Preparation method of tremella polysaccharide intelligent hydrogel for inhibiting ferroptosis and application thereof
By preparing a smart hydrogel of Tremella polysaccharide, combined with ketithiolide diamine and growth factors, the problem of difficult repair of osteoporotic bone defects was solved, and the effects of inhibiting ferroptosis and regenerating bone tissue were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF NANTONG UNIV
- Filing Date
- 2025-05-08
- Publication Date
- 2026-04-21
AI Technical Summary
In existing technologies, osteogenic differentiation is inhibited, osteoclasts are overactive, and angiogenesis is impaired, making the repair of osteoporotic bone defects more difficult, and single treatment strategies are unlikely to achieve good repair results.
A smart hydrogel with tremella polysaccharides was developed by preparing methacrylamide tremella polysaccharide TrepMA and oxidized tremella polysaccharide O-Trep, which were then combined with ketithiothiol diamine TK-NH2 to form a hydrogel with ferroptosis inhibition. Growth factors or active molecules were added, and the hydrogel was cured under ultraviolet light using a photoinitiator to form a hydrogel with a three-dimensional network structure.
Tremella polysaccharide smart hydrogel can inhibit ferroptosis, promote osteogenic differentiation, reduce the concentration of iron ions in the microenvironment, activate the endogenous antioxidant function of osteoblasts, improve osteoporosis, and promote bone tissue regeneration.
Smart Images
Figure CN120361297B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomaterial preparation technology, and in particular to a method for preparing a smart hydrogel of Tremella fuciformis polysaccharide that inhibits ferroptosis and its application. Background Technology
[0002] In recent years, the incidence of bone injuries has continued to rise, driving the increasing demand for bone repair. The bone repair process mainly includes three overlapping stages: the inflammation stage, the bone formation stage, and the bone remodeling stage. Currently, the "gold standard" for the clinical treatment of bone defects is still based on autologous / allogeneic bone transplantation or artificial bone transplantation. Due to factors such as donor limitations, immune rejection, and infection, scarring, delayed healing, and even poor bone union are inevitable. Furthermore, autologous bone transplantation has limitations in its availability and may trigger immune responses, significantly impacting clinical outcomes. With the development of biomaterials science, the design of novel tissue engineering scaffolds with biological activity and function based on natural materials has gradually become a research hotspot. Among them, hydrogels, as a promising material, can effectively promote nutrient exchange due to their porous three-dimensional network structure, which is similar to the extracellular matrix. They also possess certain mechanical properties, providing filling and support for bone defects. However, given the high incidence of osteoporosis in the aging population, the healing of bone defects associated with osteoporosis has gradually become a clinical challenge. Osteoporosis-induced bone loss not only reduces the biomechanical properties of bone tissue and increases the incidence of fractures, but also reduces bone regeneration potential, leading to further slowed healing. Currently, the development of novel materials that can improve bone defects associated with osteoporosis remains to be studied.
[0003] Studies have shown that osteoporosis is accompanied by an imbalance in iron metabolism in the body. Large amounts of iron cannot be metabolized and accumulate in the bones, causing iron overload in osteoblasts. Under iron overload conditions, osteoblasts are prone to ferroptosis. Iron overload can significantly inhibit the differentiation capacity of osteoblasts, weakening their function and thus triggering an imbalance in bone metabolism, ultimately leading to osteoporosis. Furthermore, ferroptosis can further exacerbate bone formation disorders by regulating key signaling pathways within osteoblasts. Osteoclasts are responsible for bone resorption, degrading the bone matrix by secreting acidic substances and enzymes. Under iron overload conditions, osteoclast activity increases, bone resorption intensifies, and the rate of bone loss exceeds the rate of new bone formation, thereby accelerating the progression of osteoporosis. Osteoporosis is a metabolic bone disease caused by an imbalance in bone homeostasis, mainly regulated by osteoblasts and osteoclasts. Due to inhibited osteogenic differentiation, excessive osteoclast activity, and impaired angiogenesis, the repair of osteoporotic bone defects becomes more difficult, and single treatment strategies are insufficient to achieve good repair results. Therefore, the development of bioactive materials and treatment strategies based on the concept of multiple repairs is of great significance for this type of disease. Summary of the Invention
[0004] The purpose of this invention is to address the problem in the prior art that, due to the inhibition of osteogenic differentiation, excessive activity of osteoclasts, and impaired angiogenesis, the repair of osteoporotic bone defects becomes more difficult, and a single treatment strategy is insufficient to achieve good repair results.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] A smart hydrogel of Tremella polysaccharide with ferroptosis inhibition was prepared using methacrylated Tremella polysaccharide TrepMA, oxidized Tremella polysaccharide O-Trep, and ketothiol diamine TK-NH2.
[0007] Preferably, the TrepMA is obtained by reacting Tremella polysaccharide raw material with methacrylic anhydride, and the O-Trep is synthesized based on the oxidation of Tremella polysaccharide with sodium periodate.
[0008] Preferably, the preparation method of the Tremella polysaccharide intelligent hydrogel is as follows:
[0009] S1: Synthesis of TrepMA
[0010] Tremella polysaccharide was dissolved in deionized water to prepare a Tremella polysaccharide solution. Then, methacrylic anhydride was added to the Tremella polysaccharide solution to prepare a mixed solution. The mixed solution was stirred and the pH of the mixed solution was adjusted to 8.0. Then, the mixed solution was placed in an ice bath to react. After the reaction was completed, the mixed solution was dialyzed in deionized water using a dialysis bag to remove unreacted methacrylic anhydride. After dialysis, the solution was freeze-dried to obtain solid TrepMA.
