Hydrogel for osteoporotic bone defect repair and preparation method thereof

Abstract

The invention discloses hydrogel for osteoporotic bone defect repair and a preparation method of the hydrogel. The hydrogel comprises basic hydrogel and mineralized photosynthetic microorganisms, the mineralized photosynthetic microorganism comprises a photosynthetic microorganism, a mineralized shell layer arranged on the surface of the photosynthetic microorganism, and rare earth up-conversion nano-particles loaded in cells of the photosynthetic microorganism, and the rare earth up-conversion nano-particles can convert near-infrared light into visible light. According to the present invention, the oxygen production type mineralized microalgae composite hydrogel with characteristics of deep tissue activation, controllable oxygen production and simultaneous realization of four-effect synergy of acid neutralization, continuous oxygen supply, angiogenesis promotion and osteogenesis mineralization can be provided, such that osteoblast activity can be promoted, osteoclast activity can be inhibited, in-vivo osteogenesis can be promoted, and the technical support can be provided for osteoporotic bone defect repair.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to a hydrogel for repairing osteoporotic bone defects and its preparation method. Background Technology

[0002] Osteoporosis (OP) is a systemic bone disease characterized by decreased bone mass and destruction of bone microstructure, leading to increased bone fragility and fracture risk. The pathological microenvironment of osteoporotic bone defects has the following core characteristics: 1. Acidic microenvironment: Overactive osteoclasts secrete large amounts of H+. + The local pH can be as low as 4.5-5.5, directly dissolving bone minerals and inhibiting osteoblast function; 2. Hypoxia and insufficient angiogenesis: Destruction of bone microstructure leads to a reduction in vascular network, and severe local hypoxia hinders stem cell recruitment, osteogenic differentiation and tissue regeneration; 3. Chronic inflammation and immune imbalance: A pro-inflammatory state dominated by M1 macrophages continuously releases factors such as TNF-α and IL-6, which exacerbate osteoclast activity and inhibit osteogenic formation; 4. Imbalance in bone metabolism: The coexistence of abnormally high osteoclast activity and impaired osteoblast function leads to bone resorption far exceeding bone formation.

[0003] Therefore, the local pathological microenvironment of bone defects caused by osteoporosis is complex, resulting in poor repair capacity and difficulty in healing. Current treatment strategies, such as autologous / allogeneic bone transplantation, have inherent limitations, including limited donors, immune rejection, infection risks, and the inability to regulate the pathological microenvironment. Single regulatory strategies (such as anti-resorption drugs and growth factor delivery) are insufficient to simultaneously reverse these multiple pathological processes and suffer from significant side effects and limited efficacy. Therefore, there is an urgent need to develop novel intelligent biomaterials that can synergistically regulate the complex microenvironment of osteoporosis and promote in-situ bone regeneration. Summary of the Invention

[0004] To address the above-mentioned problems, the present invention aims to provide a hydrogel for repairing osteoporotic bone defects and a method for preparing the same.

[0005] The technical solution of the present invention is as follows: On the one hand, a hydrogel for repairing osteoporotic bone defects is provided, comprising a base hydrogel and mineralized photosynthetic microorganisms, wherein the mineralized photosynthetic microorganisms include photosynthetic microorganisms, a mineralized shell disposed on the surface of the photosynthetic microorganisms, and rare earth upconversion nanoparticles loaded inside the cells of the photosynthetic microorganisms, wherein the rare earth upconversion nanoparticles are capable of converting near-infrared light into visible light.

[0006] Preferably, the base hydrogel is any one or more of methacrylamide gelatin, sodium alginate, hyaluronic acid, chitosan, polyethylene glycol derivatives, and silk fibroin; the photosynthetic microorganism is any one of Synechococcus spp., cyanobacteria, and green algae; the mineralized shell is made of any one or more of calcium carbonate, calcium phosphate, and calcium silicate; and the rare earth upconversion nanoparticles are any one or more of NaYF4:Yb,Er, NaGdF4:Yb,Er, NaLuF4:Yb,Er, LiYF4:Yb,Er, NaYF4:Yb,Tm, and NaYF4:Yb,Er@NaYF4.

[0007] Preferably, the mineralized shell is deposited on the surface of the photosynthetic microorganisms through in-situ deposition.

