Composite hydrogel loaded with rare human ginsenoside Rh4-ce nanoparticles

Abstract

The application belongs to the technical field of medical materials, and provides a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticle, wherein the rare ginsenoside Rh4 is complexed with cerium ions to form a nanoparticle, the dispersibility and stability of the rare ginsenoside Rh4 in the hydrogel system are significantly improved, and the Ce 3+ / Ce 4+ valence state is cycled to endow the material with sustained antioxidant and microenvironment regulation capabilities, the GelMA provides a three-dimensional scaffold structure and cell attachment space, the HA provides mechanical enhancement and mineralization induction, and the CeRh4NCs cooperatively improve the local inflammatory oxidative stress microenvironment and potentially promote osteogenesis and angiogenesis. The application overcomes the technical defects of conventional bone repair materials in the application of active factors, microenvironment regulation and mechanical support, forms a multifunctional craniomaxillofacial bone defect repair material with anti-inflammatory and antioxidant properties, and promotes osteogenesis and vascularization, and has application value.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and particularly relates to a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles. Background Technology

[0002] Maxillofacial bone defects are often caused by various factors such as trauma, tumor resection, congenital malformations, or infection, and their repair has always been an important clinical problem in the fields of oral and maxillofacial surgery and bone tissue engineering. Current clinical treatment methods mainly include autologous bone grafting, allogeneic bone grafting, and implantation of artificial bone substitutes. Among them, although autologous bone grafting is considered a classic method for bone defect repair, it still has problems such as limited bone source, large surgical trauma, high risk of donor site complications, high bone resorption rate, and insufficient matching for complex bone defect morphology. Although allogeneic bone and some bone substitutes can compensate for insufficient autologous bone to a certain extent, they still have defects such as limited osteogenic activity, insufficient bone integration capacity, risk of infection, and immune rejection, making it difficult to meet the comprehensive needs of craniofacial bone defects for structural reconstruction, functional restoration, and aesthetic repair.

[0003] In recent years, hydrogel scaffolds have attracted widespread attention in the field of bone defect repair due to their excellent plasticity, biocompatibility, water content, and three-dimensional network structure that mimics the extracellular matrix. Among them, GelMA (gelatin methacrylamide) hydrogels have good cell adhesion, photocrosslinking and molding capabilities, and tissue adaptability, making them an important candidate system for bone repair scaffold materials. However, single GelMA hydrogels usually suffer from insufficient mechanical strength, limited osteoinductive capacity, and weak ability to regulate the local inflammatory and oxidative stress microenvironment, making it difficult to meet the repair needs of bone defects, especially in the craniofacial region under load and in complex microenvironments. To improve these shortcomings, hydroxyapatite (HA), which has osteoconductive and inorganic mineralization characteristics, has been introduced into the GelMA system. This can improve the mechanical properties of the scaffold and enhance osteoinductive capacity, but relying solely on the combination of HA and hydrogel is still insufficient to fully regulate the inflammatory and oxidative stress microenvironment.

[0004] Ginsenosides are one of the main active ingredients in ginseng plants. Based on their content and formation process in natural ginseng, they can be divided into common ginsenosides and rare ginsenosides. Rare ginsenoside Rh4 belongs to the dammarane-type triterpenoid saponins and is commonly found in steamed or processed ginseng products. It exhibits superior biological activity compared to traditional saponins in areas such as anti-cancer activity, cardiovascular protection, metabolic regulation, anti-inflammatory effects, and antioxidant activity. It can effectively regulate inflammatory factors, improve the local microenvironment, and provide favorable conditions for tissue repair. However, to date, no literature has reported that rare ginsenoside Rh4 has a direct osteogenic effect. Furthermore, due to its low water solubility, limited local retention capacity, and insufficient stability, rare ginsenoside Rh4 has not been used as an active ingredient in traditional bone repair materials. Summary of the Invention

[0005] The purpose of this invention is to provide a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles, aiming to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] On the one hand, the present invention provides a method for preparing a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles, comprising the following steps:

[0008] Preparation of S1 and CeRh4NCs: PEG 8000 and Ce(NO3)3·6H2O were dispersed in ultrapure water and sonicated until completely dissolved to obtain a Ce-containing solution. Rare ginsenoside Rh4 was dissolved in ethanol to prepare an Rh4 ethanol solution. The Rh4 ethanol solution was slowly added to the Ce-containing solution, and the coordination self-assembly reaction was initiated by stirring at room temperature to obtain a CeRh4NCs solution. After the reaction was completed, the solution was centrifuged, washed, and then freeze-dried to obtain CeRh4NCs.

