A long-acting hydrogen-releasing stent and its preparation method and application

Abstract

A long-lasting hydrogen-releasing scaffold, its preparation method, and application, are disclosed in the medical technology field. The scaffold is a novel mesoporous bioactive glass scaffold loaded with CaSi2 nanoparticles (CSN), designed for localized and sustained high-dose hydrogen release (911 ml / g CSN). This allows for efficient reconstruction of SMEs and promotes the repair of damaged and aged bone. CSN has a hydrogen storage capacity 4.6×104 times that of water. The sustained hydrogen release capacity of polyhydroxyalkanoic acid (PHA)-coated CSN (CSN@PHA) (approximately one week) far exceeds that of hydrogen-rich water (approximately 30 minutes).

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly to a long-lasting hydrogen-releasing stent, a preparation method thereof, and an application thereof. The invention particularly relates to a novel hydrogen-releasing stent constructed by electrospraying polyhydroxyalkanoate (PHA)-coated CaSi2 nanoparticles (CSN) on a mesoporous bioactive glass (CSN@PHA-MBG) stent, which can locally and continuously (for up to 1 week) release a high dose of hydrogen (911 ml / g), and an application thereof in reshaping the aging microenvironment and improving the repair of aging bone damage. Background Art

[0002] Accelerated cellular senescence is one of the primary causes of aging and disease. Aging is characterized by persistent cell cycle arrest and a unique pro-inflammatory secretory phenotype in various cells, creating a senescence microenvironment (SME) that drives tissue deterioration in a negative feedback loop and reduces tissue self-repair capacity. Typically, the SME contributes to bone loss during aging and poses a significant challenge to fracture and defect repair in the elderly. With the increasing aging population, improving and even remodeling the SME to address impaired bone repair is becoming increasingly urgent but remains a significant challenge.

[0003] Two anti-aging strategies have been developed to manage age-related diseases, such as age-related bone disease and cancer, including the use of senolytic drugs to lyse senescent cells and the use of senolytic drugs to alter the senescence-associated secretory phenotype (SASP). Unfortunately, due to the lack of selectivity and universality of anti-aging drugs, no single drug can effectively modulate the diverse types of senescent cells involved in the SASP across the SME, resulting in significant toxic side effects and limited anti-aging effects. However, it is noteworthy that molecular hydrogen (H2) has the ability to selectively scavenge highly oxidative / toxic free radicals, such as hydroxyl radicals (OH), making it a safe, effective, and broad-spectrum anti-inflammatory agent. Because aging is closely associated with oxidative stress, molecular hydrogen (H2) has been found to have promising anti-aging effects on numerous cells (bone marrow mesenchymal stem cells, fibroblasts, and endothelial cells) and tissues (brain, periodontal tissue, aorta, and retina) without significant toxic side effects, demonstrating excellent selectivity and universality. However, aging induced by aging / damage is significantly more severe than individual aging or injury-induced aging, and the impact of H2 on aging induced by aging / damage remains unclear. In addition, the reported anti-aging hydrogen delivery methods mainly include oral administration of hydrogen-rich water and inhalation of hydrogen. Due to the low solubility and high dispersibility of hydrogen, it is impossible to achieve a high hydrogen concentration in the ward for a long time, resulting in limited anti-aging effects.

[0004] In this work, we designed a novel mesoporous bioactive glass (MBG) scaffold loaded with CaSi2 nanoparticles (CSN) for local and sustained (up to 1 week) release of high-dose H2 (911 ml / g CSN), achieving efficient reconstruction of SME and promoting the repair of damaged aged bone. The hydrogen storage capacity of CSN is 4.6×10 4 The sustained hydrogen release capacity of polyhydroxyalkanoic acid (PHA)-coated CSN (CSN@PHA) (approximately one week) is much higher than that of hydrogen-rich water (approximately 30 minutes). The study found that continuous H2 treatment can generally reduce oxidative stress in SMEs and effectively reshape the senescence-associated secretory phenotype (SASP) of various senescent cells through anti-inflammatory pathways, inducing macrophage repolarization to an anti-inflammatory phenotype, BMSC recruitment, angiogenesis, and osteogenesis, thereby supporting damaged aging bone repair. Summary of the Invention

[0005] One of the purposes of the present invention is to prepare nano-CaSi2 (CSN) by microwave-assisted chemical dissolution method, wherein the preparation method comprises the following steps:

[0006] 1.1) Completely dispersing CaSi2 raw material powder, modifier, EDTA disodium salt, and sodium hyaluronate in an alcohol solvent at a mass ratio of 100:(50-1000):(20-1000):(20-1000) in an alcohol solvent to obtain a mixture;

[0007] Among them, the preferred raw material mass ratio is CaSi2 raw powder: PVP: EDTA disodium salt: sodium hyaluronate: ethylene glycol = 100: (50-1000): (20-1000): (20-1000): 20;

[0008] CaSi2 raw powder (size range 2um-1000um, purity 90-99%), preferably 25um, purity ≥99%;

[0009] The modifier includes one or more of polyvinyl pyrrolidone (PVP), polylactic acid and polyethylene glycol;

[0010] The alcohol solvent as a dispersant includes any one or more of ethylene glycol and glycerol;

[0011] 1.2) Transfer the mixture to an autoclave and heat it using a microwave synthesizer at a temperature of 100-300°C for 0.5-10 hours.