[0008] Preferably, the base hydrogel is methacrylamide gelatin, and the rare earth upconversion nanoparticles are NaYF4:Yb,Er.

[0009] Preferably, the mass ratio of the mineralized shell to the photosynthetic microorganisms is 0.1-2:1, and the microbial density of the mineralized photosynthetic microorganisms in the hydrogel is 10. 6 -10 8 per mL.

[0010] On the other hand, a method for preparing the hydrogel for repairing osteoporotic bone defects as described in any one of the above claims is also provided, comprising the following steps: S1: Synthesize oil-soluble rare earth upconversion nanoparticles, and disperse the oil-soluble rare earth upconversion nanoparticles in a water / chloroform two-phase system containing sodium alendronate or polyacrylic acid, and ultrasonically stir to convert them into water-soluble nanoparticles to obtain water-soluble rare earth upconversion nanoparticles. S2: Obtain photosynthetic microorganisms, expand their culture to the target cell growth density, centrifuge and concentrate them to obtain cell concentrate; S3: The water-soluble rare earth upconversion nanoparticles and the cell concentrate are stirred and mixed in the dark, and after washing, photosynthetic microorganisms loaded with rare earth upconversion nanoparticles are obtained inside the cells. S4: The photosynthetic microorganisms loaded with rare earth upconversion nanoparticles inside the cells are mixed with a surface modifier to obtain a photosynthetic microorganism solution. Mineralizing materials are added to the photosynthetic microorganism solution and deposited in situ to generate a mineralized shell on the surface of the photosynthetic microorganisms to obtain mineralized photosynthetic microorganisms. S5: Prepare a basic hydrogel prepolymer solution containing a photoinitiator, add the mineralized photosynthetic microorganisms to the basic hydrogel prepolymer solution containing the photoinitiator, mix well, and irradiate with 320-405nm ultraviolet light for 1-10 minutes to obtain the hydrogel.

[0011] Preferably, in step S1, the oil-soluble upconversion nanoparticles are synthesized using a solvothermal method; in step S2, the target cell growth density is 1.5-3.

[0012] Preferably, in step S1, the ultrasonic stirring time is 6-24 hours; in step S3, the stirring and mixing time in the dark is 4-12 hours; and in step S4, the in-situ deposition time is 1-6 hours.

[0013] Preferably, in step S4, the surface modifier is any one or more of polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, chitosan, carboxymethyl chitosan, sodium alginate, and polyacrylic acid.

[0014] Preferably, in step S5, the photoinitiator is I2959 or LAP.

[0015] The beneficial effects of this invention are: This invention provides an oxygen-producing mineralizing microalgae composite hydrogel that can deeply activate tissues, control oxygen production, and simultaneously achieve a synergistic effect of "neutralizing acidity + continuous oxygen supply + promoting angiogenesis + osteogenic mineralization". It promotes osteoblast activity, inhibits osteoclast activity, promotes osteogenesis in vivo, and provides technical support for the repair of osteoporotic bone defects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 SEM image of Gel@P / U / C in Example 1; Figure 2 This is a schematic diagram showing the verification results of the calcium ion release and oxygen production performance of the Gel@P / U / C material in Example 1; where a is the calcium ion release curve at different pH levels and b is the dissolved oxygen release curve at different days. Figure 3 This is a schematic diagram illustrating the biosafety verification results of the Gel@P / U / C material in Example 1. Figure 4 This is a schematic diagram showing the in vitro validation results of the immunomodulatory properties of the Gel@P / U / C material in Example 1; where a is a schematic diagram of the IL-4 content detection results, b is a schematic diagram of the TGF-β content detection results, c is a schematic diagram of the TNF-α content detection results, and d is a schematic diagram of the IL-6 content detection results. Figure 5This is a schematic diagram illustrating the in vitro verification results of the osteogenic, osteoclast-inhibiting, and angiogenesis-promoting properties of the Gel@P / U / C material in Example 1. In this diagram, a represents the Western blot (WB) results of osteoblast markers ALP, RUNX2, BMP-2, and OPN; b represents the WB results of osteoclast markers C-fos, TRAP, and CTSK; c represents the quantitative analysis results of HUVEC migration; and d represents the quantitative analysis results of the angiogenesis experiment. Figure 6 This is a schematic diagram of the in vivo bone-promoting capacity verification results of Gel@P / U / C material in Example 1; where a is a schematic diagram of BMD quantitative results and b is a schematic diagram of BV / TV quantitative results. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and technical features described in this application can be combined with each other. It should also be pointed out that, unless otherwise indicated, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terms "comprising" or "including" and similar words used in this invention refer to elements or objects preceding the word that encompass the elements or objects listed following the word and their equivalents, without excluding other elements or objects.