[0009] S2, Construction of HA / GelMA composite precursor solution: Dissolve gelatin methacrylamide product GelMA in PBS or deionized water, heat and stir to fully dissolve it, add photoinitiator to prepare GelMA precursor solution, add nano hydroxyapatite powder HA to GelMA precursor solution, sonicate to disperse it evenly, and obtain HA / GelMA composite precursor solution.

[0010] S3. Formation of CeRh4NCs@HA / GelMA composite hydrogel: CeRh4NCs powder is dispersed in HA / GelMA composite precursor solution and ultrasonically dispersed to form CeRh4NCs / HA / GelMA composite precursor system. CeRh4NCs@HA / GelMA composite hydrogel is formed by photocrosslinking and curing, which is a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles.

[0011] On the other hand, the present invention provides a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles, which is prepared by the above-mentioned preparation method.

[0012] On the other hand, the present invention provides an application of a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles in the preparation of maxillofacial bone defect repair materials.

[0013] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0014] This invention organically combines CeRh4 metal complex nanoparticles, nano-hydroxyapatite, and GelMA hydrogel to construct a composite system that has both bioactivity and structural support functions, overcoming the shortcomings of existing single hydrogel materials with insufficient mechanical properties, single inorganic materials with poor biocompatibility, and traditional bone substitute materials with limited functions.

[0015] The CeRh4NCs prepared in this invention are obtained through the use of cerium ions (Ce). 3+ / Ce 4+The valence cycle can continuously scavenge excess reactive oxygen species locally, reducing inflammation and oxidative stress damage. Simultaneously, rare ginsenoside Rh4 exerts anti-inflammatory and osteogenic effects, effectively improving the local microenvironment of bone defects and providing favorable conditions for osteogenic and tissue regeneration. By complexing with Ce ions to form nanoparticles (CeRh4NCs), it overcomes the problems of low water solubility, poor local retention capacity, and insufficient stability of natural bioactive small molecules, achieving stable release and microenvironment regulation of rare ginsenoside Rh4 in the hydrogel. The introduction of nano-hydroxyapatite significantly improves the mechanical properties and structural stability of the composite hydrogel and provides a mineralized microenvironment similar to natural bone tissue, which is conducive to promoting osteoblast adhesion, proliferation, differentiation, and mineralization, improving the material's osteoconductivity and bone repair efficiency. Meanwhile, the three-dimensional network structure formed by GelMA... The composite hydrogel possesses excellent biocompatibility, water retention, and biodegradability, providing a suitable growth space for cells and enabling local retention and sustained release of active components at the defect site. In vitro experimental results show that the composite hydrogel provided by this invention has good cell and blood compatibility, promoting cell activity, cell spreading, and scaffold remodeling. It significantly enhances alkaline phosphatase (ALP) activity, mineralized nodule formation, and osteogenic gene expression, and promotes vascular endothelial cell migration and tube formation, indicating that the material has good osteogenic and angiogenesis-promoting capabilities. Therefore, the material provided by this invention can not only improve the inflammatory and oxidative microenvironment in the bone defect area and enhance the mechanical properties of the scaffold material, but also significantly improve the bone tissue regeneration capacity. It is particularly suitable for the repair of complex bone defects such as craniofacial bone defects, and has good clinical application prospects and translational value. Attached Figure Description

[0016] Figure 1 A flowchart illustrating the preparation process of the composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles provided in this embodiment of the invention.

[0017] Figure 2 A physical image of the CeRh4NCs solution provided in an embodiment of the present invention;

[0018] Figure 3 Transmission electron microscope images of CeRh4NCs provided in embodiments of the present invention;

[0019] Figure 4 The images shown are X-ray photoelectron spectroscopy (XPS) analysis images of CeRh4NCs provided in the embodiments of the present invention. a is the full XPS spectrum, b is the C 1s spectrum, c is the O 1s spectrum, and d is the Ce 3d spectrum.