[0012] 1.3) After cooling to 45-55°C, crush in a pulse ultrasonic instrument with a power of 100-1000W / cm 2, and work intermittently, work: rest time = 1: (0.1-10), the ultrasonic crushing time can be selected from 10 to 500 minutes;

[0013] 1.4) After centrifugation to remove large particles, the supernatant was collected, washed, and centrifuged again to disperse it in ethanol.

[0014] The washing method includes deionized water plus any one of ethanol or methanol or a mixture of any two in any proportion; such as a mixture of ethanol and water in a volume ratio of 1:1.

[0015] Another object of the present invention is to develop a novel mesoporous bioactive glass (MBG) scaffold loaded with CaSi2 nanoparticles (CSN) for local and sustained (up to 1 week) release of high-dose H2 (911 ml / g CSN), achieving efficient reconstruction of SMEs and promoting the repair of damaged and aged bones. The hydrogen storage capacity of CSN is 4.6×10 4 The sustained hydrogen release capacity of polyhydroxyalkanoic acid (PHA)-encapsulated CSN (CSN@PHA) (about one week) is much higher than that of hydrogen-rich water (about 30 minutes). The method is as follows:

[0016] A method for preparing a long-acting hydrogen-releasing stent, comprising the following steps:

[0017] 2.1) Polyether F127 (F127), Ca(NO3)2·4H2O, and TEP were sequentially dissolved in ethanol containing 0.5M hydrochloric acid; then, ethyl orthosilicate was added to the above solution, stirred vigorously, and rotary evaporated at 40-100°C to uniformly impregnate the viscous MBG sol onto a polyurethane sponge; wherein the raw materials were prepared in a mass ratio of F127:Ca(NO3)2·4H2O:TEP:0.5M hydrochloric acid:ethanol:ethyl orthosilicate = 4:(0.1-5):(0.05-5):(0.1-10):50:(1-20);

[0018] Further, for example, 4.0 g of F127, 0.76 g of Ca(NO₃)₂.4H₂O, and 0.23 g of TEP were sequentially dissolved in 50 ml of ethanol containing 1.0 g of 0.5 M hydrochloric acid. 5.2 g of ethyl orthosilicate was then added to the solution, stirred vigorously for 24 hours, and subjected to rotary evaporation at 60°C for 30 minutes. The viscous MBG sol was then uniformly impregnated onto a custom-made polyurethane sponge.

[0019] Furthermore, for example, the vigorous stirring time can be selected to be 10-100 hours, the rotation time can be selected to be 10-100 minutes, and the mold can be a customized polyurethane sponge or any other mold;

[0020] 2.2) calcining the MBG scaffold at 400-800°C for 2-10 hours; preferably calcining at 600°C for 6 hours;

[0021] 2.3) CSN@PHA-MBG scaffolds were prepared by electrostatic spraying: PHA was dissolved in a DCM / DMF solution with a volume ratio of (1-10):1. CSN and PHA were then uniformly mixed at a particle concentration of 0.05-5 mg / ml to form a CSN@PHA mixture. The CSN@PHA mixture was sprayed onto the MBG scaffold, and a voltage of 5-50 kV was applied to generate an electrostatic field within a working distance of 4-40 cm.

[0022] The molecular weight of PHA is 5000-50000 Da.

[0023] Specifically, a CSN@PHA-MBG scaffold containing 50 μg of CSN was prepared using an electrostatic spraying method. 1.5 mg of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHA, MW = 25,000 Da) was dissolved in 10 mL of DCM / DMF (8:2, v / v) solution. The CSN and PHA solutions were then uniformly mixed at a particle concentration of 0.5 mg / mL. After ultrasonic dispersion, the CSN@PHA mixture was transferred to a 5 mL syringe and sprayed onto the MBG scaffold using a 22-gauge needle at a rate of 0.05-2 mL / h for 20 minutes. An 18 kV voltage was applied to generate an electrostatic field within a working distance of 10 cm. To achieve the desired spraying effect, the MBG scaffold was rotated continuously.

[0024] The molecular weight of poly (3-hydroxybutyric acid-co-3-hydroxyvaleric acid) can be selected from 5000-50000 Da, and the solubility concentration can be selected from 5%-20%; the PHA solution of CSN can be selected from 0.05-5 mg / ml, preferably 0.5 mg / mL;

[0025] The spraying speed can be selected as 0.5mL / h, the spraying time can be selected as 5-60min, the applied voltage can be selected as 5-50 kV, and the working distance can be selected as 4-40 cm.

[0026] A third object of the present invention is to provide a long-lasting hydrogen-releasing stent manufactured by any of the above methods.

[0027] A fourth object of the present invention is to provide the use of the long-acting hydrogen-releasing stent as a medicine.

[0028] The long-acting hydrogen-releasing stent is used to improve the aging microenvironment and / or prevent the aging of bone marrow mesenchymal stem cells.