[0019] On one hand, the present invention provides a hydrogel for repairing osteoporotic bone defects, comprising a base hydrogel and mineralized photosynthetic microorganisms, wherein the mineralized photosynthetic microorganisms include photosynthetic microorganisms, a mineralized shell disposed on the surface of the photosynthetic microorganisms, and rare earth upconversion nanoparticles loaded inside the cells of the photosynthetic microorganisms, wherein the rare earth upconversion nanoparticles are capable of converting near-infrared light into visible light.

[0020] In this invention, the hydrogel, when irradiated with near-infrared light (tissue penetration depth > 10 mm) at 808-980 nm, can utilize rare-earth upconversion nanoparticles to convert the near-infrared light into visible light at 400-550 nm, thereby providing light for photosynthetic microorganisms to perform photosynthesis; when the mineralized shell contains calcium carbonate, it can react with locally excessive H+. + The reaction generates CO2, which serves as an additional carbon source for photosynthesis, thus improving oxygen production efficiency. When the mineralized shell is calcium phosphate, calcium silicate, or a mixture thereof, it can release mineral ions (such as Ca2+). + PO4 3- SiO3 2-The hydrogel neutralizes the acidic microenvironment and promotes osteogenic mineralization; the mineral ions generated by degradation can be mineralized in situ by body fluid phosphate into phosphate nanoparticles, further regulating the immune microenvironment; the exosomes continuously secreted by photosynthetic microorganisms can synergistically promote vascular endothelial cell migration and lumen formation with oxygen; in summary, the hydrogel of the present invention can achieve a four-effect synergistic effect of "neutralizing acidity + continuous oxygen supply + promoting angiogenesis + osteogenic mineralization".

[0021] In one specific embodiment, the base hydrogel is any one or more of methacrylamide gelatin, sodium alginate, hyaluronic acid, chitosan, polyethylene glycol derivative, and silk fibroin; the photosynthetic microorganism is any one of Synechocystis sp. PCC 6803, cyanobacteria (optionally, the cyanobacteria is Synechocystis sp. PCC 6803), and green algae (optionally, the green algae is Chlorella); the mineralized shell is made of any one or more of calcium carbonate, calcium phosphate, and calcium silicate; and the rare earth upconversion nanoparticles are any one or more of NaYF4:Yb,Er, NaGdF4:Yb,Er, NaLuF4:Yb,Er, LiYF4:Yb,Er, NaYF4:Yb,Tm, and NaYF4:Yb,Er@NaYF4.

[0022] In one specific embodiment, the mineralized shell is deposited on the surface of the photosynthetic microorganisms through in-situ deposition.

[0023] In one specific embodiment, the base hydrogel is methacrylamide gelatin, and the rare earth upconversion nanoparticles are NaYF4:Yb,Er. In this embodiment, the methacrylamide gelatin exhibits excellent cell compatibility and photocrosslinking properties, while the NaYF4:Yb,Er rare earth upconversion nanoparticles demonstrate superior performance in terms of upconversion efficiency and visible light emission intensity, making them particularly suitable for activating photosynthetic microorganisms.

[0024] In one specific embodiment, the NaYF4:Yb,Er rare earth upconversion nanoparticles have a specified molar content of Yb of 10-25%, a molar content of Er of 0.5-5%, and a molar content of Y as the balance (100% - Yb% - Er%), approximately 70-90%.

[0025] In one specific embodiment, the mass ratio of the mineralized shell to the photosynthetic microorganisms is 0.1-2:1, and the microbial density of the mineralized photosynthetic microorganisms in the hydrogel is 10. 6 -10 8 per mL.

