair capabilities, while Fe... 2+ By regulating hypoxia-related signaling pathways (such as HIF-1α) and cellular functional status, it further promotes osteogenic and angiogenesis processes. In addition, Fe... 2+ Both SP and Fe3O4 participate in the regulation of immune cell function, and arginine plays a key role in immune metabolism. Together, they help improve the inflammatory microenvironment and promote tissue regeneration. Therefore, the combination of SP and Fe3O4 can facilitate the interaction between arginine-NO and Fe... 2+ The "metabolic regulation" dual-pathway synergistic mechanism enables the remodeling and regeneration of the bone immune microenvironment, which has potential application value in diseases such as drug-related osteonecrosis of the jaw.

[0037] ZA, or zoledronic acid, is a bisphosphonate anti-bone resorption drug commonly used clinically to treat osteoporosis and bone metastases from tumors. In MRONJ-related studies, ZA is one of the classic inducing factors for constructing drug-related osteonecrosis of the jaw cells or animal models. It can simulate the pathological microenvironment of MRONJ by inhibiting osteoclast activity and disrupting the bone remodeling process. Therefore, it is often used as a positive stimulus or a model-building drug.

[0038] Flow cytometry detection of macrophage polarization: (1) Cell grouping treatment: Macrophages were divided into control group, LPS stimulation group, ZA treatment group and ZA+Fe / SP combined treatment group according to the experimental design, and cultured under the corresponding conditions to the set time point.

[0039] (2) Cell collection and preparation of single-cell suspension: After the cells in each group are processed, the cells are collected, washed thoroughly with PBS, and centrifuged at 300–400 g for 5 min to obtain single-cell suspension.

[0040] (3) Fc blocking treatment: Use 1% BSA or Fc receptor blocker to block the cells for about 20–30 min to reduce non-specific antibody binding.

[0041] (4) Antibody staining: Add fluorescently labeled antibodies (CD86 for M1 type labeling, CD206 for M2 type labeling) and incubate at 4°C in the dark for about 30 min.

[0042] (5) Washing and resuspending: After incubation, wash twice with PBS to remove unbound antibodies and resuspend the cells in an appropriate amount of buffer.

[0043] (6) On-machine detection: Use flow cytometer to collect sample data, set FSC / SSC to exclude debris and perform single-cell gating analysis.

[0044] (7) Data Analysis: FlowJo software was used to analyze CD86. + With CD206 + Cell proportions were determined, and the M2 / M1 ratio was calculated to assess macrophage polarization under different treatment conditions.

[0045] like Figure 3 As shown, Fe / SP can induce macrophage polarization from M1 to M2. Figure 4 As shown, ELISA results demonstrate that Fe / SP can inhibit the inflammatory factor TNF-α, increase the secretion of IL-10, and improve the pro-inflammatory environment unique to MRONJ.

[0046] Fe / SP cells were co-cultured with HUVECs, BMSCs, and RAW264.7 cells in vitro. The angiogenesis assay for HUVECs involved culturing HUVECs (4 × 10⁻⁶ cells per cell). 4 (cells / mL) were seeded in Matrigel-coated culture wells, and Fe / SP (5×10⁻⁶ cells / mL) was added to the upper chamber of the Transwell chamber. 6 BMSCs were co-cultured at a concentration of 1.2 × 10⁻⁶ cells / mL to assess their effect on endothelial cell angiogenesis. In an in vitro osteogenic differentiation assay, BMSCs were co-cultured at a concentration of 1.2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of [number] cells / ml in 24-well plates and cultured for 24 h to promote cell adhesion. Subsequently, in a Transwell co-culture system, Fe / SP (5 × 10⁻⁶) was added to the upper chamber. 6 RAW264.7 cells / mL were indirectly co-cultured with BMSCs in the lower chamber to assess its effect on osteogenic differentiation of BMSCs. In vitro anti-inflammatory assays were performed using RAW264.7 cells at a concentration of 1×10⁻⁶ cells / mL. 6 Cells were seeded at a density of [number] cells / ml in 12-well plates and cultured for 24 h to promote cell adhesion. Subsequently, in a Transwell co-culture system, Fe / SP (5 × 10⁻⁶) was added to the upper chamber. 6 Fe / SP was indirectly co-cultured with RAW264.7 cells / mL in the lower chamber to evaluate its in vitro anti-inflammatory effects. Figure 5 and Figure 6 As shown, Fe / SP can promote angiogenesis and osteogenic differentiation of BMSCs. Figure 7 As shown, Fe / SP can inhibit inflammation in vitro.