[0029] The long-acting hydrogen-releasing scaffold is used to reduce the levels of two representative SASP components IL-6 and Il-1β in SME, and / or increase the levels of representative anti-inflammatory cytokines IL-4 and IL-10, and / or induce the formation of new bone.

[0030] The beneficial effects of the present invention are:

[0031] 1. Currently, CaSi2 is typically prepared on the market by mixing elemental silicon powder and elemental calcium powder under inert gas protection, ball milling, and then vigorously calcining to synthesize calcium silicide powder. However, these methods require extremely high temperatures (>1000°C), the preparation process is complex, the production cost is high, and the powder produced is mostly micron-sized particles. The present invention overcomes these problems.

[0032] 2. The reported anti-aging hydrogen delivery methods mainly include oral administration of hydrogen-rich water and inhalation of hydrogen. Due to the low solubility and high dispersibility of hydrogen, it is impossible to achieve a high hydrogen concentration in the ward for a long time, resulting in limited anti-aging effects.

[0033] The present invention designs a novel mesoporous bioactive glass (MBG) scaffold loaded with CaSi2 nanoparticles (CSN) for local and sustained (up to 1 week) release of high-dose H2 (911 ml / g CSN), achieving efficient reconstruction of SME and promoting the repair of damaged aging bones. The hydrogen storage capacity of CSN is 4.6×10 4 times, the sustained hydrogen release capacity of polyhydroxyalkanoic acid (PHA)-encapsulated CSN (CSN@PHA) (about one week) is much higher than that of hydrogen-rich water (about 30 minutes);

[0034] 3. The senescent microenvironment (SME) is a major barrier to tissue repair in the elderly, leading to persistent inflammatory responses, increased senescence accumulation, and loss of regenerative capacity. Effectively and safely regulating the SME is crucial for repairing bone damage in aging, but remains a significant challenge. In this work, we found that long-term and sustainable hydrogen supply has the potential to reshape the SME through universal anti-inflammatory and anti-senescence mechanisms, and preserve the regenerative capacity of bone marrow mesenchymal stem cells (BMSCs) by preventing their senescence.

[0035] In the present invention, a new hydrogen-releasing scaffold capable of locally and continuously (up to 1 week) releasing high-dose hydrogen (911 ml / g) was constructed by electrospraying polyhydroxyalkanoate (PHA)-coated CaSi2 nanoparticles (CSN) on a mesoporous bioactive glass scaffold (CSN@PHA-MBG). This scaffold achieved efficient reconstruction of SME and enhanced repair of the femoral defect model in aged mice (24 months old, equivalent to 70 years old). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 (a) is a representative scanning electron microscopy image and 1(b) is the elemental map, 1(c) is the X-ray diffraction pattern of CSN and CaSi2 raw materials, 1(d) is the cytotoxicity / biocompatibility of CSN at different concentrations, and 1(e) is a representative image of BMSCs after treatment with different concentrations of CSN for 24 h.

[0037] Figure 2 CSN@PHA-MBG scaffold parameter regulation selection: 2(a) shows the hydrogen release curves of CSN and CSN@PHA-MBG in PBS. The inset shows the reaction of CSN in aqueous solution, with a high hydrogen production rate. 2(b) shows the SEM images of PHA solutions after electrostatic spraying at different concentrations. In order to obtain better morphological results, a concentration of 150 μg mL -1 2(c) is a PHA solution (150 μg mL -1 ) SEM images of CNS@PHA after electrostatic spraying at different CNS concentrations; 2(d) is a representative elemental analysis image of CSN@PHA; 2(e) is the SEM images of CSN@PHA-MBG scaffolds at different magnifications.

[0038] Figure 3 Figure 3(a) shows the biosafety verification results of PHA-MBG and CSN@PHA-MBG scaffolds: 3(a) is the live / dead fluorescence image of BMSCs seeded on PHA-MBG or CSN@PHA-MBG scaffolds; 3(b) is the SA-β-GAL staining of bone marrow mesenchymal stem cells on PHA-MBG or CSN@PHA-MBG scaffolds; 3(c) is the statistical result corresponding to Figure b.

[0039] Figure 4 H2 concentration at different times after filling the bone defect site with CSN@PHA-MBG scaffold.

[0040] Figure 5 Effects of CSN@PHA-MBG scaffold on senescence of damaged aged bone cells and macrophage polarization.

[0041] Schematic diagram of the in vivo treatment and measurement scheme (a), representative immunostaining images (b), and corresponding quantitative analysis of F4 / 80, iNOS, and ARG1 levels at the implantation site (c), representative flow cytometry images (d, f, h, j) and corresponding quantitative analysis (e, g, i, k) of total senescent cells (d, e), senescent myeloid cells (f, g), senescent macrophages (H, I), and macrophage polarization (J, K). Scale bar in panel b, 100 μm.

[0042] Figure 6Figure 7(a) shows the schematic diagram of the experimental design for cytokine array analysis, 7(b) and (c) show representative mouse cytokine array images and corresponding quantification, and 7(d) shows the cytokine levels measured by ELISA.

