A double-layer stent and a preparation method thereof

Abstract

The application provides a double-layer stent and a preparation method thereof, which comprises the following steps: dissolving L-polylactic acid in a solvent, adding calcium salt to the L-polylactic acid, and mixing to obtain a precursor solution; then electrospinning the precursor solution to obtain a fiber membrane; drying the fiber membrane to obtain a fiber layer of the double-layer stent; dissolving polyvinyl alcohol, gelatin and polyamide in a binary solvent, adding barium titanate to the binary solvent, and mixing to obtain a hydrogel solution; placing the fiber membrane at the bottom of a mold, then placing the hydrogel solution in the mold, and then performing freeze-crosslinking to shape the hydrogel in the double-layer stent; and finally immersing the hydrogel in the double-layer stent after demolding in salt water to obtain the double-layer stent. The fiber membrane and the hydrogel solution are combined through freeze-crosslinking to obtain the double-layer stent, and the double-layer stent can accelerate the formation of new bone when applied to osteoporotic bone defects and under the stimulation of ultrasonic waves.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically a double-layer scaffold and its preparation method. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by a decrease in bone mass per unit volume, a reduced ratio of mineral salts and bone matrix, and deterioration of bone microstructure, leading to increased bone fragility and susceptibility to fractures. Regeneration of osteoporotic bone defects is particularly challenging, resulting in prolonged bone healing periods and unsatisfactory outcomes from conventional treatments. Electrical stimulation has emerged as an effective treatment modality, accelerating bone repair by regulating cell proliferation and differentiation and promoting extracellular matrix deposition. However, clinical translation has often been hampered by the reliance on external power sources and the mismatch between these factors and rigid implantable materials. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to prepare a double-layer scaffold that can promote osteoporotic bone healing.

[0004] The present invention solves the above-mentioned technical problems through the following technical means:

[0005] The first aspect of this invention provides a method for preparing a double-layer scaffold, comprising the following steps: S1 dissolves L-polylactic acid in a solvent, adds calcium salt and mixes to obtain a precursor solution, then electrospins the precursor solution to obtain a fiber membrane; the fiber membrane is dried to obtain a fiber layer of a bilayer scaffold; polyvinyl alcohol, gelatin and polyamide are dissolved in a binary solvent, add barium titanate and mix to obtain a hydrogel solution; S2 places the fiber membrane at the bottom of the mold, then places the hydrogel solution in the mold, and then performs freeze crosslinking to form the hydrogel in the double-layer scaffold. After demolding, the hydrogel in the double-layer scaffold is immersed in salt water to finally obtain the double-layer scaffold.

[0006] Beneficial Effects: A bilayer scaffold is obtained by cryo-crosslinking a fibrous membrane with a hydrogel solution. When applied to osteoporotic bone defects, this scaffold accelerates new bone formation under ultrasound stimulation. The fibrous membrane provides physical support and bone conduction, and generates electrical signals in response to piezoelectricity under ultrasound stimulation. The flexible and viscous ion-conducting hydrogel connects the bilayer scaffold to cells and tissues to transmit these signals. Through the synergistic effect of polylactic acid (PLA) and barium titanate, endogenous mechanical energy induced by ultrasound from physiological activity is converted into a strong electric field. This electric field potential is effectively propagated and amplified through the ion network of the hydrogel, directly establishing a continuous, non-electrically stimulated microenvironment at the bone defect site. Under ultrasound stimulation, the bilayer scaffold exhibits excellent ROS scavenging and electrical signal transmission capabilities. It enhances osteoblast proliferation and differentiation, increases the expression of key osteogenic proteins, and simultaneously inhibits osteoclast activity.

[0007] Preferably, the mass ratio of L-polylactic acid to calcium salt is 10:1.

[0008] Preferably, the calcium salt is calcium chloride.

[0009] Preferably, the solvent is dichloroethane.

[0010] Preferred electrospinning process parameters are: voltage of 16kV~20kV, receiving distance of 13cm~17cm, injection speed of 0.8mL / h~1.2mL / h, roller speed of 1400rpm~1600rpm, and spinning time of 4h.