[0011] S2: Synthesis of O-Trep:
[0012] Prepare a Tremella polysaccharide solution, then add sodium periodate to the Tremella polysaccharide solution to make a mixed solution. Stir the mixed solution and then place it at room temperature for reaction. Use a dialysis bag to dialyze the mixed solution in deionized water for 3 days to remove sodium periodate. After dialysis, freeze dry to obtain solid O-Trep.
[0013] S3: Preparation of hydrogel precursor solution:
[0014] Dissolve the TrepMA synthesized in S1 and the O-Trep synthesized in S2 in deionized water, then add the photoinitiator, TK-NH2 and active molecules, and sonicate for 5 minutes to mix thoroughly. The resulting mixed solution is the hydrogel precursor solution. Subsequent operations must be performed within 1 hour after the precursor solution is prepared.
[0015] S4: Preparation of DTrep
[0016] The hydrogel precursor solution obtained in S3 is loaded into a syringe and injected into a mold or bone defect site. After irradiating the solution with ultraviolet light at a wavelength of 405nm for 1-60 seconds, the solidified DTrep composite hydrogel is obtained.
[0017] Preferably, the molecular weight of the tremella polysaccharide selected in S1 and S2 is above 3000 Da, and the tremella polysaccharide is dissolved in deionized water by heating in a 50°C water bath to promote dissolution. The concentration of the tremella polysaccharide solution prepared in S1 and S2 is 0.5%-6%wt.
[0018] Preferably, the concentration of methacrylic anhydride in the mixed solution in S1 is 1%-20%wt, the concentration of sodium periodate in the mixed solution in S2 is 0.5-3mg / mL, and the stirring rate of the mixed solutions in S1 and S2 is 300rpm.
[0019] Preferably, in step S1, a 1M sodium hydroxide solution is used to adjust the pH of the mixed solution.
[0020] Preferably, the concentration of TrepMA added in S3 is 3%-8%wt, and the mass ratio of TrepMA to O-Trep is 1:1-1:3.
[0021] Preferably, the photoinitiator in S3 is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate with a concentration of 1%-5%wt, the concentration of TK-NH2 is 0.05-0.2 mg / mL, and the active molecule is at least one of EGF, FGF, PDGF, TGF-β, VEGF, BMP-2, BMP-4, BMP-7, and OGP, with a content of 0.01-0.5 μg / mL in the mixed solution.
[0022] This application also provides the application of Tremella fuciformis polysaccharide smart hydrogel with ferroptosis inhibition in the preparation of products for repairing bone defects, wherein the Tremella fuciformis polysaccharide smart hydrogel with ferroptosis inhibition is the Tremella fuciformis polysaccharide smart hydrogel described above.
[0023] Compared with the prior art, this application has the following beneficial effects:
[0024] This application ingeniously utilizes Tremella fuciformis polysaccharide derived from natural plants. Firstly, by reacting the hydroxyl groups of Tremella fuciformis polysaccharide with methacrylic anhydride, carbon-carbon double bonds are introduced, synthesizing TrepMA. Secondly, aldehyde-modified Tremella fuciformis polysaccharide O-Trep is prepared based on the oxidation of sodium periodate. With the assistance of a photoinitiator and under blue or ultraviolet light excitation, the carbon-carbon double bonds of both react to form a hydrogel from the precursor solution. The amino groups at both ends of TK-NH2 react with the aldehyde groups in O-Trep, acting as a secondary cross-linking agent. Adding growth factors or active molecules to the precursor solution in the hydrogel allows for drug loading, ultimately forming a smart hydrogel of Tremella fuciformis polysaccharide. Compared to other processes, this method is simple, the components are controllable, and the antioxidant properties of Tremella fuciformis polysaccharide are preserved. Furthermore, the TK in the hydrogel can intelligently respond to high concentrations of ROS in the microenvironment, accelerating the release of active molecules under high ROS conditions, thus accelerating the healing of severely damaged areas.
[0025] The Tremella polysaccharide smart hydrogel prepared by this invention exhibits a variety of biological activities. It not only provides certain support for bone regeneration, but also, through its excellent biocompatibility, bioactivity, and drug controlled release, inhibits ferroptosis, improves local osteoporosis, has antioxidant / anti-inflammatory effects, and induces osteogenic differentiation, thus effectively promoting bone tissue regeneration. Attached Figure Description
[0026] Figure 1 These are the equations and schematic diagrams involved in the preparation process of the intelligent hydrogel of Tremella polysaccharide in this invention.
[0027] Figure 2 These are the characterization data and physical photographs of a Tremella polysaccharide smart hydrogel with ferroptosis inhibition effect according to Example 1 of the present invention. (A) shows the 1H-NMR spectra of Tremella fuciformis polysaccharide (TP), O-Trep, and TrepMA. The characteristic peaks marked in blue boxes indicate that the aldehyde group in O-Trep and the methacryloyl group in TrepMA have been successfully modified. (B) shows the infrared spectra of Tremella fuciformis polysaccharide (TP), O-Trep, and TrepMA, which also confirm that O-Trep and TrepMA have been successfully modified. (C) A photograph of the OGP@DTrep hydrogel in Example 1 of this invention. The precursor solution solidifies after being irradiated with ultraviolet light and is called a hydrogel. (D) Photographs and scanning electron microscope images of the OGP@DTrep hydrogel, TrepMA, and DTepp hydrogel in Example 1 of this invention show that OGP@DTrep exhibits a three-dimensional network structure with clearly visible pores. (E) Rheological test of the OGP@DTrep hydrogel in Example 1 of this invention confirms its hydrogel properties. (F) Creep rate test of the OGP@DTrep hydrogel in Example 1 of this invention.