[0020] Figure 5 X-ray diffraction (XRD) images of CeRh4NCs provided in embodiments of the present invention;

[0021] Figure 6 Fourier transform infrared (FTIR) images of rare ginsenosides Rh4 and CeRh4NCs provided for embodiments of the present invention;

[0022] Figure 7 The following are scanning electron microscope images of four different hydrogels provided in the embodiments of the present invention: A is GelMA hydrogel, B is Rh4 / GelMA composite hydrogel, C is CeRh4NCs / GelMA composite hydrogel, and D is CeRh4NCs@HA / GelMA composite hydrogel.

[0023] Figure 8 The following are the performance results of four different hydrogels provided in the embodiments of the present invention: a is the compression performance, b is the swelling rate, c is the degradation rate, and d is the water retention rate.

[0024] Figure 9 The images show four groups of live / dead staining images of BMSC cells after different hydrogel treatments provided in this embodiment of the invention. A is GelMA hydrogel, B is Rh4 / GelMA composite hydrogel, C is CeRh4NCs / GelMA composite hydrogel, and D is CeRh4NCs@HA / GelMA composite hydrogel.

[0025] Figure 10 The images show cytoskeleton staining of BMSCs after four different hydrogel treatments provided in this embodiment of the invention. A is GelMA hydrogel, B is Rh4 / GelMA composite hydrogel, C is CeRh4NCs / GelMA composite hydrogel, and D is CeRh4NCs@HA / GelMA composite hydrogel.

[0026] Figure 11 The images show alkaline phosphatase (ALP) staining of BMSC cells treated with different hydrogels at 7 days after osteogenic induction, as provided in the embodiments of the present invention. A is GelMA hydrogel, B is Rh4 / GelMA composite hydrogel, C is CeRh4NCs / GelMA composite hydrogel, and D is CeRh4NCs@HA / GelMA composite hydrogel.

[0027] Figure 12 The images show Alizarin Red (ARS) staining of BMSC cells treated with different hydrogels at 21 days after osteogenic induction, according to four embodiments of the present invention. A is GelMA hydrogel, B is Rh4 / GelMA composite hydrogel, C is CeRh4NCs / GelMA composite hydrogel, and D is CeRh4NCs@HA / GelMA composite hydrogel. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0030] Example 1: A composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles, the preparation process of which is as follows: Figure 1 As shown, the specific steps include:

[0031] S1. CeRh4NCs were prepared via a metal-organic coordination self-assembly method, specifically as follows:

[0032] ① Disperse PEG 8000 and Ce(NO3)3·6H2O in ultrapure water and sonicate until completely dissolved to obtain a Ce-containing solution, wherein the concentration of PEG 8000 is 20 mg / mL. -1 The concentration of Ce(NO3)3·6H2O was 16.6 mg / mL. -1 ;

[0033] ② A rare ginsenoside Rh4 was dissolved in ethanol to prepare an Rh4 ethanol solution with a concentration of 5 mg / mL. -1 ;

[0034] ③ Slowly add the Rh4 ethanol solution to the Ce-containing solution (volume ratio 1:1), stir at room temperature for 30 min to initiate the coordination self-assembly reaction, allowing the rare ginsenoside Rh4 to react with Ce. 3+ Sufficient complexation was performed to obtain a stable CeRh4NCs solution, such as... Figure 2 As shown;

[0035] ④ After the reaction is complete, remove unreacted impurities by high-speed centrifugation (8000g, 10 min) and wash three times with ultrapure water;

[0036] ⑤ The obtained material was freeze-dried to obtain stable CeRh4NCs powder, which was stored at -20℃ for later use;

[0037] Construction of S2 and HA / GelMA composite precursor solutions:

[0038] Gelatin methacrylamide (GelMA) was dissolved in sterile phosphate-buffered saline (PBS) or deionized water and heated and stirred in a water bath at 40-50°C to ensure complete dissolution, preparing a 10% (w / v) GelMA solution. Then, approximately 0.25% (w / v) of photoinitiator LAP (phenyl(2,4,6-trimethylbenzoyl) lithium phosphate) was added to form a homogeneous and transparent GelMA precursor solution. Nano-hydroxyapatite powder (HA) was then added to the GelMA precursor solution and uniformly dispersed by ultrasonication to obtain an HA / GelMA composite precursor solution, where the HA concentration was 10% (w / v). In this process, HA not only serves as an active component for bone repair but also provides a foundation for improving the mechanical properties of the subsequent gelled material.