[0026] On the other hand, the present invention also provides a method for preparing the hydrogel for repairing osteoporotic bone defects as described in any one of the above claims, comprising the following steps: S1: Synthesize oil-soluble rare earth upconversion nanoparticles, and disperse the oil-soluble rare earth upconversion nanoparticles in a water / chloroform two-phase system containing sodium alendronate or polyacrylic acid, and ultrasonically stir to convert them into water-soluble nanoparticles to obtain water-soluble rare earth upconversion nanoparticles. S2: Obtain photosynthetic microorganisms, expand their culture to the target cell growth density, centrifuge and concentrate them to obtain cell concentrate; S3: The water-soluble rare earth upconversion nanoparticles and the cell concentrate are stirred and mixed in the dark, and after washing, photosynthetic microorganisms loaded with rare earth upconversion nanoparticles are obtained inside the cells. S4: The photosynthetic microorganisms loaded with rare earth upconversion nanoparticles inside the cells are mixed with a surface modifier to obtain a photosynthetic microorganism solution. Mineralizing materials are added to the photosynthetic microorganism solution and deposited in situ to generate a mineralized shell on the surface of the photosynthetic microorganisms to obtain mineralized photosynthetic microorganisms. S5: Prepare a basic hydrogel prepolymer solution containing a photoinitiator, add the mineralized photosynthetic microorganisms to the basic hydrogel prepolymer solution containing the photoinitiator, mix well, and irradiate with 320-405nm ultraviolet light for 1-10 minutes to obtain the hydrogel.

[0027] In one specific embodiment, in step S1, the oil-soluble upconversion nanoparticles are synthesized using a solvothermal method. It should be noted that the synthesis of oil-soluble rare-earth upconversion nanoparticles is prior art. In this embodiment, a solvothermal method is used for synthesis, and the specific operating conditions (such as reaction temperature, time, precursor ratio, etc.) can be referenced from conventional processes in the art, and will not be elaborated further here. Furthermore, besides the solvothermal method, other existing methods suitable for synthesizing oil-soluble rare-earth upconversion nanoparticles with similar upconversion luminescence properties (such as thermal decomposition, co-precipitation, microemulsion, etc.) are also applicable to this invention.

[0028] In one specific embodiment, in step S2, the target cell growth density is 1.5-3. It should be noted that expanding the photosynthetic microorganisms to the target cell growth density ensures a sufficient number of cells for subsequent centrifugation concentration and loading with rare earth upconversion nanoparticles and mineralization treatment. This guarantees that the loading density of mineralized photosynthetic microorganisms in the final hydrogel is sufficient to achieve sustained and efficient oxygen production, immune regulation, and osteopromoting effects. A higher target cell growth density results in a higher concentration of the cell concentrate obtained after subsequent concentration, and a higher density of mineralized photosynthetic microorganisms in the final hydrogel, which is beneficial for improving the oxygen production capacity and bioactivity of the material. However, excessively high density may lead to intercellular nutrient competition or decreased cell viability. (Except for the OD used in this embodiment...)680 Besides 1.5-3.0, other target cell growth densities in the prior art that can achieve similar cell harvest and viability can also be applied to this invention.

[0029] In one specific embodiment, in step S1, the ultrasonic stirring time is 6-24 hours; in step S3, the stirring and mixing time in the dark is 4-12 hours; and in step S4, the in-situ deposition time is 1-6 hours.

[0030] In one specific embodiment, in step S4, the surface modifier is any one or more of polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, chitosan, carboxymethyl chitosan, sodium alginate, and polyacrylic acid.

[0031] In one specific embodiment, in step S5, the photoinitiator is I2959 or LAP. It should be noted that the photoinitiator is used to generate free radicals under ultraviolet or blue light irradiation, initiating the double bond polymerization reaction in the basic hydrogel prepolymer, thereby achieving rapid in-situ crosslinking and molding of the hydrogel while maintaining low phototoxicity to the supported mineralized photosynthetic microorganisms, thus preserving their cell viability and photosynthetic activity. Besides I2959 or LAP used in this embodiment, other photoinitiators in the prior art that can achieve similar photoinitiated polymerization and have good biocompatibility (such as TPO (2,4,6-trimethylbenzoyl-diphenylphosphine oxide)) are also applicable to this invention.