[0011] Preferably, the average diameter of the fiber membrane is 500±30nm, and the pore size of the hydrogel is 10um~30um.

[0012] Preferably, the concentration of polyvinyl alcohol is 8 wt%.

[0013] Preferably, the concentration of gelatin is 4 wt%.

[0014] Preferably, the concentration of polyamide is 2 wt%.

[0015] Preferably, the concentration of barium titanate nanoparticles is 5 wt%.

[0016] Preferably, the freezing temperature is -18℃ to -22℃, and the freezing time is 24 hours.

[0017] Preferably, the brine is a phosphate-buffered brine, and the soaking time is 24 hours.

[0018] Preferably, the binary solvent is a mixture of glycerol and water, wherein the volume ratio of glycerol to water is 1:4.

[0019] The second aspect of the present invention provides a method for preparing the above-mentioned double-layer stent to obtain the double-layer stent. Attached Figure Description

[0020] Figure 1 This is a SEM image of the PLLA@Ca nanofiber membrane in Example 1 of this invention; Figure 2 This is the FTIR spectrum of polylactic acid (PLA) in Example 1 of this invention; Figure 3 This is the FTIR spectrum of the PLLA@Ca nanofiber membrane in Example 1 of this invention; Figure 4 This is the XPS analysis diagram of the PLLA@Ca nanofiber membrane in Example 1 of the present invention; Figure 5 This is the piezoelectric response diagram of the PLLA@Ca nanofiber membrane in Example 1 of the present invention; Figure 6 This is a piezoelectric response mapping diagram of the PLLA@Ca nanofiber membrane in Example 1 of the present invention; Figure 7 This is a SEM image of barium titanate nanoparticles in the PLLA@Ca / Hydrogel@BTO composite material in Example 1 of this invention; Figure 8 This is the energy dispersive spectroscopy (EDS) elemental diagram of barium titanate nanoparticles in the PLLA@Ca / Hydrogel@BTO composite material in Example 1 of this invention; Figure 9 This is the XRD pattern of barium titanate nanoparticles in the PLLA@Ca / Hydrogel@BTO composite material in Example 1 of this invention; Figure 10 This is a morphology diagram of the PLLA@Ca / Hydrogel@BTO composite material in Example 1 of the present invention; Figure 11 These are the XRD spectra of PVA, gelatin, PVA gel prepared in Comparative Example 3, and PLLA@Ca / Hydrogel@BTO composite material prepared in Example 1 in this invention. Figure 12 These are the FTIR spectra of PVA, gelatin, PVA gel prepared in Comparative Example 3, and PLLA@Ca / Hydrogel@BTO composite material prepared in Example 1 in this invention. Figure 13 This is a viscosity transition diagram of the PLLA@Ca / Hydrogel@BTO composite material in Example 1 of this invention; Figure 14 This is a diagram showing the adhesion behavior of the PLLA@Ca / Hydrogel@BTO composite material on different matrices in Example 1 of this invention; Figure 15 These are fluorescence imaging images of cultured cells of control, PLLA@Ca, and PLLA@Ca / Hydrogel@BTO in the experimental examples of this invention; Figure 16 This is a bar graph showing the quantitative analysis of bone mineral density after 8 weeks of treatment in the normal control group, model group, PLLA@Ca group, and PLLA@Ca / Hydrogel@BTO+US group in the experimental examples of this invention. Figure 17 These are staining images of rat bone tissue after 8 weeks of treatment in the normal control group, model group, PLLA@Ca group, and PLLA@Ca / Hydrogel@BTO+US group in the experimental examples of this invention. Figure 18 This is a schematic diagram of bone treatment using the PLLA@Ca / Hydrogel@BTO composite material in Embodiment 1 of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0023] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0024] Example 1 This embodiment provides a method for preparing a double-layer scaffold, specifically including the following steps: S1 dissolved 1.0 g of polylactic acid (PLLA) in 5 ml of dichloroethane and magnetically stirred at 37 °C for 6 h to form a clear solution. Then, 0.1 g of CaCl2 nanoparticles were added to the solution, stirred for 2 h, and sonicated (200 W) for 15 min to disperse CaCl2 evenly, thus obtaining the PLLA@Ca precursor solution.