[0039] Formation of S3, CeRh4NCs@HA / GelMA composite hydrogel:

[0040] CeRh4NCs were dissolved in an HA / GelMA composite precursor solution and ultrasonically dispersed to form a CeRh4NCs / HA / GelMA composite precursor system. The CeRh4NCs / HA / GelMA composite precursor system was injected into a mold, defect area, or pre-designed molding space. Under irradiation with a specific wavelength light source (405 nm ultraviolet light, irradiation time of 45-75 s), the system was photocrosslinked and cured to form a three-dimensional crosslinked network structure CeRh4NCs@HA / GelMA composite hydrogel (wherein, based on rare ginsenoside Rh4, the equivalent final concentration of rare ginsenoside Rh4 in the composite hydrogel of CeRh4NCs is 25 μM), that is, a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles. CeRh4NCs and HA are stably embedded in the GelMA network, with uniform structure and stable component distribution.

[0041] Performance testing:

[0042] 1. The CeRh4NCs prepared in Example 1 were analyzed, and the transmission electron microscope images were obtained as follows: Figure 3 As shown, CeRh4NCs are spherical or nearly spherical with a particle size of approximately 10-50 nm. They are uniformly distributed, without obvious agglomeration, and have a uniform internal density, indicating that CeRh4NCs are spherical or nearly spherical. 3+ It successfully coordinates with rare ginsenoside Rh4 to form stable nanoparticles with good dispersibility and stability, and can achieve sustained-release activity, antioxidant and osteogenic functions.

[0043] 2. X-ray photoelectron spectroscopy (XPS) images of CeRh4NCs are shown below. Figure 4 As shown, XPS full spectrum ( Figure 4(a) confirms that the material contains elements such as Ce, C, and O; the high-resolution spectrum further shows that the C 1s spectrum ( Figure 4 (b) and O 1s spectrum ( Figure 4 The presence of characteristic peaks such as CO, C=O, and Ce-O in the 3d spectrum (c) indicates that the oxygen-containing functional groups in the rare ginsenoside Rh4 participate in the coordination with Ce ions; Ce 3d spectrum ( Figure 4 Ce can be seen in (d) 3+ and Ce 4+ The coexistence of cerium indicates that the cerium element in the material has the ability to undergo reversible valence state transformation, which provides a structural basis for its antioxidant and reactive oxygen species scavenging functions; in summary, CeRh4NCs have a stable chemical structure and are suitable for constructing functional bone repair scaffolds.

[0044] 3. X-ray diffraction (XRD) images of CeRh4NCs are shown below. Figure 5 As shown in the spectrum, CeRh4NCs exhibit clear characteristic diffraction peaks that correspond roughly to relevant standard peaks. The peak widths are moderate, indicating that the particle size is at the nanoscale and that the crystallinity is limited. The absence of obvious impurity peaks demonstrates the high purity of the nanoparticles and the presence of rare ginsenoside Rh4 and Ce. 3+ The complexation is stable, and the particles are easily and uniformly distributed in the HA / GelMA hydrogel, ensuring the synergy between the material structure and function;

[0045] 4. Fourier transform infrared (FTIR) images of rare ginsenosides Rh4 and CeRh4NCs are shown below. Figure 6 As shown in the comparison, the -OH peak of rare ginsenoside Rh4 (3416 cm⁻¹) is... -1 It shifts to approximately 3376 cm in CeRh4NCs. -1 The retention of characteristic peaks for CO, C=O, and CH indicates that the hydroxyl groups of rare ginsenoside Rh4 participate in Ce. 3+ Coordination forms nanoparticles while maintaining an intact organic framework. This stable structure facilitates incorporation into HA / GelMA hydrogels, achieving antioxidant, anti-inflammatory, and potential bone-promoting functions.