[0032] In a specific embodiment, when the base hydrogel is methacrylamide gelatin, step S5, preparing the base hydrogel prepolymer containing the photoinitiator includes the following sub-steps: dissolving methacrylamide gelatin in PBS buffer to prepare a solution with a mass concentration of 5-20%, then adding a photoinitiator with a mass concentration of 0.01-0.5%, heating to dissolve, and obtaining the base hydrogel prepolymer containing the photoinitiator.

[0033] It should be noted that, in addition to the photocrosslinking preparation method described above, other existing thermal crosslinking, ionic crosslinking, or enzymatic crosslinking methods can also be applied to the preparation of the hydrogel described in this invention.

[0034] Example 1 A hydrogel for repairing osteoporotic bone defects is prepared by the following steps: (1) Oil-soluble rare earth upconversion nanoparticles NaYF4:Yb,Er (Y 77 mol%; Yb 21 mol%; Er 2 mol%) (UCNPs) were synthesized by solvothermal method. The oil-soluble UCNPs were dispersed in a mixed solution containing 40 mL of chloroform and 400 mg of sodium alendronate and stirred vigorously at room temperature for 12 h to convert them into water-soluble rare earth upconversion nanoparticles. (2) Synechococcus elongatus PCC 7942 was cultured in BG-11 medium until OD 680 = 2.5, centrifuge at 7000 rpm for 5 min to collect cells, resuspend in sterile PBS and concentrate to a cell density of approximately 102. 9 cells / mL, to obtain concentrated cell solution; (3) The water-soluble rare earth upconversion nanoparticles and the cell concentrate were stirred and mixed in the dark for 6 hours, washed three times with RO water, and stored in RO water for later use; (4) Add 2 mg / mL of PVP K30 to the mixture in step (3) to obtain algal solution, and add 0.33 mol / L CaCl2 and equimolar Na2CO3 to the algal solution. React for 6 h to deposit calcium carbonate on the surface of algal cells in situ and mineralize it in situ to obtain mineralized Synechococcus. (5) Weigh out methacrylamide gelatin (70% degree of substitution) and dissolve it in PBS to prepare a 10% solution. Add 0.01% photoinitiator I2959 and dissolve at 50°C to obtain GelMA prepolymer solution. (6) Add the mineralized Synechococcus to the GelMA prepolymer solution, mix well, and irradiate with 320-405nm ultraviolet light for 5min to obtain the hydrogel, denoted as Gel@P / U / C.

[0035] Example 2 Unlike Example 1, in step (1) of this example, the rare earth upconversion nanoparticle doping ratio is Y 78 mol%, Yb 20 mol%, Er 2 mol%, and polyacrylic acid (PAA, molecular weight 2000 Da, 200 mg) is used instead of sodium alendronate during phase transfer. The final hydrogel is denoted as Gel@P / U / C-PAA.

[0036] Example 3 Unlike Example 1, in step (2) of this example, Chlorella vulgaris is used instead of Synechococcus slenderus, and the culture is carried out until OD. 680= 2.0 after concentration; in step (4), the mineralizing material was changed to 0.33 mol / L CaCl2 and equimolar Na2HPO4, and the reaction was carried out at room temperature for 4 h to deposit a calcium phosphate shell in situ. The final hydrogel is denoted as Gel@C / U / P (in this example, C represents Chlorella and P represents calcium phosphate).

[0037] Comparative Example 1 Unlike Example 1, this comparative example omits the calcium carbonate mineralization step in step (4) and directly embeds the microalgae concentrate obtained in step (3) into the GelMA hydrogel in steps (5) and (6) to obtain a hydrogel without rare earth upconversion nanoparticles, denoted as Gel@P / U.

[0038] Test Example 1 The morphology of Example 1 was observed using a scanning electron microscope, and the results are as follows: Figure 1 As shown. From Figure 1 It can be seen that Synechococcus elongatus PCC 7942 (green) is uniformly distributed in the microporous network structure of Gel@P / U / C.

[0039] Test Example 2 The material's ability to release calcium ions under acidic conditions was verified using a calcium ion detection kit, and the results are as follows: Figure 2 As shown in a. From Figure 2 As can be seen, the material can continuously release calcium ions under acidic conditions. Next, a dissolved oxygen meter was used to verify the material's oxygen production performance under light irradiation, and the results are as follows: Figure 2 As shown in b. From Figure 2 b shows that the material can continuously produce oxygen under 980 nm light irradiation, indicating that the material has the ability to produce oxygen under light irradiation.