[0025] The PLLA@Ca precursor solution was loaded into a 20mL syringe containing a stainless steel needle with an inner diameter of 0.9mm. The stainless steel needle was connected to a high-voltage DC power supply. The specific parameters for electrospinning are as follows: Environment: 25±2℃, 40±5% relative humidity; injection rate: 1.0 mL / h; voltage: 18 kV; receiving distance: 15 cm; spinning time: 4 h; then the nanofiber membrane was collected on a rotating drum at 1500 rpm. The nanofiber membrane was then placed in a vacuum oven and dried at 30℃ for 24 h to remove residual dichloroethane, yielding the PLLA@Ca nanofiber membrane, which serves as the fiber layer of the bilayer scaffold. The average diameter of the PLLA@Ca nanofiber membrane was 500±30 nm.

[0026] S2 dissolves polyvinyl alcohol, gelatin, and polyamide in a glycerol-water binary solvent and stirs magnetically at 70°C for 2 hours until a transparent and homogeneous polymer solution is formed. The volume ratio of glycerol to water is 1:4, and the concentration of polyvinyl alcohol in the polymer solution is 8 wt%, the concentration of gelatin is 4 wt%, and the concentration of polyamide is 2 wt%.

[0027] Then, barium titanate nanoparticles (BTO) were added to the polymer solution and stirred at 70°C for 1 h. The mixture was then sonicated (300W, working time 3 s, interval 3 s) for 30 min to eliminate agglomeration and ensure uniform dispersion of barium titanate, resulting in a hydrogel solution with a barium titanate concentration of 5 wt%.

[0028] S3 lays the PLLA@Ca nanofiber membrane flat on the bottom of a polytetrafluoroethylene mold (10mm×10mm×2mm), then adds a hydrogel solution to the polytetrafluoroethylene mold, removes air bubbles from the hydrogel solution by gently shaking, and then freezes and crosslinks at -20℃ for 24h to form the hydrogel in the double-layer scaffold.

[0029] After demolding, the hydrogel was immersed in phosphate-buffered saline (PBS, pH 7.4) at 37°C for 24 hours to remove unreacted small molecules (such as polyamide), resulting in the PLLA@Ca / Hydrogel@BTO composite material, which is the bilayer scaffold.

[0030] Comparative Example 1 This comparative example provides a method for preparing a PLLA nanofiber membrane. The difference between this comparative example and step S1 of Example 1 is that CaCl2 nanopowder was not added, while the other steps are the same as step S1 of Example 1, and a PLLA nanofiber membrane is finally obtained.

[0031] Comparative Example 2 This comparative example provides a method for preparing Hydrogel@BTO composite hydrogel. The difference between this comparative example and Example 1 is that step S1 is omitted, and in S3, PLLA@Ca nanofiber membrane is not added to the polytetrafluoroethylene mold. The other steps are the same as in Example 1, and Hydrogel@BTO composite hydrogel is finally obtained.

[0032] Comparative Example 3 This comparative example provides a method for preparing PVA gel. The difference between this comparative example and Example 1 is that only steps S2 and S3 are used, and barium titanate nanoparticles are not added in step S2, and PLLA@Ca nanofiber membrane is not added in step S3. All other steps are the same as in Example 1, and PVA gel is finally obtained.

[0033] according to Figure 1 As shown, the PLLA@Ca nanofiber membrane possesses a uniform, continuous, and interconnected nanofiber network structure. This nanofiber network structure exhibits a high porosity of approximately 85%, which promotes nutrient transport and cell infiltration, a key prerequisite for bone tissue engineering scaffolds.