[0046] The above characterization results collectively demonstrate that the embodiments of the present invention successfully prepared structurally stable and uniformly dispersed CeRh4NCs. Furthermore, these nanoparticles possess the dual characteristics of rare organic active ginsenoside Rh4 and reversibly regulated valence states of Ce ions, providing a reliable foundation for the subsequent construction of CeRh4NCs@HA / GelMA composite hydrogels and their anti-inflammatory, antioxidant, and bone-repairing effects.

[0047] 5. Four material systems were analyzed: GelMA hydrogel, Rh4 / GelMA composite hydrogel, CeRh4NCs / GelMA composite hydrogel, and CeRh4NCs@HA / GelMA composite hydrogel. The Rh4 / GelMA composite hydrogel was prepared by dissolving rare ginsenoside Rh4 in DMSO (dimethyl sulfoxide) to obtain an Rh4 solution with a concentration of 100 mmol / L. -1 The Rh4 solution was added to the GelMA precursor solution and mixed thoroughly to obtain the Rh4 / GelMA composite precursor solution. The final equivalent concentration of rare ginsenoside Rh4 in the CeRh4NCs hydrogel was 25 μM, based on rare ginsenoside Rh4. The solution was then cured. The CeRh4NCs / GelMA composite hydrogel was prepared by adding CeRh4NCs to the GelMA precursor solution and dispersing them uniformly by ultrasound. The final equivalent concentration of rare ginsenoside Rh4 in the CeRh4NCs hydrogel was 25 μM, based on rare ginsenoside Rh4. The solution was then cured.

[0048] Obtain scanning electron microscope (SEM) images as follows Figure 7As shown, four sets of SEM images illustrate the influence of different compositions of the composite hydrogel on the three-dimensional pore network structure: The first set is a GelMA hydrogel with a continuous network of pores, large and irregular pore sizes, and a smooth scaffold surface, providing basic three-dimensional support for cell attachment; the second set is a Rh4 / GelMA composite hydrogel, whose pore network is similar to that of GelMA hydrogel, but the pore wall surface is rougher, with visible sheet-like or granular attachment structures, indicating that the rare ginsenoside Rh4 is relatively uniformly dispersed in the scaffold without obvious aggregation, which to some extent changes the microstructure of the scaffold surface; the third set is a CeRh4NCs / GelMA composite hydrogel, where high-magnification observation shows that CeRh4NCs are attached to the scaffold framework surface and pore walls, with a particle size of about 10-50 nm, regular morphology, and uniform distribution, indicating that CeRh4NCs are successfully embedded in the hydrogel. The first group, CeRh4NCs@HA / GelMA composite hydrogel, endows the material with antioxidant and sustained-release functions of active substances. The fourth group is CeRh4NCs@HA / GelMA composite hydrogel, which presents a continuous and uniform three-dimensional network of pores. HA particles and CeRh4NCs are distributed on the surface of the framework, indicating that the multi-component composite synergistically forms a stable scaffold structure. The pore structure is uniform and continuous, which can provide an ideal microenvironment for osteoblast attachment, migration and mineralization deposition, while ensuring the stable release of CeRh4NCs in the composite system, realizing antioxidant, anti-inflammatory and potential osteogenic functions. In summary, with the increase of composite components, the hydrogel scaffold gradually forms a functional composite system from a single GelMA, and the pore uniformity and microstructure stability are significantly improved. CeRh4NCs@HA / GelMA composite hydrogel realizes the synergistic effect of multiple components, supporting mechanical properties, microenvironment regulation and osteogenic potential.

[0049] 6. Test results of compression properties, swelling rate, degradation rate, and water retention rate of different material systems are as follows: Figure 8 As shown, the fracture stress and strain of the CeRh4NCs@HA / GelMA group were higher than those of the GelMA group and the Rh4 / GelMA group. This is because HA and CeRh4NCs synergistically optimize the scaffold structure. The swelling experiment showed that the CeRh4NCs@HA / GelMA group had a uniform pore network, high water absorption ratio, and rapid stability, which is conducive to the diffusion of active molecules and cell migration. The degradation experiment showed that the CeRh4NCs@HA / GelMA group degraded slowly, which can ensure long-term support. The water retention experiment showed that the CeRh4NCs@HA / GelMA group had a high and stable water retention rate, which is conducive to osteoblast proliferation and mineralization deposition. Overall, the results show that the CeRh4NCs@HA / GelMA group has continuous pores and uniform component distribution, achieving synergy between mechanical support and microenvironment function, which is suitable for the repair of maxillofacial bone defects.