[0040] Test Example 3 Cell viability at the standard working concentration (1000 μg / mL) was assessed using CCK-8 counting assays, and the results are as follows: Figure 3 As shown. From Figure 3 It can be seen that the cell survival rate of the material is still above 80% at the conventional working concentration (1000 μg / mL), and there is no significant cytotoxicity, which can be further investigated.

[0041] Test Example 4 To investigate the material's ability to reverse the osteoporosis immune microenvironment, the material was co-incubated with RAW264.7, and the results are as follows: Figure 4 As shown. From Figure 4It can be seen that the material induces the upregulation of M2 macrophage markers IL-4 and TGF-β. Simultaneously, the material also induces the downregulation of M1 markers TNF-α and IL-6, indicating that both calcium ion release (Gel@P / U / C group) and oxygen production (Gel@P / U+NIR group) can partially promote macrophage polarization towards the M2 type, and the combination of the two (Gel@P / U / C+NIR group) exhibits a significantly enhanced synergistic effect, revealing the synergistic amplifying effect of calcium ion signaling and oxygen abundance in activating the M2 polarization pathway.

[0042] Test Example 5 To verify the in vitro osteogenic and osteoclast-inhibiting properties of the material, RAW264.7 cells were first co-incubated with Gel@P / U / C under NIR irradiation. The calcium ions released by the material and the dissolved oxygen generated by PCC7942 photosynthesis effectively drove the polarization of RAW264.7 macrophages towards the anti-inflammatory M2 phenotype. The supernatant was then collected to prepare "M2 macrophage conditioned medium (M2 medium)". Osteogenic and osteoclast-related proteins were then detected by Western blotting. The results are as follows: Figure 5 As shown in Figure a, compared with the control group, Gel@P / U / C, M2 macrophage conditioned medium (M2 medium), and their combined treatment group (Gel@P / U / C + M2 medium) all significantly upregulated the expression levels of osteoblast markers ALP, RUNX2, BMP-2, and OPN. Notably, the Gel@P / U / C + M2 medium group exhibited the best osteogenic induction activity, revealing that the Gel@P / U / C + NIR system promotes bone regeneration through a dual mechanism: on the one hand, the released calcium ions can directly increase intracellular calcium concentration and provide mineralization raw materials; on the other hand, the released calcium... 2+ It synergistically induces macrophage M2 polarization with O2, thereby constructing a regenerative microenvironment rich in anti-inflammatory factors.

[0043] Overactivation of osteoclasts and their mediated bone resorption are the core pathological mechanisms leading to the poor healing of osteoporotic bone defects. Therefore, an M2 macrophage conditioned medium (M2 medium) was prepared using the previously established Gel@P / U / C+NIR system to investigate the synergistic inhibitory effect of the material-mediated physicochemical microenvironment (pH regulation) and immune microenvironment (M2 factor) on osteoclast differentiation. Figure 5Western blot results showed that the expression levels of osteoclast-related proteins (c-Fos, TRAP, and CTSK) were significantly downregulated in the Gel@P / U / C, M2, and Gel@P / U / C+M2medium groups, with the Gel@P / U / C+M2medium group exhibiting the greatest inhibition. This suggests that Gel@P / U / C significantly inhibits osteoclast activity by neutralizing the acidic microenvironment, while M2 exerts a similar effect by secreting anti-inflammatory factors. This Gel@P / U / C-mediated immune-microenvironment co-regulation strategy demonstrates great potential for treating osteoporotic bone defects.

[0044] In areas of bone defects caused by osteoporosis, there is a reduction in local vascular networks. Therefore, the effects of Gel@P / U photosynthetically produced oxygen and secreted EVs on scratching and tubular formation were investigated. Results are as follows: Figure 5 Quantitative analysis showed that oxygen produced by photosynthesis effectively promoted the migration of HUVECs. Furthermore, the migration rates of the oxygen + GW4869 (EV inhibitor) and Gel@P / U (EVs) groups were lower than those of the Gel@P / U + NIR group. These results indicate that EVs secreted by PCC 7942 and oxygen produced by PCC7942 through photosynthesis jointly promote the migration of HUVECs. The quantitative results of the angiogenesis assay were consistent with those of the scratch assay. Figure 5 d).