[0034] according to Figure 2 and Figure 3 As shown, this indicates that there is a molecular interaction between calcium ions and polylactic acid (PLA). Figure 2 The FTIR spectrum of L-polylactic acid shows a depth of 1750 cm⁻¹. -1 (C=O stretching), 1260 cm -1 (Asymmetric COC stretching) and 1080 cm -1 Characteristic peaks of (asymmetric COC stretching). Figure 3 In the FTIR spectrum of the PLLA@Ca nanofiber membrane, the characteristic peak of C=O (1742 cm⁻¹) is observed. -1 ) and COC characteristic peak (1075 cm⁻¹) -1 Both peaks show a significant red shift. This red shift indicates a coordination interaction between calcium ions and oxygen atoms in the ester and ether groups of the L-type polylactic acid chain, thereby altering the electron cloud density of these chemical bonds. Furthermore, the PLLA@Ca nanofiber membrane spectrum shows a significant red shift at 3450 cm⁻¹. -1 A weak, broad peak appeared, corresponding to the adsorbed hydroxyl groups, confirming that calcium ions react chemically with polylactic acid (PLLA) rather than simply mixing physically. The PLLA@Ca nanofiber membrane enhances the bone conductivity of the bilayer scaffold through calcium ion release and promotes a more ordered crystal structure, which is beneficial for triggering piezoelectricity under ultrasonic stimulation.

[0035] according to Figure 4As shown, the elemental composition and chemical state within the PLLA@Ca nanofiber membrane were further confirmed. Spectroscopy confirmed the successful binding of Ca, C, and O, with C1s and O1s peaks consistent with the PLLA polymer backbone. The additional O1s component at 533.5 eV is attributed to the Ca-O bond. The Ca 2p spectrum exhibited the characteristic dual state 2p / 2 with a binding energy of 347.2 eV (Ca) and 350.8 eV (Ca 2p / 2), confirming the presence of calcium in a divalent cation state. These results collectively confirm the chemical integration of calcium ions into the PLLA matrix, a structure that ensures controlled ion release to support biomineralization without inducing sudden cytotoxicity.

[0036] according to Figure 5 As shown, the PLLA@Ca nanofiber membrane exhibits a well-defined symmetrical butterfly ring and a sharp phase-switching ring of nearly 180 degrees, indicating the presence of stable ferroelectric domains.

[0037] Effective piezoelectric coefficient = ΔZ / ΔV (ΔZ: piezoelectric displacement, ΔV: applied voltage). The effective piezoelectric coefficient of the PLLA@Ca nanofiber membrane is approximately 8.5 pm / V, which is significantly improved compared to the PLLA nanofiber membrane of Comparative Example 1 (approximately 5.2 pm / V). This is attributed to the introduction of calcium ions, which disrupts the random orientation of the PLLA chains, promotes the alignment of molecular dipoles, and thus increases net polarization and piezoelectric activity.

[0038] according to Figure 6 As shown, the PLLA@Ca nanofiber membrane exhibits a uniform color distribution, indicating a uniform piezoelectric response across the entire surface of the PLLA@Ca nanofiber membrane. This spatially uniform electromechanical coupling is crucial for generating reliable and predictable electrical signals under physiological and mechanical loads.

[0039] according to Figure 7 and 10 As shown, the barium titanate nanoparticles in the PLLA@Ca / Hydrogel@BTO composite material are spherical with a uniform size distribution of approximately 50–80 nm and exhibit good dispersion. The PLLA@Ca / Hydrogel@BTO composite material is a highly interconnected three-dimensional porous network with a pore size range of 10–30 μm. The barium titanate nanoparticles are uniformly embedded within the framework of the PLLA@Ca / Hydrogel@BTO composite material, and the hydrogen bonding interaction between glycerol and the surface of the barium titanate nanoparticles promotes their dispersion. The porous structure of the PLLA@Ca / Hydrogel@BTO composite material is beneficial for ion transport and cell integration.

[0040] according to Figure 8As shown, Ba, Ti, and O are uniformly distributed in the PLLA@Ca / Hydrogel@BTO composite material, ensuring isotropic ultrasonic response to piezoelectric stimulation.

[0041] according to Figure 9 and 11 As shown, barium titanate nanoparticles were successfully incorporated into polymers of polyvinyl alcohol, gelatin, and polyamide. The XRD spectrum of the PLLA@Ca / Hydrogel@BTO composite material shows both the amorphous halo of the polymer and the sharp characteristic peaks of the perovskite BTO phase, indicating that the barium titanate nanoparticles maintained their crystal structure during the preparation of the PLLA@Ca / Hydrogel@BTO composite material.