[0028] Figure 3This is a test of the chelation of iron ions by the OGP@DTrep hydrogel in Example 1 of this invention. (A) is a molecular docking simulation diagram of the chelation of iron ions by Tremella fuciformis polysaccharide molecules. It can be seen that the binding energy between Tremella fuciformis polysaccharide molecules and iron ions reaches -11.74 kcal / mol, which has excellent binding ability; (B) is a photograph of the hydrogel adsorbing iron ions. The hydrogel is immersed in ferric chloride solution, and the solution can be clarified in a short time, that is, it chelates the free iron ions in the solution; (C) is the elemental mapping analysis of the hydrogel immersed in ferric chloride solution and (D) is the energy dispersive spectroscopy analysis. It can be seen that iron ions are successfully adsorbed in the hydrogel. Therefore, the above results show that the hydrogel proposed in this invention has excellent ability to chelate iron ions.
[0029] Figure 4 This demonstrates the biocompatibility and osteoblast differentiation-promoting effects of the OGP@DTrep hydrogel in Example 1 of this invention. (A) Fluorescent staining of osteoblast MC3T3 cells seeded on OGP@DTrep hydrogel showed that the hydrogel increased the level of OPN in the cells; (B) Expression of osteoblast differentiation-related genes in MC3T3 cells seeded on OGP@DTrep hydrogel showed high expression of osteoblast differentiation-related genes OCN, OPN, Runx2, and COL-1; (C) Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining of MC3T3 cells seeded on OGP@DTrep hydrogel; (D) Quantitative analysis of ARS staining; (E) Quantitative analysis of ALP staining. The above staining data suggest that the hydrogel promotes mineralization and deposition in MC3T3 cells, indicating that the hydrogel can promote bone matrix secretion; (F) Tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts co-cultured with the hydrogel showed that the hydrogel inhibited osteoclast activity; (G) Western blotting. Blot analysis of the expression of osteogenic differentiation-related proteins in MC3T3 cells seeded on OGP@DTrep hydrogel and their (H) quantitative data further demonstrate that the OGP@DTrep hydrogel in Example 1 of this invention can promote osteogenic differentiation of osteoblasts.
[0030] Figure 5This invention illustrates the effect of OGP@DTrep hydrogel on osteoblast ferroptosis in Example 1. After OGP@DTrep hydrogel was seeded into MC3T3 cells, ferroptosis pathways were activated by stimulation with ferroptosis activator ferric(III)ammonium citrate (AIC). (A) A fluorescent image of intracellular ROS labeled with the DCFH-DA fluorescent probe after AIC stimulation, and (BC) Flow cytometry and quantitative data analysis, showing that OGP@DTrep hydrogel significantly reduced ROS levels in cells; (D) Fluorescence image of lipid peroxides detected in cells using the C11 BODIPY fluorescent probe; (E) Fluorescence image of intracellular iron ion levels analyzed using FerroGreen staining; (F) Fluorescence staining of ferroptosis-related antioxidant enzyme GPX4 in cells cultured in each hydrogel group; (GH) Western blotting and quantitative analysis verified the effect of OGP@DTrep hydrogel on the expression of osteoblast ferroptosis-related proteins; (I) Transmission electron microscopy (TEM) images showing the morphology and structure of mitochondria in stimulated MC3T3 cells. The results above show that the hydrogel reduced ROS levels, lipid peroxide levels, and iron ion concentration in MC3T3 cells after AIC stimulation. In particular, the hydrogel inhibited the activation of the ferroptosis signaling pathway and protected the mitochondrial structure and morphology, thus fully demonstrating that the OGP@DTrep hydrogel has a good inhibitory effect on osteoblast ferroptosis. (*P<0.05, **P<0.01 or ***P<0.001).
[0031] Figure 6 This invention illustrates the repair effect of OGP@DTrep hydrogel in repairing bone defects in rats with osteoporosis, as described in Example 1. An osteoporosis animal model was established in rats through ovariectomy, followed by creating a 2mm diameter bone defect in the femoral condyle. The OGP@DTrep hydrogel was then used to repair the bone defect. After 4 and 8 weeks of repair of femoral condyle defects with the hydrogel, histopathological staining analysis was performed. (AB) are images of pathological sections stained with hematoxylin and eosin (H&E), Masson's and toluidine blue (TB). (CD) MicroCT images show quantitative analysis of bone mass at the defect site after 4 and 8 weeks of repair with the hydrogel. Subsequently, bone mass analysis was performed based on the MicroCT images, including (E) bone connectivity density (Conn.D), (F) number of trabeculae (Tb.N), (G) intertrabecular spacing (Tb.Sp), and (H) total volume ratio (Tb.BV / TV). These results show that the hydrogel accelerates the healing of bone defects with osteoporosis in rats and the regeneration of trabeculae. Detailed Implementation
[0032] The present invention will be further described in detail below with reference to specific embodiments.
[0033] A smart hydrogel of Tremella polysaccharide with ferroptosis inhibition was prepared using methacrylated Tremella polysaccharide (TrepMA), oxidized Tremella polysaccharide (O-Trep), and ketithiodiamine (TK-NH2).
[0034] The TrepMA is obtained by reacting Tremella polysaccharide raw material with methacrylic anhydride, and the O-Trep is synthesized based on the oxidation of Tremella polysaccharide with sodium periodate.