[0045] Test Example 6 Based on in vitro experiments, the promoting effect of the material on in vivo osteogenicity was further investigated. Eight weeks after osteoporosis modeling of C57, bone defect modeling was performed, followed by treatment. Micro-CT technology is a highly invasive method for quantitative assessment of bone tissue healing. At weeks eight and twelve, bone defect repair was assessed using micro-CT via three-dimensional reconstruction. Trabeculae are crucial for supporting hematopoiesis within the medullary cavity and play a key role in bone regeneration. Quantitative analysis results of bone microparameters are as follows: Figure 6 As shown. From Figure 6 It can be seen that the final material group showed an increase in both BMD and BV / TV, verifying the material's role in promoting osteogenesis in vivo.

[0046] It should be noted that the basic hydrogel is designed to provide a biocompatible three-dimensional network structure for encapsulating and immobilizing mineralized photosynthetic microorganisms, enabling the material to be injected and molded in situ. Simultaneously, it provides a suitable microenvironment for the mineralized photosynthetic microorganisms to maintain their long-term vitality, photosynthetic activity, and material exchange. Besides the methacryloyl gelatin used in the above embodiments, other hydrogels with similar encapsulation, cross-linking, and biocompatibility properties (such as sodium alginate, hyaluronic acid, chitosan, polyethylene glycol derivatives, silk fibroin, etc.) in the prior art can also be applied to this invention.

[0047] The photosynthetic microorganisms are designed to produce oxygen through photosynthesis under visible light irradiation and to secrete active substances such as exosomes to promote angiogenesis. Besides *Synechococcus elongatus* PCC 7942 used in the above embodiments, other microorganisms in the prior art capable of photosynthetic oxygen production and similar biological activities (such as other cyanobacteria, green algae, *Chlorella*, *Synechocystis* sp. PCC 6803, etc.) can also be applied to this invention.

[0048] The mineralized shell is designed to protect photosynthetic microorganisms from external stress and to respond to the acidic conditions of the osteoporosis pathological microenvironment (which consumes H+). + Neutralizes acidity and releases Ca 2+ Mineral ions promote osteogenic mineralization, and when calcium carbonate is used, it reacts with H+. + The reaction produces CO2, which serves as an additional carbon source for photosynthesis to enhance oxygen production efficiency. Besides the calcium carbonate used in the above embodiments, other inorganic materials with similar acid-responsiveness, mineral ion release, and protective functions (such as calcium phosphate, calcium silicate, or mixtures thereof) in the prior art can also be applied to this invention.

[0049] The rare-earth upconversion nanoparticles are designed to be loaded inside photosynthetic microbial cells, converting near-infrared light (tissue penetration depth > 10 mm) into visible light (400-700 nm), thereby activating photosynthetic microorganisms in deep tissues to perform photosynthesis, achieving controllable oxygen production and deep activation. Besides the NaYF4:Yb,Er rare-earth upconversion nanoparticles used in the above embodiments, other rare-earth nanoparticles with similar upconversion luminescence properties in the prior art (such as NaGdF4:Yb,Er, NaYF4:Yb,Tm, LiYF4:Yb,Er, core-shell structure NaYF4:Yb,Er@NaYF4, etc.) are also applicable to this invention.

[0050] The surface modifier mixed with photosynthetic microorganisms in step S4 serves as a dispersant, stabilizer, and mineralization template to promote uniform in-situ deposition of mineralized materials on the surface of photosynthetic microorganisms. Besides PVP K30 used in the above embodiments, other polymers with similar dispersing, stabilizing, and template functions (such as polyethylene glycol, polyvinyl alcohol, chitosan, polyacrylic acid, sodium alginate, etc.) in the prior art can also be applied to this invention.

[0051] The above description is merely a representative embodiment of the present invention and is not intended to limit the present invention in any way. Any embodiment made by those skilled in the art without departing from the scope of the present invention and utilizing the disclosed technical content is an equivalent embodiment of the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A hydrogel for repairing osteoporotic bone defects, characterized in that, It includes a basic hydrogel and mineralized photosynthetic microorganisms. The mineralized photosynthetic microorganisms include photosynthetic microorganisms, a mineralized shell disposed on the surface of the photosynthetic microorganisms, and rare earth upconversion nanoparticles loaded inside the cells of the photosynthetic microorganisms. The rare earth upconversion nanoparticles can convert near-infrared light into visible light.