[0042] according to Figure 12 As shown, the intermolecular interactions are further elucidated, revealing the characteristic peaks of PVA, gelatin, the PVA gel prepared in Comparative Example 2, and the PLLA@Ca / Hydrogel@BTO composite material. The PLLA@Ca / Hydrogel@BTO composite material shows a peak at 580 cm⁻¹. -1 A distinct Ti-O-Ti bending vibration peak is observed. The OH tensile band of the PLLA@Ca / Hydrogel@BTO composite material (~3300 cm⁻¹) is also visible. -1 The gradual redshift indicates a strong hydrogen bond between the polymer chain and the barium titanate nanoparticles, which contributes to the mechanical integrity of the PLLA@Ca / Hydrogel@BTO composite.

[0043] according to Figure 13 As shown, the PLLA@Ca / Hydrogel@BTO composite material can transition from a pre-bonded state to a firmly bonded state within 2 minutes, indicating that the Hydrogel@BTO composite hydrogel can adhere to bone tissue quickly and strongly. Strong tissue adhesion is crucial for ensuring effective electrical signal transduction at the bone-implant interface. The PLLA@Ca / Hydrogel@BTO composite material was bonded to porcine bone, and the interfacial adhesion force was measured to be approximately 15 kPa using a quantitative 180-degree peel test. This strong adhesion mainly originates from the multiple hydrogen bonds and electrostatic interactions between the gelatin and the bone surface, as well as the Ca²⁺ released from the PLLA@Ca nanofiber membrane. + The metal ion bridging effect formed at the interface enables ternary coordination crosslinking of gelatin, calcium ions, and bone tissue.

[0044] according to Figure 14 As shown, the PLLA@Ca / Hydrogel@BTO composite material can effectively adhere to various matrices, including glass and metal, highlighting its stable adhesion ability in complex physiological environments.

[0045] Experimental Example The PLLA@Ca / Hydrogel@BTO composite material and PLLA@Ca nanofiber membrane prepared in Example 1 were used for osteoporosis treatment testing.

[0046] Before evaluating osteoporosis tests, the biosafety of the ultrasound stimulation protocol needs to be confirmed. Control, PLLA@Ca, and PLLA@Ca / Hydrogel@BTO protocols were set up; specifically, MC3T3-E1 cells were cultured in culture dishes and subjected to daily ultrasound stimulation (1 MHz, 1 W / cm²). 2 Cells were cultured on PLLA@Ca nanofiber membranes for 5 minutes, and dead and live cells were identified by staining and finally displayed by fluorescence imaging (i.e., the control). Cells were cultured on PLLA@Ca / Hydrogel@BTO composite material (double-layer scaffold) for 5 minutes, and all other aspects were the same as the control (i.e., PLLA@Ca / Hydrogel@BTO).

[0047] according to Figure 15 As shown in the figure, dead and live cells exhibit excellent cell compatibility and are evenly distributed throughout the interconnected porous structure under daily ultrasound stimulation, demonstrating that the ultrasound activation protocol does not impair cell viability and results in excellent cell compatibility.

[0048] The specific tests for osteoporosis treatment are as follows: Animal culture: Female SD rats, 3 months old, were housed in the Animal Center of the Beijing Institute of Nanoenergy and Nanosystems in a pathogen-free environment at 25°C. Female SD rats were randomly divided into a sham-operated group and an OVX group.

[0049] OVX group: SD rats underwent bilateral ovariectomy under intraperitoneal injection of 3 ml / kg of 10% chloral hydrate for anesthesia. Three months later, femoral bone density (BMD) was measured by micro-CT.

[0050] Sham-operated group: SD rats were anesthetized by intraperitoneal injection of 3 ml / kg of 10% chloral hydrate, and only adipose tissue was removed. Three months later, femoral bone density (BMD) was measured by micro-CT. The femoral BMD of the OVX group rats was ≤70% of that of the sham-operated group rats, therefore the OVX group rats were diagnosed with osteoporosis.