[0043] Figure 7 Effects of CSN@PHA-MBG scaffolds on BMSC recruitment and senescence: representative immunostaining images (a) and corresponding quantitative data (b) of p16 (white) and LepR (red) expression at aged bone defect sites on days 3 and 7 after indicated treatment, representative flow cytometry images (e, g) and corresponding quantitative analysis (f, h) of recruited MSCs (e, f) and senescent MSCs (g, h) at defect sites on days 3 and 7 after indicated treatment, representative immunostaining images (i) and corresponding quantitative data (j, k) of Osterix (white), CD31 (red) and Emcn (green) at defect sites on days 7 and 14 after indicated treatment, the scale bars in panels a and i correspond to 100 μm.

[0044] Figure 8 Results of repairing aged bone defects using CSN@PHA-MBG scaffolds: sequential fluorescent labeling of bone formation with calcein (green) and Alizarin Red S (red) (a), and corresponding quantitative analysis of mineral apposition rate (b), histological examination of new tissues at aged bone defect sites after treatment on days 14 and 28 (c), representative SRμCT images of new tissues on day 28 after control, PHA-MBG or CSN@PHA-MBG treatment (d) and corresponding quantification of BV / TV (e), Tb.N (f), and Tb.Th (g), representative immunostaining images (h ) and corresponding quantitative data (i) p16 (red), Vpp3 (red), Osterix (white), and Emcn (green) expression in new tissues at day 28 after the indicated treatments, demonstrating the H2-mediated mechanism of regeneration in damaged aged bone. The CSN@PHA-MBG scaffolds locally and continuously release H2 by inducing potent anti-inflammatory (macrophage polarization) and general anti-aging remodeling of the SME, thereby promoting MSC recruitment and maintaining its regenerative capacity (J); BV / TV, bone volume to total volume ratio; Tb.N, trabecular number; Tb.Th, trabecular thickness; scale bar in panel h, 100 μm. In panel d, yellow, blue, red, and green arrows point directly to the bone defect site, hypertrophic chondrocytes, fibrous tissue, new bone, and scaffold, respectively. DETAILED DESCRIPTION

[0045] The present invention adopts microwave-assisted chemical dissolution method to prepare nano-CaSi2 (CSN). Ethylene glycol is used as dispersant and microwave mechanical stripping method is used to prepare CSN: First, 100mg CaSi2 raw material powder, 400mg polyvinyl pyrrolidone (PVP), 140mg EDTA disodium salt and 100mg sodium hyaluronate are completely dispersed in 20mL ethylene glycol; then the mixture is transferred to a 20mL autoclave and heated at 160℃ for 3h using a microwave synthesizer. After cooling to 50℃, the mixture is heated in a pulse ultrasonicator (425W / cm -2 The product was pulverized for 90 minutes at 4000 rpm (on / off, 6.6 s / 2.2 s). To remove large particles, the product was centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected. The product was washed three times with an ethanol / water (1:1, v / v) mixture, centrifuged at 12,000 rpm for 20 minutes, and then dispersed in 20 ml of ethanol.

[0046] CaSi2 raw powder, size is 25um, purity of raw powder is ≥99% (size range can be 2-1000um, purity can be 90-99%).

[0047] Based on its hydrolysis product Ca 2+ and SiO3 2- Due to its easy metabolism and osteoinductivity, CaSi2 is considered a potential hydrogen prodrug with high H2 production rate (104.2 mg / g or 1.28 L / g) and high biocompatibility. However, CaSi2 bulk and micronized powders hardly decompose under physiological conditions. Therefore, we hypothesized that nanosize can enhance the hydrolysis activity of CaSi2 to release hydrogen. CaSi2 nanoparticles (CSN) were synthesized by microwave-assisted chemical exfoliation; SEM image ( Figure 1 a) shows that the size of the synthesized CSN is about 500 nm. EDS elemental mapping ( Figure 1 b) and XRD data ( Figure 1 c) It was clearly demonstrated that chemical exfoliation did not alter the chemical composition and crystal structure of CaSi2; moreover, CNS did not induce observable cytotoxicity to BMSCs over a wide concentration range, demonstrating high cytocompatibility ( Figure 1 d,1e).

[0048] Reported anti-aging hydrogen delivery methods mainly include oral administration of hydrogen-rich water and inhaled hydrogen. Due to the low solubility and high dispersibility of hydrogen, it is impossible to achieve a high hydrogen concentration in the diseased area for a long time, resulting in limited anti-aging effects. Therefore, the present invention designed a novel mesoporous bioactive glass (MBG) scaffold loaded with nano-CaSi2 nanoparticles (CSN) for local and sustained (up to 1 week) high-dose H2 (911 ml / g CSN), achieving efficient reconstruction of SME and promoting the repair of damaged aged bones. The hydrogen storage capacity of CSN is 4.6×10 4 The sustained hydrogen release capacity of polyhydroxyalkanoic acid (PHA)-encapsulated CSN (CSN@PHA) (about one week) is much higher than that of hydrogen-rich water (about 30 minutes). The method is as follows:

[0049] 4.0 g of F127, 0.76 g of Ca(NO3)2·4H2O, and 0.23 g of TEP were dissolved sequentially in 50 mL of ethanol containing 1.0 g of 0.5 M hydrochloric acid. 5.2 g of tetraethyl orthosilicate was then added to the solution, stirred vigorously for 24 h, and rotary evaporated at 60°C for 30 min. The viscous MBG sol was then uniformly impregnated onto a custom-made polyurethane sponge and calcined at 600°C for 6 h to obtain the MBG scaffold. The CSN@PHA-MBG scaffold was prepared by electrostatic spraying. Taking the CSN@PHA-MBG scaffold containing 50 μg of CSN as an example, 1.5 mg of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHA, molecular weight 25,000 Da) was dissolved in 10 mL of dimethylformamide (8:2, v / v) solution. The CSN and PHA solution were then mixed at a ratio of 0.5 mg mL -1 After ultrasonic dispersion, the CSN@PHA mixture was transferred into a 5 mL syringe and injected with a 22-gauge needle at 0.5 mL / min. -1 The solution was sprayed on the MBG bracket at a speed of 20 min, and a voltage of 18 kV was applied to generate an electrostatic field within a working distance of 10 cm.

[0050] The results showed that, as expected, CSN could indeed be hydrolyzed in PBS at pH 7.4 to release a large amount of H2, but it was completely degraded within one day ( Figure 2However, early inflammation in bone damage can persist for several days, and a long-term H2 supply lasting several days has also been shown to be beneficial for the repair of aged bone damage. Therefore, we encapsulated CSN (CSN@PHA) with polyhydroxyalkanoate (PHA, a natural hydrophobic biopolymer) to reduce the hydrolysis / degradation rate of CSN. The CSNs were then deposited on the porous surface of MBG substrates by electrospraying. In addition to high biocompatibility and good biodegradability, the main reasons for choosing PHA are its hydrophobicity and thermoplasticity, which support the hindrance of CSN hydrolysis to achieve sustained H2 release and enhance the electrospraying practicality of CSN-encapsulated polymer nanoparticles, respectively. Moreover, its degradation products exhibit higher tissue compatibility.

[0051] Using a common electrospraying method, CSN@PHA with adjustable incorporation ratio can be easily produced on a large scale and immobilized on a porous scaffold. Under optimized parameters, CSN@PHA can be electrosprayed into regular particles with uniform particle size ( Figure 2 b, 2c). The element mapping pattern shows that CSN can be embedded into PHA in an encapsulated manner ( Figure 2 d). By comparing the SEM images before and after electrospraying, it can be observed that CSN@PHA is uniformly coated on the porous surface of MBG ( Figure 2 e, 2f). Notably, the synthesized CSN@PHA-MBG exhibited an ultra-sustained H2 release profile, with a release time of up to 7 days ( Figure 2 a) Such prolonged H2 release duration would be able to cover the inflammation (acute and chronic) and new tissue formation phases of bone regeneration, and would also be particularly beneficial for SME remodeling in aged bone lesions.

[0052] Furthermore, live / dead cell staining revealed that both PHA-MBG and CSN@PHA-MBG scaffolds with different CSN contents had high biocompatibility, which had no significant effect on the viability of BMSCs ( Figure 3 a). More importantly, after 7 days of culture, the SA-β-gal expression in BMSCs of CSN@PHA-MBG scaffolds was significantly reduced compared with that of PHA-MBG scaffolds without H2 supply, showing approximately 70 μg g -1 The minimum effective CSN content (or hydrogen dose) ( Figure 3 b) We selected a CSN content of 560 μg g -1 The developed CSN@PHA-MBG scaffold for subsequent in vivo therapeutic experiments enables the local and sustainable supply of high-dose H2, providing the possibility of promoting the regeneration of injured aging bones.

[0053] The senescent microenvironment (SME) is a major barrier to tissue repair in the elderly, leading to persistent inflammation, increased senescence accumulation, and loss of regenerative capacity. Effectively and safely modulating the SME is crucial for repairing bone damage in aging but remains a significant challenge. In this work, we discovered that a long-term and sustainable supply of hydrogen has the potential to remodel the SME through universal anti-inflammatory and anti-senescence mechanisms and preserve the regenerative capacity of bone marrow mesenchymal stem cells (BMSCs) by preventing their senescence. Therefore, we constructed a novel hydrogen-releasing scaffold capable of localized and sustained (up to 1 week) release of high-dose hydrogen (911 mL / g) by electrospraying polyhydroxyalkanoate (PHA)-coated CaSi2 nanoparticles (CSN) onto a mesoporous bioactive glass (CSN@PHA-MBG) scaffold. This scaffold achieved efficient SME reconstruction and enhanced repair of femoral plug defects in aged mice (24 months, equivalent to 70 years).

[0054] Methods: This study established a femoral plug defect model in mice. C57BL / 6 male mice (22-24 months of age) were randomly divided into three groups, each consisting of eight mice: a control group (no implant), a PHA-MBG group, and a CSN@PHA-MBG group. Briefly, under isoflurane anesthesia, an 8 mm long straight incision was made in the distal femur of each hind leg. A 1.2 mm diameter circular defect was then created vertically in the distal femoral shaft using a trephine. After irrigating the wound with saline, a cylindrical stent was implanted and the wound was sutured with silk suture.