[0035] In addition, this application also provides a method for preparing a Tremella fuciformis polysaccharide smart hydrogel with ferroptosis inhibition; please refer to [link to relevant documentation]. Figure 1 In the figure, TP represents Tremella fuciformis polysaccharide, NaIO4 represents sodium periodate, MA represents methacrylic anhydride, and OGP represents growth factors or active molecules loaded in the hydrogel. The specific steps of the preparation method using OGP as an example are shown below:
[0036] S1: Synthesis of Tremella polysaccharide modified with methacrylic anhydride (TrepMA)
[0037] Tremella polysaccharide was dissolved in deionized water and heated in a 50°C water bath to promote dissolution, resulting in a Tremella polysaccharide solution with a concentration of 0.5%-6%wt. A certain amount of methacrylic anhydride was then added to the Tremella polysaccharide solution to prepare a mixed solution with a concentration of 1%-20%wt. The mixed solution was stirred at 300 rpm, and the pH was adjusted to 8.0 with a 1M sodium hydroxide solution. The mixed solution was then reacted in an ice bath for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water for 3 days using a dialysis bag to remove unreacted methacrylic anhydride. In one embodiment, the dialysis bag had a molecular weight cutoff of 500-20000 Da. After dialysis, the solution was freeze-dried to obtain solid TrepMA.
[0038] S2: Synthesis of O-Trep (Oxidized Tremella Polysaccharide):
[0039] The polysaccharide of Tremella fuciformis was dissolved in deionized water and heated in a 50°C water bath to promote dissolution, thus preparing a Tremella fuciformis polysaccharide solution with a concentration of 0.5%-6%wt. Then, a certain amount of sodium periodate was added to the Tremella fuciformis polysaccharide solution to prepare a mixed solution with a sodium periodate concentration of 0.5-3 mg / mL. The mixed solution was stirred at 300 rpm and then reacted at room temperature for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water for 3 days using a dialysis bag to remove sodium periodate. In one embodiment, the molecular weight cutoff of the dialysis bag was 500-20000 Da. After dialysis, the solution was freeze-dried to obtain solid O-Trep.
[0040] The tremella polysaccharides selected in S1 and S2 have a molecular weight of 3000 Da or higher.
[0041] S3: Preparation of hydrogel precursor solution:
[0042] The TrepMA synthesized in S1 and the O-Trep synthesized in S2 are dissolved in deionized water according to a certain ratio and concentration. In one embodiment, the concentration of TrepMA is 3%-8%wt, and the mass ratio of TrepMA to O-Trep is 1:1-1:3. A certain amount of photoinitiator, ketithiolide diamine (TK-NH2), and active molecules are further added. The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate with a concentration of 1%-5%wt. The concentration of TK-NH2 is 0.05-0.2 mg / mL. The active molecule is at least one selected from EGF, FGF, PDGF, TGF-β, VEGF, BMP-2, BMP-4, BMP-7, and OGP, and its content in the mixed solution is 0.01-0.5 μg / mL. The mixture is ultrasonically vibrated for 5 minutes to thoroughly mix. The resulting mixed solution is the hydrogel precursor solution. Subsequent operations must be performed within one hour after the precursor solution is prepared.
[0043] S4: Preparation of Tremella fuciformis polysaccharide smart hydrogel (DTrep):
[0044] The hydrogel precursor solution obtained in S3 is loaded into a syringe and injected into a mold or bone defect site. After irradiating the solution with ultraviolet light at a wavelength of 405nm for 1-60s, the solidified DTrep composite hydrogel is obtained.
[0045] Based on the above preparation method, the Tremella fuciformis polysaccharide smart hydrogel with ferroptosis inhibition is prepared. This application also provides the application of the Tremella fuciformis polysaccharide smart hydrogel with ferroptosis inhibition in the preparation of products for repairing bone defects.
[0046] The Tremella fuciformis polysaccharide smart hydrogel provided in this application can inhibit ferroptosis and promote the healing of bone defects accompanied by osteoporosis through the following mechanisms:
[0047] (1) The ketithiolide group in DTrep can react rapidly with reactive oxygen free radicals (ROS), playing a role in intelligent response and scavenging ROS, anti-inflammatory and controlling the release of loaded drugs. The released drugs can promote osteogenic differentiation of osteoblasts.
[0048] (2) The carboxyl groups in the polysaccharide of Tremella fuciformis in DTrep can form a highly efficient chelation with iron ions, thereby inhibiting the activation of cell ferroptosis-related signaling pathways by reducing the concentration of iron ions in the microenvironment.
[0049] (3) DTrep can activate the Nrf2 signaling pathway in osteoblasts, activate the endogenous antioxidant activity of osteoblasts, and inhibit osteoblast apoptosis.
[0050] The above content will be described below with reference to specific embodiments.
[0051] Experimental materials and their sources:
[0052]
[0053] Example 1:
[0054] (1) Synthesis of Tremella polysaccharide modified with methacrylic anhydride (TrepMA): 3g of Tremella polysaccharide (molecular weight 50kDa) was dissolved in 100mL of deionized water and heated in a 50℃ water bath to fully dissolve it, so as to prepare a Tremella polysaccharide solution with a concentration of 3%wt. Then, 5g of methacrylic anhydride was added to the Tremella polysaccharide solution to prepare a mixed solution. The mixed solution was stirred at 300rpm and the pH of the mixed solution was adjusted to 8.0 with a 1M sodium hydroxide solution. The mixed solution was then reacted in an ice bath for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water for 3 days using a 5000Da dialysis bag to remove unreacted methacrylic anhydride. After dialysis, the solution was freeze-dried to obtain solid TrepMA.
[0055] (2) Synthesis of oxidized tremella polysaccharide (O-Trep): 3g of tremella polysaccharide (molecular weight 50kDa) was dissolved in 100mL of deionized water and heated in a 50℃ water bath to fully dissolve it, so as to prepare a tremella polysaccharide solution with a concentration of 3%wt. Then, sodium periodate was added to the tremella polysaccharide solution to make the sodium periodate concentration in the mixed solution 1mg / mL. At this time, the mixed solution was stirred at 300rpm. Then, the mixed solution was reacted at room temperature for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water for 3 days using a dialysis bag to remove sodium periodate. After dialysis, it was freeze-dried to obtain solid O-Trep.