[0051] The OVX group rats represent a pathological bone microenvironment in postmenopausal osteoporosis, characterized by chronic oxidative stress, impaired osteoblast function, and increased osteoclast activity—conditions where conventional treatments typically fail. We overcame these impaired healing conditions by using ultrasound stimulation to clear ROS and deliver bioactive cues. In our experimental study, critical-sized femoral defects (2 mm in diameter, 3 mm in depth) were created in the osteoporotic OVX group rats, and treatments were administered in four groups, as detailed below: Rats in the OVX group were randomly divided into four groups (n=6 per group): a normal control group, a model group, a PLLA@Ca group, and a PLLA@Ca / Hydrogel@BTO+US group. The normal control group had no femoral defect; the model group had a femoral defect (2 mm in diameter, 3 mm in depth) without implantation of material; the PLLA@Ca group had a PLLA@Ca nanofiber membrane implanted in the femoral defect; and the PLLA@Ca / Hydrogel@BTO+US group had a PLLA@Ca / Hydrogel@BTO composite material implanted in the femoral defect. The rats underwent daily ultrasound stimulation (1 MHz, 1 W / cm²). 2 (5 min), for 8 consecutive weeks.

[0052] Rats with implanted materials had their wounds sutured and were given penicillin (200,000 U / kg, intramuscular) for the first 3 days to prevent infection. After 8 weeks, rats in the normal control group, PLLA@Ca group, model group, and PLLA@Ca / Hydrogel@BTO+US group were anesthetized, and their femurs were scanned (70 kV, 114 μA, 18 μm resolution). Bone mineral density (BMD), BV / TV, Tb, Th (trabecular thickness), and N (trabecular number) in the defect area were analyzed using CTAn software.

[0053] Bone mineral density of rats after 8 weeks of treatment Figure 16 As shown, the therapeutic effect became very significant in the challenging OVX group rats. Eight weeks after implantation, micro-CT scans revealed a significant recovery in bone mineral density (BMD) in the PLLA@Ca / Hydrogel@BTO+US group, indicating that the BMD of rats with the PLLA@Ca / Hydrogel@BTO composite material plus ultrasound stimulation significantly exceeded that of the PLLA@Ca group and the normal control group, approaching that of healthy bone. This demonstrates that the complete PLLA@Ca / Hydrogel@BTO composite material, combined with ultrasound stimulation, effectively overcomes the inherent impaired healing capacity of osteoporosis through electrical stimulation and redox modulation, far more effectively than systems without ultrasound activation.

[0054] Histological analysis: Rats were sacrificed 8 weeks post-surgery, and femoral specimens were obtained. Soft tissue was removed, and the medullary cavity was rinsed with physiological saline to obtain bone tissue. The bone tissue was fixed in 4% paraformaldehyde at 4°C for 48-72 hours. After fixation, the bone tissue was thoroughly rinsed with PBS and then transferred to 10% EDTA decalcification solution (pH 7.4, containing 0.5% PFA) for decalcification at room temperature for 3-4 weeks, changing the decalcification solution twice a week until there was no resistance when puncturing the bone cortex. After decalcification, the bone tissue was rinsed with running water for 24 hours, and then sequentially dehydrated in a gradient of 70%, 80%, 95%, and 100% ethanol (2 hours each), cleared with xylene (twice, 1 hour each), and impregnated with paraffin at 60°C (3 times, 2 hours each), finally embedded and molded. Then, a rotary microtome was used to cut continuous sections with a thickness of 5 μm. After spreading the sections in a 42℃ water bath, they were mounted on glass slides treated with 0.1% poly-L-lysine and baked at 60℃ for 12 hours. The sections were then used for various staining processes.

[0055] H&E staining: After dewaxing and hydration, the sections are stained with hematoxylin for 8-10 minutes, differentiated with 1% hydrochloric acid and ethanol, blued with 0.5% ammonia, stained with 0.5% eosin for 1-2 minutes, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin for observation of new bone formation.

[0056] Masson trichrome staining: After hematoxylin staining, the sections are stained with Ponceau S and acid fuchsin for 5-10 minutes, treated with phosphomolybdic acid for 5 minutes, stained with aniline blue for 5 minutes, and differentiated with 0.2% glacial acetic acid to evaluate collagen deposition.