[0050] 7. After co-culturing different material systems with BMSC cells for 1 day, staining was performed. The live / dead staining images of the treated BMSC cells are shown below. Figure 9As shown in the figure, green fluorescence represents live cells, and red fluorescence represents dead cells. The different columns correspond to the experimental groups in order: GelMA hydrogel, Rh4 / GelMA composite hydrogel, CeRh4NCs / GelMA composite hydrogel, and CeRh4NCs@HA / GelMA composite hydrogel. Overall observation revealed that cells covered the scaffold surface in each group of materials, with uniform distribution of green fluorescence and very little red fluorescence, indicating that the composite hydrogel scaffold has good cell compatibility and no obvious toxicity. The CeRh4NCs@HA / GelMA group showed high cell survival rate and uniform distribution, indicating that the nanoparticles and HA composite did not cause cell damage, and the scaffold provided a suitable microenvironment for attachment and growth. The overall results demonstrate that the composite hydrogel material provided in this embodiment is safe and conducive to the proliferation and attachment of osteogenic cells, providing a reliable basis for subsequent in vitro osteogenic induction and in vivo bone repair applications.

[0051] 8. Staining images of the cytoskeleton of BMSCs after treatment with different material systems are shown below. Figure 10 As shown in the figure, green fluorescence represents the cytoskeleton structure, and blue fluorescence represents the cell nucleus. The different columns correspond to the experimental groups in order: GelMA hydrogel, Rh4 / GelMA composite hydrogel, CeRh4NCs / GelMA composite hydrogel, and CeRh4NCs@HA / GelMA composite hydrogel. Overall observation shows that the cells in each group exhibit an extended morphology on the hydrogel scaffold, with an intact cytoskeleton structure and abundant pseudopodia, indicating that the scaffold surface provides a good attachment environment. The cells in the CeRh4NCs@HA / GelMA group are evenly distributed, and their extensibility is better than other experimental groups, indicating that its porous structure and surface chemical environment are conducive to the attachment of osteogenic cells and cytoskeleton development. The cell nuclei are regularly distributed, and no abnormal staining or aggregation was observed, further proving that it has no obvious toxicity to cells. The overall results show that the composite hydrogel scaffold provided in this embodiment of the invention can support cell attachment, expansion, and cytoskeleton remodeling, providing an ideal microenvironment for subsequent in vitro osteogenic induction and in vivo bone regeneration.

[0052] 9. Alkaline phosphatase (ALP) staining images of BMSCs treated with different material systems at 7 days after osteogenic induction are shown below. Figure 11As shown, the alkaline phosphatase activity of osteoblasts on the surface of different groups of hydrogels is displayed. All groups of cells showed purple-blue deposition, representing ALP activity. The GelMA group showed less deposition, indicating that the basic scaffold has limited osteogenic induction. The Rh4 / GelMA and CeRh4NCs / GelMA groups showed significantly increased deposition, indicating that single-component activity or mineralization components can enhance osteogenic signals. The CeRh4NCs@HA / GelMA group showed the most uniform deposition and the deepest color, indicating that the synergistic effect of CeRh4NCs and HA significantly promotes early osteoblast differentiation and ALP expression. Overall, the results demonstrate that the composite hydrogel provided in this invention not only supports cell growth but also significantly enhances osteogenic activity through the synergistic effect of CeRh4NCs and HA, providing an ideal foundation for subsequent mineralization deposition and bone regeneration.