[0056] (3) Preparation of hydrogel precursor solution: The TrepMA synthesized in step (1) and the O-Trep synthesized in step (2) were dissolved in 10 mL of deionized water at a mass ratio of 1:2. The concentration of TrepMA was 3% wt. The photoinitiator LAP, ketithiolide (TK-NH2) and osteogenic peptide OGP were added with concentrations of 0.02% wt, 0.1 mg / mL and 0.1 μg / mL, respectively. The mixture was ultrasonically vibrated for 5 minutes to mix thoroughly. The resulting mixed solution is the hydrogel precursor solution. Subsequent operations were performed within 1 hour after the precursor solution was prepared.
[0057] (4) Preparation of Tremella polysaccharide intelligent hydrogel: The hydrogel precursor solution obtained in step (3) is loaded into a syringe, the solution is injected into the mold or bone defect site, and after being irradiated with ultraviolet light at a wavelength of 405nm for 30 seconds, the solution is solidified to obtain Tremella polysaccharide intelligent hydrogel, which is named OGP@DTrep.
[0058] Example 2
[0059] (1) Synthesis of Tremella polysaccharide modified with methacrylic anhydride (TrepMA): 5g of Tremella polysaccharide (molecular weight 50kDa) was dissolved in 100mL of deionized water and heated in a water bath at 50℃ to fully dissolve it, thus preparing a Tremella polysaccharide solution. Then, 6g of methacrylic anhydride was added to the Tremella polysaccharide solution to prepare a mixed solution. The mixed solution was stirred at 300rpm and the pH of the mixed solution was adjusted to 8.0 with a 1M sodium hydroxide solution. The mixed solution was then reacted in an ice bath for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water for 3 days using a 5000Da dialysis bag to remove unreacted methacrylic anhydride. After dialysis, the solution was freeze-dried to obtain solid TrepMA.
[0060] (2) Synthesis of oxidized tremella polysaccharide (O-Trep): 5g of tremella polysaccharide (molecular weight 50kDa) was dissolved in 100mL of deionized water and heated in a 50℃ water bath to fully dissolve it, thus preparing a tremella polysaccharide solution. Then, sodium periodate was added to the tremella polysaccharide solution to make the sodium periodate concentration in the mixed solution 1.5mg / mL. At this time, the mixed solution was stirred at 300rpm. Then, the mixed solution was reacted at room temperature for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water for 3 days using a dialysis bag to remove sodium periodate. After dialysis, the solution was freeze-dried to obtain solid O-Trep.
[0061] (3) Preparation of hydrogel precursor solution: The TrepMA synthesized in step (1) and the O-Trep synthesized in step (2) were dissolved in 10 mL of deionized water at a mass ratio of 1:1. The concentration of TrepMA was 5% wt. The photoinitiator LAP, ketithiolide (TK-NH2) and growth factor VEGF were added with concentrations of 0.02% wt, 0.1 mg / mL and 0.1 μg / mL, respectively. The mixture was ultrasonically vibrated for 5 minutes to mix thoroughly. The resulting mixed solution is the hydrogel precursor solution. Subsequent operations were performed within 1 hour after the precursor solution was prepared.
[0062] (4) Preparation of Tremella polysaccharide intelligent hydrogel: The hydrogel precursor solution obtained in step (3) is loaded into a syringe, the solution is injected into the mold or bone defect site, and after being irradiated with ultraviolet light at a wavelength of 405nm for 30 seconds, the solution is solidified to obtain Tremella polysaccharide intelligent hydrogel loaded with VEGF.
[0063] Example 3
[0064] (1) Synthesis of Tremella polysaccharide modified with methacrylic anhydride (TrepMA): 3g of Tremella polysaccharide (molecular weight 50kDa) was dissolved in 100mL of deionized water and heated in a 50℃ water bath to fully dissolve it, so as to prepare a Tremella polysaccharide solution with a concentration of 3%wt. Then, 5g of methacrylic anhydride was added to the Tremella polysaccharide solution to prepare a mixed solution. The mixed solution was stirred at 300rpm and the pH of the mixed solution was adjusted to 8.0 with a 1M sodium hydroxide solution. The mixed solution was then reacted in an ice bath for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water for 3 days using a 5000Da dialysis bag to remove unreacted methacrylic anhydride. After dialysis, the solution was freeze-dried to obtain solid TrepMA.
[0065] (2) Synthesis of oxidized tremella polysaccharide (O-Trep): 3g of tremella polysaccharide (molecular weight 50kDa) was dissolved in 100mL of deionized water and heated in a 50℃ water bath to fully dissolve it, so as to prepare a 3% tremella polysaccharide solution. Then sodium periodate was added to the tremella polysaccharide solution to make the sodium periodate concentration in the mixed solution 1mg / mL. At this time, the mixed solution was stirred at 300rpm. Then the mixed solution was reacted at room temperature for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water for 3 days using a dialysis bag to remove sodium periodate. After dialysis, it was freeze-dried to obtain solid O-Trep.
[0066] (3) Preparation of hydrogel precursor solution: The TrepMA synthesized in step (1) and the O-Trep synthesized in step (2) were dissolved in 10 mL of deionized water at a mass ratio of 1:2. The concentration of TrepMA was 3% wt. Photoinitiator LAP, ketithiolide (TK-NH2) and growth factor BMP-2 were added with concentrations of 0.02% wt, 0.1 mg / mL and 0.1 μg / mL, respectively. The mixture was ultrasonically vibrated for 5 minutes to mix thoroughly. The resulting mixed solution is the hydrogel precursor solution. Subsequent operations were performed within 1 hour after the precursor solution was prepared.