[0047] Addressing the significant inter-patient variation in defect morphology within the MRONJ jawbone defect model, this invention further integrates 3D printing technology. This allows for the creation of personalized scaffolds that perfectly match the bone defect morphology based on the patient's CBCT data. This achieves precise matching and close fit between the material and the specific jawbone defect area, maximizing repair effectiveness and minimizing foreign body reaction, thereby improving repair outcomes and clinical translation potential. In some embodiments, LAP powder is dissolved in sterile pure water using vortexing to prepare a 0.25% (w / v) LAP photoinitiator solution. Lyophilized methacrylamide gelatin (GelMA) is added to the LAP photoinitiator solution to achieve a final concentration of 10% (w / v). The mixture is stored in the dark and incubated in a 65 °C water bath for 30 minutes, with intermittent vortexing to ensure complete dissolution. After cooling, the GelMA solution is filtered through a 0.22 μm syringe filter for sterilization. After the sterile solution cooled to below 37 °C, Spirulina cells (i.e., engineered Spirulina carriers) with Fe3O4 nanoparticles coated with polyethyleneimine on their surface were added, with a final density of 1 × 10⁻⁶. 6 Cells / mL, forming printable bio-ink. The bio-ink was loaded into sterile 5 mL low-temperature brown cartridges and printed using a bioprinter (Bio Architect SR, Hangzhou Genofe Biotechnology Co., Ltd., China). The scaffold was stacked and constructed under the following printing parameters: nozzle diameter 200 μm, layer height 200 μm, printing speed 2 mm / s, extrusion pressure 0.15 MPa, nozzle temperature 28 °C, and plateau temperature 4 °C. Each layer was immediately illuminated with 405 nm blue-violet light (1 W / cm²) after deposition. 2 Cross-linking was achieved by irradiation for 10 seconds. After printing, the entire scaffold was further irradiated under the same light source for 1 minute to ensure complete curing. All operations were performed under sterile, light-protected conditions. The scaffold for in vivo implantation was cylindrical, with dimensions of Ø3 mm × 2 mm. Two sets of scaffolds were prepared: the Gel group (containing only GelMA) and the Fe / SP@Gel group (GelMA containing Spirulina cells with Fe3O4 nanoparticles coated with polyethyleneimine on the surface).

[0048] In summary, the composite material of the present invention comprises a hydrogel matrix and an engineered spirulina carrier dispersed in the hydrogel matrix; wherein, the engineered spirulina carrier is obtained by loading polyethyleneimine-coated Fe3O4 nanoparticles onto the surface of spirulina, and the engineered spirulina highly expresses arginine. On the one hand, the present invention uses a "sulfur-limited-ion-compensated" culture strategy to metabolically reconstruct spirulina, significantly increasing its intracellular L-arginine content by approximately 2.5 times. L-arginine can simultaneously improve vascular function through the iNOS pathway and promote macrophage polarization towards repair-type M2 and tissue repair through the Arg-1 pathway. By improving the vascular and immune microenvironment, it intervenes in the core obstacle of bone defects from a metabolic perspective. On the other hand, the surface of spirulina is modified with Fe3O4 nanoparticles, which can controllably release Fe3O4 nanoparticles in an inflammatory environment. 2+ It can be taken up by bone marrow mesenchymal stem cells, effectively activating the expression of osteogenic-related genes and driving new bone formation. This invention obtains a composite material that synergistically regulates anti-inflammatory, angiogenesis-promoting, and osteogenic effects by enriching L-arginine and loading Fe3O4 nanoparticles into Spirulina. This material precisely matches the complex pathological microenvironment requirements of bone defects, thereby effectively treating bone defects, especially drug-induced osteonecrosis of the jaw.

[0049] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A composite material based on engineered spirulina, characterized in that, The composite material comprises a hydrogel matrix and an engineered spirulina carrier dispersed in the hydrogel matrix; wherein, the engineered spirulina carrier is obtained by loading Fe3O4 nanoparticles coated with polyethyleneimine onto the surface of spirulina, and the engineered spirulina highly expresses arginine.

2. The composite material as described in claim 1, characterized in that, The arginine in question is L-arginine.

3. The composite material as described in claim 1, characterized in that, The hydrogel matrix comprises any one of the following: methacrylamide gelatin, sodium alginate composite hydrogel, sodium alginate hydrogel, hyaluronic acid hydrogel, methacrylamide hyaluronic acid hydrogel, collagen hydrogel, fibroin hydrogel, chitosan hydrogel, polyethylene glycol hydrogel, polyethylene glycol diacrylate hydrogel, and silk fibroin hydrogel.

4. A method for preparing the composite material according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step S1: FeCl2·4H2O solution is mixed with ammonia water, and then polyethyleneimine aqueous solution is added to it. The mixture is reacted under high pressure to obtain Fe3O4 nanoparticles coated with polyethyleneimine. Step S2: Spirulina is cultured in a 50% sulfur-deficient spirulina basal medium to obtain a spirulina solution; Step S3: Disperse the polyethyleneimine-coated Fe3O4 nanoparticles in the spirulina solution, collect the precipitate by centrifugation, and obtain the engineered spirulina carrier modified with polyethyleneimine-coated Fe3O4 nanoparticles. Step S4: Disperse the engineered spirulina carrier in a hydrogel solution to obtain the composite material.

5. The preparation method according to claim 4, characterized in that, The Fe3O4 nanoparticles have a nanoparticle size of 10 nm to 200 nm.

6. The preparation method according to claim 4, characterized in that, In step S1, the reaction under high pressure specifically refers to a high-pressure reaction at 120℃~160℃ for 2h~5h in a high-pressure reactor.

7. The preparation method according to claim 4, characterized in that, In step S2, the cultivation conditions are a temperature of 20℃~35℃ and a light intensity of 3000~6000 1x.

8. The preparation method according to claim 4, characterized in that, The method for preparing the 50% sulfur-deficient Spirulina basal culture medium includes: reducing K2SO4 and MgSO4·7H2O in the Spirulina basal culture medium by 50% respectively, and supplementing KCl and MgCl2·6H2O in equal molar amounts to make the sulfur concentration 106 mg S / L.

9. An application of the composite material as described in any one of claims 1 to 3, characterized in that, The composite material is used to prepare drugs for treating bone defects.

10. The application as described in claim 9, characterized in that, The bone defect includes at least one of the following: traumatic bone defect, infectious bone defect, post-tumor resection bone defect, radiation-induced osteonecrosis-related defect, alveolar bone resorption or bone defect, drug-related bone defect, congenital or developmental bone defect.

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