[0053] 10. Alizarin Red (ARS) staining images of BMSCs treated with different material systems at 21 days of osteogenic induction are shown below. Figure 12 As shown, the osteoblast mineralization deposition in different groups of hydrogels is illustrated. The GelMA group showed the least red deposition, indicating that the basic scaffold has a limited effect on mineralization induction. The Rh4 / GelMA group and the CeRh4NCs / GelMA group showed a significant increase in red deposition, indicating that single-component active or mineralizing components can promote cell mineralization deposition. The CeRh4NCs@HA / GelMA group showed the richest and most uniform red deposition, indicating that CeRh4NCs and HA synergistically enhance the osteoblast mineralization capacity. Overall, the results show that the composite hydrogel provided in this embodiment can effectively induce osteoblast calcification deposition in the scaffold network, providing a solid foundation for in vitro osteoogenesis and future maxillofacial bone defect repair, while also demonstrating the multifunctional synergy and innovation of the material.

[0054] The composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles provided in this invention can be applied to bone defect sites through local filling or implantation, and is particularly suitable for the repair of craniofacial bone defects. After implantation, the material can promote cell adhesion, spreading and migration due to its porous or loose three-dimensional structure. On the other hand, CeRh4NCs and HA in the material can continuously regulate the local microenvironment, promote osteoblast differentiation, mineralized nodule formation and vascular endothelial cell migration and lumen formation, thereby accelerating new bone formation and functional reconstruction of the defect area. As the repair process progresses, the GelMA matrix can be gradually degraded, providing space for new tissue ingrowth and defect area remodeling, ultimately achieving effective repair of the bone defect area.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles, characterized in that, Includes the following steps: Preparation of S1 and CeRh4NCs: PEG 8000 and Ce(NO3)3·6H2O were dispersed in ultrapure water and sonicated until completely dissolved to obtain a Ce-containing solution. Rare ginsenoside Rh4 was dissolved in ethanol to prepare an Rh4 ethanol solution. The Rh4 ethanol solution was slowly added to the Ce-containing solution, and the coordination self-assembly reaction was initiated by stirring at room temperature to obtain a CeRh4NCs solution. After the reaction was completed, the solution was centrifuged, washed, and then freeze-dried to obtain CeRh4NCs. S2, Construction of HA / GelMA composite precursor solution: Dissolve gelatin methacrylamide product GelMA in PBS or deionized water, heat and stir to fully dissolve it, add photoinitiator to prepare GelMA precursor solution, add nano hydroxyapatite powder HA to GelMA precursor solution, sonicate to disperse it evenly, and obtain HA / GelMA composite precursor solution. S3. Formation of CeRh4NCs@HA / GelMA composite hydrogel: CeRh4NCs are dispersed in HA / GelMA composite precursor solution and ultrasonically dispersed to form CeRh4NCs / HA / GelMA composite precursor system. CeRh4NCs@HA / GelMA composite hydrogel is formed by photocrosslinking and curing, which is a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles.

2. The method for preparing the composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles according to claim 1, characterized in that, In S1, the concentration of PEG 8000 in the Ce-containing solution is 20 mg / mL. -1 The concentration of Ce(NO3)3·6H2O was 16.6 mg / mL. -1 ; The concentration of rare ginsenoside Rh4 in the Rh4 ethanol solution is 5 mg / mL. -1 ; The volume ratio of Rh4 ethanol solution to Ce solution is 1:

1.

3. The method for preparing the composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles according to claim 1, characterized in that, In S1, the specific centrifugation process is as follows: centrifuge at a high speed of 8000g for 10 minutes.

4. The method for preparing the composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles according to claim 1, characterized in that, In S2, the concentration of GelMA in the GelMA precursor solution is 10% (w / v), and the concentration of the photoinitiator is 0.05-0.5% (w / v). The concentration of nano-hydroxyapatite in the HA / GelMA composite precursor solution is 10% (w / v).

5. The method for preparing the composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles according to claim 1, characterized in that, In S3, the specific operation of photocrosslinking curing is as follows: irradiation with 405 nm ultraviolet light for 45-75 s.

6. The method for preparing the composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles according to claim 1, characterized in that, In S3, based on rare ginsenoside Rh4, the equivalent final concentration of rare ginsenoside Rh4 in CeRh4NCs in the composite hydrogel is 25 μM.

7. A composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles, characterized in that, It is prepared using the preparation method described in any one of claims 1-6.

8. The application of a composite hydrogel loaded with rare ginsenoside Rh4-Ce nanoparticles as described in claim 7 in the preparation of maxillofacial bone defect repair materials.

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