[0067] (4) Preparation of Tremella polysaccharide intelligent hydrogel: The hydrogel precursor solution obtained in step (3) is loaded into a syringe, the solution is injected into a mold or bone defect site, and after being irradiated with ultraviolet light at a wavelength of 405nm for 30 seconds, the solution is solidified to obtain Tremella polysaccharide intelligent hydrogel loaded with BMP-2.
[0068] Verification Experiment 1:
[0069] This application employed nuclear magnetic resonance spectroscopy (¹H-NMR), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), rheological and creep tests to characterize the properties of materials derived from Tremella fuciformis polysaccharides. The results are as follows: Figure 2As shown, the 1H-NMR spectrum of Trep-MA exhibits characteristic peaks of the methacryloyl group at 5.1 and 5.6 ppm. Figure 2 A). The 1H-NMR spectrum of O-Trep showed characteristic peaks for aldehyde groups at 5.2 and 5.6 ppm. For example... Figure 2 As shown in Figure B, FT-IR spectroscopy confirmed the successful synthesis of these two components. Compared to unmodified Tremella polysaccharide (TP), the Trep-MA spectrum at 1735 cm⁻¹... -1 The characteristic peak gradually increases, representing the C=O group in the methacryloyl structure. In the FT-IR spectrum of O-Trep, the peak at 1735 cm⁻¹ represents the aldehyde group (C=O). -1 The intensity of characteristic peaks also increased. Furthermore, all samples showed increases at 1636, 1422, 3431, 2927, and 1074 cm⁻¹. -1 The typical characteristic peaks of Tremella fuciformis polysaccharides appeared at the [specific locations], corresponding to OH stretching vibration, CH stretching vibration, carbonyl CO stretching vibration in uronic acid, CH angle-changing vibration, and COC stretching vibration, respectively. Therefore, the above results indicate that the monomer constituting the Tremella fuciformis polysaccharide-based OGP@DTrep dual-network hydrogel was successfully prepared. Subsequently, three types of hydrogels were constructed: Trep-MA (without O-Trep and TK), DTrep hydrogel, and the OGP@DTrep hydrogel constructed in Example 1. Figure 2 As shown in Figure D, DTrep exhibits excellent moldability and injectability even in humid environments and can be cured into a hydrogel under UV-induced conditions. Figure 2 SEM images of D showed that the OGP@DTrep hydrogels constructed in Example 1 all exhibited a porous microstructure. This typical hydrogel structural feature is conducive to cell migration and tissue ingrowth. Furthermore, rheological experiments showed that ( Figure 2 E) Each hydrogel group exhibited a high storage modulus (G') and a low loss modulus (G”); that is, the hydrogels displayed solid-like viscoelasticity, indicating that OGP@DTrep successfully transitioned to a gel state. Rheological data also showed that, due to their dual-network structure, the elastic moduli of OGP@DTrep and DTrep were greater than those of Trep-MA. Subsequently, the stability of the various hydrogels was evaluated by swelling ratio testing. Figure 2 As shown in Figure F, OGP@DTrep reached a stable state in a liquid environment within 6 hours and exhibited a low swelling rate. However, within 48 hours, the swelling rate of Trep-MA increased further, while the creep change of OGP@DTrep was minimal, indicating that the OGP@DTrep hydrogel has better stability. Maintaining the stability of the hydrogel structure helps to preserve shape and integrity during bone regeneration, while minimizing pressure on surrounding tissues or separation from the target organ.
[0070] Verification Experiment 2
[0071] The Tremella fuciformis polysaccharide-based smart hydrogel can block osteoblast ferroptosis by reducing the concentration of free iron ions in the microenvironment. This application verifies the chelating ability of the above-mentioned Tremella fuciformis polysaccharide-based hydrogel and iron ions. Figure 3 As shown in Figure A, for TP molecules and Fe 3+ Molecular docking simulations were performed to determine the coordination of Fe ions. The results showed that Fe... 3+ The binding energy between Fe ions and TP is -11.47 kcal / mol, indicating that Fe 3+ It can efficiently bind to the polysaccharide molecular chain of Tremella fuciformis. Interaction analysis showed that iron ions form salt bridges with the carboxyl groups in TP and hydrogen bonds with the hydroxyl groups in TP. The distance of the hydrogen bonds is 2.8 Å. Subsequently, the adsorption of Fe by the hydrogel was tested in this application. 3+ The ability of ions. For example... Figure 3 As shown in Figure B, after immersion in FeCl3 solution for only 1 minute, Trep-MA rapidly turned yellow due to the adsorption of a large amount of iron ions, while the initially yellow supernatant solution turned colorless. Subsequently, the iron content in the central portion of the hydrogel was verified in this application. Figure 3 As shown in the C&D diagram, the mapping elemental analysis and EDS energy dispersive spectroscopy results indicate that after Trep-MA was immersed in FeCl3 solution, a large amount of iron was enriched in the gel network within a very short time (1 minute). This demonstrates that the hydrogel prepared based on TP exhibits excellent chelating ability for iron ions, suggesting that it may be able to block ferroptosis by regulating the concentration of iron ions in the microenvironment.