[0057] TRAP staining: Incubate the sections at 37°C in a solution (containing acid phosphatase substrate, potassium sodium tartrate and Fast Red Violet LB salt, pH 5.0) in the dark for 30-60 minutes, then counterstain with 1% methyl green for 1-2 minutes for observation of osteoclasts.

[0058] Immunohistochemical staining: After dewaxing and hydration, the sections were subjected to antigen retrieval in a 95°C water bath with 0.01 M sodium citrate buffer for 20 minutes. After cooling to room temperature, they were incubated in 3% H2O2 at room temperature for 10 minutes to block endogenous peroxidase. The sections were then blocked in 5% BSA (containing 0.1% Triton X-100) at room temperature for 1 hour. Rabbit anti-BMP2 antibody (1:200) and rabbit anti-Runx2 antibody (1:150) were then added, and the sections were incubated overnight (12-16 hours) at 4°C. After rinsing with PBS, the sections were incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (1:500) at room temperature for 1 hour. DAB staining solution (0.03% DAB + 0.01% H2O2) was used for staining at room temperature for 3-5 minutes, followed by hematoxylin counterstaining for 1 minute. The sections were then dehydrated and mounted.

[0059] Histological quantification: ImageJ software was used to analyze positive cells (n=3). All data are expressed as mean ± standard deviation. One-way ANOVA and Tukey's post-hoc test were used for statistical analysis. P < 0.05 was considered statistically significant.

[0060] according to Figure 17 As shown, H&E staining revealed that rat bone defects treated with the PLLA@Ca / Hydrogel@BTO composite material under ultrasound stimulation were almost completely bridged by well-tissued, mature new bone, characterized by dense, interconnected trabeculae seamlessly integrated with the host bone margin. In stark contrast, the control group exhibited incomplete healing, characterized by persistent fibrous tissue and disordered, immature woven bone, directly reflecting suboptimal mineralization without ultrasound activation.

[0061] Masson trichrome staining further confirmed these findings, revealing abundant, well-arranged collagen deposits in the newly formed matrix under ultrasound activation, indicating strong tissue maturation and mechanical capacity. This high-quality matrix formation is associated with minerals that accelerate nodule maturation, confirming that piezoelectric cues not only drive calcium deposition but also drive organized, functional bone matrix synthesis.

[0062] TRAP staining revealed a significant inhibition of osteoclast numbers, particularly in the ultrasound-activated group, resolving the pathological bone resorption associated with osteoporosis. This anti-catabolic effect is related to the piezoelectric catalytic ROS scavenging mechanism of the PLLA@Ca / Hydrogel@BTO composite: by neutralizing mitochondrial superoxide and H2O2, the PLLA@Ca / Hydrogel@BTO composite disrupts the rankl-induced cascade reaction of osteoclasts amplified in the oxidative microenvironment. Simultaneously, piezoelectric stimulation of osteoblasts generates an anabolic advantage, effectively rebalancing bone remodeling towards net formation. This dual-action therapeutic effect, driven by the combination of ultrasound-activated electrical signals and ROS scavenging, underscores the immense potential of this self-powered strategy in treating challenging osteoporotic bone defects—ultrasound serving as a non-invasive trigger to maximize therapeutic efficacy.

[0063] according to Figure 18 As shown, an ultrasound-stimulated self-powered bone regeneration system based on the "dual piezoelectric synergy and ion-electron amplification" strategy is used for synergistic osteogenic treatment of osteoporotic bone defects.

[0064] In summary, this invention successfully designed a self-powered, integrated bone regeneration PLLA@Ca / Hydrogel@BTO composite material, a bilayer scaffold, for ultrasound-stimulated bone regeneration by synergistically coupling a PLLA@Ca nanofiber membrane (ultrasound-responsive) with an ion-conductive hydrogel loaded with barium titanate (ultrasound-responsive). The layered structure of this PLLA@Ca / Hydrogel@BTO composite material coordinates a multi-layered therapeutic cascade under ultrasound stimulation. The PLLA@Ca nanofiber membrane provides necessary physical support and osteoconductive cues by mimicking the natural bone matrix, while its ultrasound-responsive piezoelectricity generates electrical signals upon ultrasound exposure. The flexible and viscous Hydrogel@BTO ensures seamless integration of the device with the tissue (crucial for stable ultrasound-induced signal transmission) and serves as a secondary ultrasound-responsive piezoelectric module.