[0055] Given the profound effects of sustained H2 release on general anti-inflammatory / aging in vitro, the in vivo therapeutic principle and performance of the CSN@PHA-MBG scaffold were further evaluated using a critical-size femoral plug defect model (1.2 mm diameter circular defect) in aged mice (24 months old). Since chronic inflammation in SME greatly hinders the healing of aged tissues, early anti-inflammatory treatment of bone damage is crucial for timely and effective bone repair. Therefore, we first studied the in vivo hydrogen release behavior of the CSN@PHA-MBG scaffold and the effect of the CSN@PHA-MBG scaffold on local inflammation within 7 days after treatment. Figure 4 The CSN@PHA-MBG scaffolds released hydrogen for up to nine days in vivo, even longer than in PBS (pH 7.4), likely due to the slower diffusion of hydrogen molecules in bone. This sustained hydrogen release behavior could be beneficial for local anti-aging and anti-inflammatory effects.

[0056] from Figure 5In the ac, strong green fluorescence of F4 / 80 (a biomarker of macrophages) appeared at the implantation site on days 3 and 7, reflecting the typical inflammatory phenomenon when macrophages are massively recruited during the initial stage of bone repair. Although there was no significant difference in the degree of macrophage infiltration, the CSN@PHA-MBG scaffolds showed a strong anti-inflammatory effect compared with the control group and PHA-MBG carriers, as shown by lower iNOS expression (a biomarker of the M1 macrophage phenotype) and higher ARG1 levels (a biomarker of the M2 macrophage phenotype), mainly due to the sustained release of hydrogen.

[0057] In addition, flow cytometric analysis of cells from aged bone defects was performed to evaluate the role of local sustained H2 release in regulating SME aging and inflammation ( Figure 5 dk). Figure 5 d, 5e, p16-positive cells occupied a high proportion, which increased over time (16.7% on day 3 and 23.2% on day 7), showing a typical bystander effect of senescence gradually spreading to surrounding cells. Even so, CSN@PHA-MBG significantly and stably alleviated the progression of senescence / damage superimposed senescence, but PHA-MBG as a carrier was always less significant ( Figure 5 d-5g), indicating that the sustained H2 release from CSN@PHA-MBG can indeed locally and continuously prevent cellular senescence in aged bone defects SME. In particular, according to previous reports, macrophages in SME exhibited a high p16-positive phenotype (over 80%), which could be significantly reduced by CSN@PHA-MBG on day 3 ( Figure 5 h, 5i). Although the decrease in the proportion of p16-positive macrophages induced by H2 on day 7 did not seem as significant as on day 3, sustained H2 release plays an important role in mediating immune responses in a macrophage phenotype-dependent manner. Figure 5 j, 5k As can be seen in the control group, the number of M1 phenotype macrophages was much higher than that of M2 phenotype macrophages (about 2 times on day 3), reflecting a highly pro-inflammatory response in the inflammatory stage. Compared with the control group (1.96 on day 3, 1.54 on day 7) and PHA-MBG (1.29 on day 3, 0.64 on day 7), CSN@PHA-MBG more significantly and stably reduced the ratio of macrophage numbers between M1 phenotype and M2 phenotype (0.71 on day 3, 0.57 on day 7), indicating that according to the in vitro results, sustained H2 release made a significant contribution to anti-inflammatory polarization, especially on day 7, CSN@PHA-MBG induced a high proportion of M2 phenotype macrophage infiltration (about 40%, Figure 5 j, 5k), which means that the effective transformation of the microenvironment from pro-inflammatory to anti-inflammatory is beneficial to SME remodeling and repair of aging bone defects.

[0058] Cytokines and chemokines derived from local cells play an important role in manipulating the microenvironment and immune response. As senescent cells accumulate with age, they can develop the SASP and extend their harmful paracrine secretions to surrounding cells, leading to chronic inflammation and hindering tissue repair. Given this, we analyzed the expression of cytokines in damaged tissues collected during the initial inflammatory phase using cytokine arrays and ELISA ( Figure 6 a) If Figure 6 As shown in Figures 6b and 6c, the significant differences in cytokine expression between the control group and the scaffold-treated group can be mainly divided into three categories, including the downregulation of SASP components (purple panel), the upregulation of anti-inflammatory factors (green panel), and the improvement of repair / regeneration-related factors (blue panel). It can be clearly seen that the CSN@PHA-MBG scaffold has a significant inhibitory effect on typical SASP components including IL-6, IL-7, IL-1α, IL-1β, IL-13, and IL-15, while the CSN@PHA-MBG scaffold regulates representative anti-inflammatory factors such as IL-4, IL-10, and IL-1ra / IL-1F3. CSN@PHA-MBG generally showed a stronger and more active regulatory effect than PHA-MBG, which means that the sustained release of hydrogen made a significant contribution compared with the carrier. In addition, from Figure 6 c It can be seen that compared with PHA-MBG, CSN@PHA-MBG more significantly induced the upregulation of typical angiogenic (C1qR1 / CD93, Endoglin / CD105 and endostatin) and osteogenic (Reg3G, MMP-2, Fetuin A and osteopontin) factors, and also inhibited the expression of typical osteoclast factors (MMP-9 and myeloperoxidase). To obtain more accurate quantification, ELISA was used to analyze the prototype components of SASP, anti-inflammatory cytokines and osteogenesis-related proteins from aged bone defect sites. Continuous H2 supply in SME can significantly attenuate SASP ( Figure 6 d) and acted as an anti-inflammatory regulator of increased IL-4 and IL-10 levels. Furthermore, CSN@PHA-MBG showed significantly higher expression of endoglin / CD105, osteopontin, and the anti-bone-breaking cytokine osteoprotectin compared to the control and PHA-MBG, indicating that localized sustained H2 release from CSN@PHA-MBG can indeed modulate the secretome of SMEs, favoring bone regeneration. Combined analyses of general cellular senescence, inflammation, and the SASP confirmed that the SMEs in aged bone defects were remodeled by the sustained H2 release from the CSN@PHA-MBG scaffold.