[0072] Verification Experiment 3:
[0073] This application further evaluated the effect of the OGP@DTrep hydrogel constructed in Example 1 on osteoblast differentiation under ferroptosis. The hydrogel was prepared into a disc-shaped sample with a diameter of 1.5 cm and a height of 3 mm, placed at the bottom of a 24-well plate, and then osteoblast MC3T3 cells were introduced at a rate of 2 × 10⁻⁶. 5 Inoculated into 24-well plates and cultured in DMEM medium for subsequent experiments. Figure 4 Immunofluorescence staining images in A showed that OGP@DTrep significantly increased the level of osteopontin (OPN) in osteoblast MC3T3 cells. Figure 4 As shown in Figure B, RT-PCR results further support this finding: after co-culturing MC3T3 cells with OGP@DTrep, the osteogenic differentiation-related genes Runx2, OCN, OPN, and COL-1 were significantly highly expressed. Figure 4The results of Alizarin Red (ARS) staining and alkaline phosphatase (ALP) staining in C showed that MC3T3 cells co-cultured with OGP@DTrep exhibited the highest levels of alkaline phosphatase and mineralization, which was confirmed by quantitative analysis. Figure 4 D&E). This indicates that OGP@DTrep can stimulate osteoblasts to secrete ECM to form new bone. Furthermore, Western blot (WB) data showed that OGP@DTrep increased the protein levels of wnt7b, Runx2, and OPN, which are associated with bone regeneration. Figure 4 G&H). In addition, such as Figure 4 As shown in Figure F, tartrate-resistant acid phosphatase (TRAP) staining results indicate that OGP@DTrep can inhibit osteoclast activity. These data suggest that OGP@DTrep helps restore the osteoblast / osteoclast balance in the pathological microenvironment of osteoporosis.
[0074] Subsequently, this application stimulated ferroptosis in MC3T3 cells, verifying the regulatory effect of the OGP@DTrep hydrogel constructed in Example 1 on the ferroptosis pathway in osteoblast MC3T3 cells. Ferrous ammonium citrate (AIC) was used as a ferroptosis activator to stimulate the ferroptosis pathway in MC3T3 cells. Figure 5 As shown in Figure A, after stimulation with AIC, MC3T3 cells co-cultured with OGP@DTrep hydrogel exhibited lower levels of green fluorescence compared to the control group, indicating a decrease in intracellular reactive oxygen species (ROS) levels. Figure 5 As shown in B&C, flow cytometry and related quantitative analyses revealed that OGP@DTrep possessed the greatest ROS scavenging ability, indicating that the hydrogel can remove excess ROS from cells. Furthermore, the accumulation of lipid peroxides and iron ions are important indicators of ferroptosis. Subsequently, the C11 BODIPY fluorescent probe was used in this application to verify the level of intracellular lipid peroxides. Figure 5 As shown in Figure D, OGP@DTrep exhibited the lowest levels of lipid peroxides. Furthermore, FerroGreen staining results showed that MC3T3 cells co-cultured with OGP@DTrep also showed the lowest levels of iron enrichment. Western blot bands and quantitative statistics of ferroptosis-related proteins are shown in Figure D. Figure 5 As shown in G&H, OGP@DTrep hydrogel significantly promoted the high expression of FTH1, GSTP1, GPX4 and Nrf2 proteins in MC3T3 cells. In addition, the expression of ACSL4 and Cox-2 was significantly reduced, indicating that the intracellular ferroptosis pathway activated by AIC stimulation was blocked. Figure 5The results showed that the OGP@DTrep hydrogel protected the structural integrity of mitochondria, with both the outer cell membrane and inner cristae exhibiting relatively intact morphology. Furthermore, the hydrogel activated the intracellular Nrf2 / Gpx4 pathway, implying enhanced endogenous antioxidant capacity.
[0075] Verification Experiment 4:
[0076] This application further evaluates the effect of the OGP@DTrep hydrogel constructed in Example 1 on bone defect sites and promoting new bone regeneration in a rat osteoporosis model. Animal modeling: All animal experimental procedures were ethically and scientifically approved by the Animal Care and Use Committee of Nantong University (Approval No.: S20240617-003). All experimental procedures complied with the ARRIVE guidelines and were conducted in accordance with the UK Animal (Scientific Procedures) Act 1986 and related guidelines. The rat osteoporotic bone defect model was established using 12-week-old female SD rats weighing 300-400 grams. Bilateral ovariectomy was performed on the female rats. Estrogen levels began to decline 15 days after surgery. Estrogen levels reached their lowest level after one month. The osteoporosis model was established after 4 to 8 weeks. Then, bone defect surgery was performed on the ovariectomized group and the sham-operated control group. The subcutaneous area of the lateral femoral condyle of the rat was incised with a scalpel to expose the bone surface. A bone defect model with a diameter of 2.5 mm and a depth of 3 mm below the lateral condyle was then created. A hydrogel precursor solution was injected to fill the defect and cured by UV crosslinking. The joint capsule and skin incision were then sutured. Mice were allowed free movement in their cages. Animals were randomly assigned to each group.
[0077] The results showed that 4 weeks post-surgery, H&E Masson and toluidine blue (TB) staining results indicated ( Figure 6 In groups A and B, OGP@DTrep significantly promoted inward growth of osteoid tissue at the defect site and a certain amount of trabecular bone formation. Other groups still showed some bone loss and less trabecular bone formation at the defect site. At 8 weeks post-operation, the blank OVX group produced a small amount of trabecular bone but still had a large cavity, while the OGP@DTrep group showed the highest level of trabecular bone formation. Figure 6 As shown in the CH diagram, MicroCT results revealed that untreated osteoporotic rats (OVX group) exhibited a low number of trabeculae, poor bone structure, and low bone content, with pathological characteristics similar to osteoporosis models in previous studies. The OGP@DTrep group showed the highest number of trabeculae in the defect areas. Bone mineral density (BMD) and total volume ratio (Tb.BV / TV) reflect bone quality and strength. Trabeculae number (Tb.N) and trabecular spacing (Tb.Sp) are the main indicators for evaluating the spatial morphology and structure of trabeculae. Figure 6Statistical results from EH showed that the BMD, Tb.N, Tb.Sp, and BV / TV indices of the OGP@DTrep group were significantly higher than those of other groups, indicating that OGP@DTrep significantly promoted new bone regeneration and improved bone strength. In summary, OGP@DTrep significantly promoted bone regeneration and trabecular bone formation at the defect sites in the rat model.