[0065] The piezoelectric materials of polylactic acid (PLA) and barium titanate synergistically harvest endogenous mechanical energy induced by ultrasound from physiological activity, converting it into a powerful electric field far more effective than passive piezoelectricity without ultrasound. This potential is then effectively propagated and amplified by the ion-electron network of Hydrogel@BTO, directly establishing a continuous, electrically neutral microenvironment at the defect site. The piezoelectric catalytic generation of low-level reactive oxygen species (ROS) triggered by ultrasound stimulation further enhances this process; these low-level ROS synergistically regulate local redox balance and promote a bone-forming environment.

[0066] Ultrasound stimulation significantly enhanced osteoblast proliferation and differentiation, increased the expression of key osteoblastic proteins, and suppressed osteoclast activity—far exceeding the effects of systems without ultrasound—thus restoring metabolic homeostasis. When applied to a preclinical model of osteoporotic bone defects under ultrasound stimulation, the system significantly accelerated new bone formation, achieving higher bone volume fraction and trabecular density compared to using PLLA@Ca nanofiber membranes alone or PLLA@Ca / Hydrogel@BTO composites without ultrasound. This work pioneers a novel ultrasound-driven, energy-autonomous therapeutic paradigm, circumventing key limitations of conventional electrical stimulation therapies (e.g., external power sources) and passive piezoelectric systems. By providing an effective, self-regulating strategy to reactivate the damaged healing cascade under osteoporotic conditions—ultrasound as a non-invasive method—this dual-ultrasound-responsive piezoelectric system holds great promise for clinical use.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a double-layer scaffold, characterized in that, Includes the following steps: S1 dissolves L-polylactic acid in a solvent, adds calcium salt and mixes to obtain a precursor solution, then electrospins the precursor solution to obtain a fiber membrane; the fiber membrane is dried to obtain a fiber layer of a bilayer scaffold; polyvinyl alcohol, gelatin and polyamide are dissolved in a binary solvent, add barium titanate and mix to obtain a hydrogel solution; S2 places the fiber membrane at the bottom of the mold, then places the hydrogel solution in the mold, and then performs freeze crosslinking to form the hydrogel in the double-layer scaffold. After demolding, the hydrogel in the double-layer scaffold is immersed in salt water to finally obtain the double-layer scaffold.

2. The method for preparing a double-layer scaffold according to claim 1, characterized in that, The mass ratio of L-polylactic acid to calcium salt is 10:1, and the solvent is dichloroethane.

3. The method for preparing a double-layer scaffold according to claim 1, characterized in that, Electrospinning process parameters: voltage 16kV~20kV, receiving distance 13cm~17cm, injection speed 0.8mL / h~1.2mL / h, roller speed 1400rpm~1600rpm, spinning time 4h.

4. The method for preparing a double-layer scaffold according to claim 1, characterized in that, The average diameter of the fiber membrane is 500±30nm, and the pore size of the hydrogel is 10~30um.

5. The method for preparing a double-layer scaffold according to claim 1, characterized in that, The concentration of polyvinyl alcohol is 8 wt%; the concentration of gelatin is 4 wt%; and the concentration of polyamide is 2 wt%.

6. The method for preparing a double-layer scaffold according to claim 1, characterized in that, The concentration of barium titanate is 5 wt%.

7. The method for preparing a double-layer scaffold according to claim 1, characterized in that, The freezing temperature is -18℃ to -22℃, and the freezing time is 24 hours.

8. The method for preparing a double-layer scaffold according to claim 1, characterized in that, The brine is a phosphate-buffered brine, and the soaking time is 24 hours.

9. The method for preparing a double-layer scaffold according to claim 1, characterized in that, The binary solvent is a mixture of glycerol and water, wherein the volume ratio of glycerol to water is 1:

4.

10. A method for preparing a double-layer stent according to any one of claims 1-9.

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