[0059] In general, bone regeneration is a coordinated process that involves a significant overlap of several typical events, including hemostasis and inflammation, new tissue formation (recruitment of endogenous cells), and tissue remodeling. In addition, there is a close connection between cellular senescence and tissue regeneration. Since the local and sustained release of H2 by CSN@PHA-MBG can indeed reshape the SME in the initial stage of bone repair and play an anti-inflammatory role in the SME, we next sought to explore whether this effect would form a positive feedforward loop to promote regeneration by preventing endogenous stem cell aging and activating their regenerative function. To examine this, we co-stained the injured tissue with p16 and LepR (a biomarker of BMSCs) on days 3 and 7. BMSCs (LepR+) were visible throughout the defect, and there was no significant difference in LepR levels between the control group and PHA-MBG ( Figure 7 a, b). In contrast, on day 3, the proportion of BMSCs enriched in CSN@PHA-MBG implants was significantly higher compared with the control group. Meanwhile, a significant decrease in p16 expression was observed in CSN@PHA-MBG on days 3 and 7 compared with the control group and PHA-MBG group ( Figure 7 a, c), which is consistent with the results of flow cytometry analysis ( Figure 7 d, e). In addition, as expected, the defects implanted with CSN@PHA-MBG showed the lowest proportion of senescent BMSCs (LepR+, P16+, Figure 7 Next, we analyzed the expression of two additional BMSC markers (CD73+CD90+, Figure 7 e, f) confirmed this trend again. According to the above immunohistochemical results, compared with the control group and PHA-MBG ( Figure 7 g, h) Compared with CSN@PHA-MBG, more BMSCs can be recruited ( Figure 7 e, f), while locally preventing its aging.

[0060] Bone regeneration is highly correlated with angiogenesis, so we further examined the vasculature network formation in the scaffolds. We performed co-immunostaining of osterix+ osteoprogenitor cells, which can differentiate into osteoblasts and osteocytes, and CD31+, Emcn+ type H endothelial cells, which can maintain perivascular osteoprogenitor cells and couple angiogenesis to osteogenesis. As shown in the figure, CD31+, Emcn+ type H endothelial cells and associated Osterix+ osteoprogenitor cells were sparse at the defect site in the control group at both day 7 and day 14. In contrast, CSN@PHA-MBG scaffolds significantly increased the number of osteoprogenitor cells and type H endothelial cells, but PHA-MBG did not. In addition, Osterix+ osteoprogenitor cells were enriched around type H vessels ( Figure 7i). In summary, it can be concluded that the sustained H2 release from CSN@PHA-MBG creates a favorable niche for BMSC recruitment and anti-aging, greatly supporting angiogenesis and osteogenesis for regeneration of aged bone defects.

[0061] To further validate the above findings on the remodeling, immunomodulatory, and regenerative effects of SMEs with a sustainable H2 supply in vivo and to understand their contribution to therapeutic outcomes, bone formation rate, tissue morphology, senescent cell load, and anti-bone fragmentation potential were comprehensively analyzed. To assess bone integration, newly formed tissue was labeled sequentially with calcein (green fluorescence) on day 3 and alizarin red S (red fluorescence) on day 17. The almost complete red / green fluorescence overlap in the control group indicated that bone formation in aged mice was slow and their self-regeneration capacity was limited ( Figure 6 a), PHA-MBG provided some improvement compared with the control group, but there was no significant difference between them ( Figure 8 a, b). By comparison, the clear continuous fluorescence bands of calcein and Alizarin Red S indicated that CSN@PHA-MBG significantly improved the bone attachment rate ( Figure 8 a, b). In addition, Figure 8 Histological evidence in c showed that there were significant differences between the control group, PHA-MBG group, and CSN@PHA-MBG group. A larger defect gap with obvious fibrous tissue infiltration was visible in the control group on day 14, and the ingrowth tissue was mostly fibrous with minimal bone formation after 28 days. In contrast, PHA-MBG and CSN@PHA-MBG treatments showed significant amounts of bone formation. In particular, we observed that bone formation in CSN@PHA-MBG-treated mice was faster than that in the PHA-MBG group. In particular, the bone defect sites of PHA-MBG-treated mice were filled with abundant chondrocytes on day 14, and then trabecular bone was formed on day 28, and CSN@PHA-MBG induced increased new bone formation ( Figure 8 c).