[0078] This application utilizes Tremella fuciformis polysaccharide derived from natural plants. Firstly, by reacting the hydroxyl groups of Tremella fuciformis polysaccharide with methacrylic anhydride, carbon-carbon double bonds are introduced to synthesize TrepMA. Secondly, aldehyde-modified Tremella fuciformis polysaccharide O-Trep is prepared based on the oxidation of sodium periodate. With the assistance of a photoinitiator and under blue or ultraviolet light excitation, the carbon-carbon double bonds of both react to form a hydrogel from the precursor solution. The amino groups at both ends of TK-NH2 react with the aldehyde groups in O-Trep, acting as a secondary cross-linking agent. Adding growth factors or active molecules to the hydrogel from the precursor solution allows for drug loading, ultimately forming a smart hydrogel of Tremella fuciformis polysaccharide. Compared to other processes, this method is simple, allows for controllable composition, preserves the antioxidant properties of Tremella fuciformis polysaccharide, and the TK in the hydrogel can intelligently respond to high concentrations of ROS in the microenvironment, accelerating the release of active molecules under high ROS conditions, thus accelerating the healing of severely damaged areas.
[0079] The Tremella polysaccharide smart hydrogel prepared by this invention exhibits a variety of biological activities. It not only provides certain support for bone regeneration, but also, through its excellent biocompatibility, bioactivity, and drug controlled release, inhibits ferroptosis, improves local osteoporosis, has antioxidant / anti-inflammatory effects, and induces osteogenic differentiation, thus effectively promoting bone tissue regeneration.
Claims
1. A tremella polysaccharide smart hydrogel with ferroptosis inhibition, characterized by: The intelligent hydrogel of tremella polysaccharide is prepared by using methacrylated tremella polysaccharide TrepMA, oxidized tremella polysaccharide O-Trep and ketone thioalcohol diamine TK-NH2, and the preparation method of the intelligent hydrogel of tremella polysaccharide is as follows. S1: synthesis of TrepMA The deionized water is used to dissolve the tremella polysaccharide to prepare a tremella polysaccharide solution, then the methacrylic anhydride is added into the tremella polysaccharide solution to prepare a mixed solution, the mixed solution is stirred and the pH of the mixed solution is adjusted to 8.0, then the mixed solution is placed in an ice bath for reaction, after the reaction is completed, the mixed solution is dialyzed in deionized water by using a dialysis bag to remove the unreacted methacrylic anhydride, and then freeze-drying is performed after the dialysis is completed, so that the solid TrepMA is obtained. S2: synthesis of O-Trep The deionized water is used to dissolve the tremella polysaccharide to prepare a tremella polysaccharide solution, then the methacrylic anhydride is added into the tremella polysaccharide solution to prepare a mixed solution, the mixed solution is stirred and the pH of the mixed solution is adjusted to 8.0, then the mixed solution is placed in an ice bath for reaction, after the reaction is completed, the mixed solution is dialyzed in deionized water by using a dialysis bag to remove the unreacted methacrylic anhydride, and then freeze-drying is performed after the dialysis is completed, so that the solid TrepMA is obtained. S3: preparation of a hydrogel precursor solution The TrepMA synthesized in S1 and the O-Trep synthesized in S2 are dissolved in deionized water, then a photoinitiator, TK-NH2 and an active molecule are added, and ultrasonic oscillation is performed for 5 minutes to fully mix, so that the obtained mixed solution is a hydrogel precursor solution, and the precursor solution is prepared within 1 hour before subsequent operations are performed. S4: preparation of DTrep The hydrogel precursor solution obtained in S3 is loaded into a syringe, the solution is injected into a mold or a bone defect site, and then the solution is irradiated by ultraviolet light with a wavelength of 405 nm for 1-60 seconds, so that the cured DTrep composite hydrogel is obtained.
2. The Tremella polysaccharide smart hydrogel with ferroptosis inhibition according to claim 1, characterized in that: The molecular weight of the tremella polysaccharide selected in S1 and S2 is greater than 3000 Da, and the tremella polysaccharide is dissolved in deionized water by heating in a 50℃ water bath to promote dissolution, and the concentration of the tremella polysaccharide solution prepared in S1 and S2 is 0.5%-6%wt.
3. The Tremella polysaccharide smart hydrogel with ferroptosis inhibition according to claim 1, characterized in that: The concentration of the methacrylic anhydride in the mixed solution in S1 is 1%-20%wt, the concentration of the sodium periodate in the mixed solution in S2 is 0.5-3 mg / mL, and the stirring rate of the mixed solution in S1 and S2 is 300 rpm.
4. The Tremella polysaccharide smart hydrogel with ferroptosis inhibition according to claim 1, characterized in that: In S1, the pH of the mixed solution is adjusted by using a 1M sodium hydroxide solution.
5. The Tremella polysaccharide smart hydrogel with ferroptosis inhibition according to claim 1, characterized in that: In S3, the concentration of the added TrepMA is 3%-8%wt, and the mass ratio of TrepMA to O-Trep is 1:1-1:
3.
6. The Tremella polysaccharide smart hydrogel with ferroptosis inhibition according to claim 1, characterized in that: In S3, the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl) phosphate, and the concentration is 1%-5%wt, the concentration of TK-NH2 is 0.05-0.2 mg / mL, and the active molecule is at least one of EGF, FGF, PDGF, TGF-β, VEGF, BMP-2, BMP-4, BMP-7 and OGP, and the content of the active molecule in the mixed solution is 0.01-0.5 μg / mL.
7. The use of a tremella polysaccharide intelligent hydrogel with ferroptosis inhibition in the preparation of a product for repairing bone defects, characterized in that: The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition is the tremella polysaccharide intelligent hydrogel according to any one of claims 1-6.