2. The hydrogel for repairing osteoporotic bone defects according to claim 1, characterized in that, The base hydrogel is any one or more of methacrylamide gelatin, sodium alginate, hyaluronic acid, chitosan, polyethylene glycol derivatives, and silk fibroin; the photosynthetic microorganism is any one of Synechococcus spp., cyanobacteria, and green algae; the mineralized shell is made of any one or more of calcium carbonate, calcium phosphate, and calcium silicate; and the rare earth upconversion nanoparticles are any one or more of NaYF4:Yb,Er, NaGdF4:Yb,Er, NaLuF4:Yb,Er, LiYF4:Yb,Er, NaYF4:Yb,Tm, and NaYF4:Yb,Er@NaYF4.

3. The hydrogel for repairing osteoporotic bone defects according to claim 1 or 2, characterized in that, The mineralized shell is deposited on the surface of the photosynthetic microorganisms through in-situ deposition.

4. The hydrogel for repairing osteoporotic bone defects according to claim 2, characterized in that, The basic hydrogel is methacrylamide gelatin, and the rare earth upconversion nanoparticles are NaYF4:Yb,Er.

5. The hydrogel for repairing osteoporotic bone defects according to any one of claims 1-4, characterized in that, The mass ratio of the mineralized shell to the photosynthetic microorganisms is 0.1-2:1, and the microbial density of the mineralized photosynthetic microorganisms in the hydrogel is 10. 6 -10 8 per mL.

6. The method for preparing the hydrogel for repairing osteoporotic bone defects as described in any one of claims 1-5, characterized in that, Includes the following steps: S1: Synthesize oil-soluble rare earth upconversion nanoparticles, and disperse the oil-soluble rare earth upconversion nanoparticles in a water / chloroform two-phase system containing sodium alendronate or polyacrylic acid, and ultrasonically stir to convert them into water-soluble nanoparticles to obtain water-soluble rare earth upconversion nanoparticles. S2: Obtain photosynthetic microorganisms, expand their culture to the target cell growth density, centrifuge and concentrate them to obtain cell concentrate; S3: The water-soluble rare earth upconversion nanoparticles and the cell concentrate are stirred and mixed in the dark, and after washing, photosynthetic microorganisms loaded with rare earth upconversion nanoparticles are obtained inside the cells. S4: The photosynthetic microorganisms loaded with rare earth upconversion nanoparticles inside the cells are mixed with a surface modifier to obtain a photosynthetic microorganism solution. Mineralizing materials are added to the photosynthetic microorganism solution and deposited in situ to generate a mineralized shell on the surface of the photosynthetic microorganisms to obtain mineralized photosynthetic microorganisms. S5: Prepare a basic hydrogel prepolymer solution containing a photoinitiator, add the mineralized photosynthetic microorganisms to the basic hydrogel prepolymer solution containing the photoinitiator, mix well, and irradiate with 320-405nm ultraviolet light for 1-10 minutes to obtain the hydrogel.

7. The method for preparing the hydrogel for repairing osteoporotic bone defects according to claim 6, characterized in that, In step S1, the oil-soluble upconversion nanoparticles are synthesized using a solvothermal method; in step S2, the target cell growth density is 1.5-3.

8. The method for preparing the hydrogel for repairing osteoporotic bone defects according to claim 6, characterized in that, In step S1, the ultrasonic stirring time is 6-24 hours; in step S3, the stirring and mixing time in the dark is 4-12 hours; in step S4, the in-situ deposition time is 1-6 hours.

9. The method for preparing the hydrogel for repairing osteoporotic bone defects according to claim 6, characterized in that, In step S4, the surface modifier is any one or more of polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, chitosan, carboxymethyl chitosan, sodium alginate, and polyacrylic acid.

10. The method for preparing a hydrogel for repairing osteoporotic bone defects according to any one of claims 6-9, characterized in that, In step S5, the photoinitiator is I2959 or LAP.

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