[0062] At the end of treatment (day 28), the results of CSN@PHA-MBG scaffolds for aged bone defect repair were further evaluated by synchrotron radiation microcomputed tomography (SRμCT) and immunostaining analysis of the neoplastic tissue. Figure 8 d As can be seen, the aged bone defects in the control group without scaffold treatment were not repaired satisfactorily, and even unpredictable fractures often occurred. The corresponding quantitative SRμCT analysis showed that the bone volume / total volume ratio (BV / TV, Figure 8 e) The number of trabeculae is the least (Tb.N, Figure 8 f) and the thinnest trabecular thickness (Tb.Th, Figure 8g), confirming the limited innate regenerative capacity of aged bone. In contrast, both PHA-MBG and CSN@PHA-MBG improved regenerative outcomes and avoided fractures, as revealed by the gradual bone bridging of the defect ( Figure 8 d). Notably, CSN@PHA-MB with sustained H2-releasing ability significantly improved the repair outcome of PHA-MBG, as evidenced by the higher new bone formation (BV / TV, Figure 8 e) and better new bone microarchitecture ( Figure 8 d, c) as indicated.

[0063] In summary, we have drawn a mechanism diagram, such as Figure 8 As shown in Figure 1, macrophage repolarization reverses the inflammatory microenvironment into an anti-inflammatory microenvironment and generally slows down the progression of cellular senescence, thereby timely and effectively releasing SMEs locally and continuously. In turn, SMEs reshape the favorable microenvironment, promote the recruitment of BMSCs and osteoprogenitor cells, and enhance their regenerative capacity locally by reducing their senescence burden, thereby improving the outcome of aged bone defect regeneration.

Claims

1. A method for preparing a long-acting hydrogen-releasing stent, characterized in that: The preparation method comprises the following steps: Step 1: Preparation of CSN 1.1) Completely disperse CaSi2 raw material powder, modifier, EDTA disodium salt, and sodium hyaluronate in an alcohol solvent at a mass ratio of 100:(50-1000):(20:1000):(20-1000) to obtain a mixture; The modifier is any one or more of polyvinyl pyrrolidone, polylactic acid and polyethylene glycol; 1.2) Transfer the mixture to an autoclave and heat using a microwave synthesizer at a temperature of 100-300°C for 0.5-10 hours. 1.3) After cooling to 45-55°C, crush in a pulsed ultrasonic machine with a power of 100-1000 W / cm 2 , and work intermittently, work: rest time = 1: (0.1-10), ultrasonic crushing time is 10~500 min; 1.4) After removing large particles, wash and then disperse in ethanol by centrifugation; Step 2: Preparation of CSN@PHA-MBG scaffold based on mesoporous bioactive glass scaffold Polyhydroxyalkanoate (PHA) was dissolved in a DCM / DMF solution with a volume ratio of (1-10):1 at a concentration of 10-500 μg / mL. CSN and PHA were then uniformly mixed at a particle concentration of 0.05-5 mg / mL to form a CSN@PHA mixture. The CSN@PHA mixture was sprayed onto an MBG scaffold and a voltage of 5-50 kV was applied to generate an electrostatic field within a working distance of 4-40 cm. The molecular weight of PHA is 5000-50000 Da.

2. The method for preparing a long-acting hydrogen-releasing stent according to claim 1, wherein the alcohol solvent is one or more of ethylene glycol and glycerol.

3. The method for preparing a long-acting hydrogen-releasing stent according to claim 1, wherein: The spraying rate in the second step was 0.05-2 mL / h, the spraying time was 5-60 min, and the MBG stent was rotated during the spraying process.

4. A long-lasting hydrogen-releasing stent, characterized in that: The long-acting hydrogen-releasing stent is prepared by the preparation method of the long-acting hydrogen-releasing stent according to any one of claims 1 to 3.

5. Use of the long-acting hydrogen-releasing stent according to claim 4 for preparing a drug for improving the aging microenvironment and preventing the aging of bone marrow mesenchymal stem cells through sustained hydrogen release.

6. Use of the long-acting hydrogen-releasing stent according to claim 5 for preparing a drug for improving the aging microenvironment and preventing the aging of bone marrow mesenchymal stem cells through sustained hydrogen release, characterized in that: The long-acting hydrogen-releasing stent is used to inhibit typical SASP components including IL-6, IL-7, IL-1α, IL-1β, IL-13 and IL-15; and / or increase the levels of representative anti-inflammatory cytokines IL-4, IL-10, and IL-1ra / IL-1F3; and / or induce canonical angiogenesis; and / or upregulate osteogenic factors; and / or inhibit the expression of typical osteoclast factors.

7. Use of the long-acting hydrogen-releasing stent according to claim 6 for preparing a drug for improving the aging microenvironment and preventing the aging of bone marrow mesenchymal stem cells through sustained hydrogen release, characterized in that: The typical blood vessels include C1qR1 / CD93, Endoglin / CD105 and / or endostatin; the osteogenic factors are Reg3G, MMP-2, Fetuin A and / or osteopontin; and the typical osteoclast factors are MMP-9 and myeloperoxidase.

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