Negative pressure therapy electroactive dressing for promoting bone cell differentiation and bone regeneration and manufacturing method

Abstract

The invention discloses an electroactive dressing used for promoting bone cell differentiation and bone regeneration and used for negative pressure therapy and a manufacturing method. The manufacturing method of the electroactive dressing comprises the steps that S1, a PLLA / BG spinning solution is prepared; s2, preparing a PLLA / BG nanofiber membrane through electrostatic spinning; s3, vacuum drying; s4, PAMAM-NH2 surface modification, the negative pressure treatment dressing relates to the technical field of negative pressure treatment dressings, when mechanical deformation is generated during negative pressure work, the piezoelectric biological film generates continuous electric stimulation, and the electric stimulation has positive and beneficial effects on osteoblasts and bone tissue physiological processes. The generated electric effect continuously collects functional electrolyte in surrounding tissues, and the functional electrolyte is separated in the wound by the dendrimer PAMAM-NH2, so that excessive exudation caused by negative pressure treatment is effectively made up. Meanwhile, the flow of the electrolyte further strengthens a local electric signal, so that cell migration and osteoblast proliferation and differentiation are effectively promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of negative pressure therapy dressings, and in particular to an electroactive dressing for negative pressure therapy for promoting bone cell differentiation and bone regeneration and a preparation method thereof. Background Art

[0002] The piezoelectric effect is an important component of bioelectricity in tissues. Tissue piezoelectricity refers to the phenomenon that when tissues containing polar molecules and orderly arranged fibrous structures (such as collagen, microtubules, etc.) are subjected to mechanical stress, electrical signals can be generated. When an open fracture occurs, the piezoelectric network structure of the local tissue of the trauma is often destroyed, resulting in the loss of bioelectric signals. As an important biophysical signal, bioelectricity is widely present in various cells and tissues of the human body and is crucial to wound healing. It promotes cell migration, proliferation and differentiation through the action of electric fields, and plays an important role in all stages of wound repair. Changes in the local bioelectric environment limit the physiological functions of cells, thereby affecting the healing process of bones and tissues.

[0003] Negative pressure therapy has been widely used in the clinical treatment of open fractures and complex traumas due to its significant advantages in wound sealing, drainage, and promoting blood circulation. However, although negative pressure therapy can effectively treat wounds, in some cases, it fails to fully mobilize and maintain the bioelectric environment required for healing, thereby affecting the efficiency of tissue repair. At the same time, the absorption of tissue fluid and electrolyte loss in the wound site caused by negative pressure drainage is one of the main factors that damage the local electrical microenvironment. Under the action of negative pressure, the tissue fluid in the wound area is continuously drained, resulting in sodium ions (Na + ), potassium ion (K + ), calcium ions (Ca 2+ ) and other key electrolytes. These ions are not only key players in the generation of transmembrane potential and action potential, but their flow gradient distribution also enables efficient transmission of electrical signals between tissue cells. The loss of these functional electrolyte ions further exacerbates the loss of the electric field at the site of injury, adding to the burden on the bone and tissue healing process.

[0004] At present, there have been studies to restore the loss of local electric fields caused by factors such as trauma. On the one hand, researchers have considered supplementing the exogenous electric field to remedy the lost endogenous electric field. Commonly used methods include exogenous electrical stimulation therapy, conductive or piezoelectric biomaterials (such as biobatteries, conductive polymers, piezoelectric nanomaterials, etc.). On the other hand, for the loss of electrolyte ions in tissue fluid caused by drainage during negative pressure therapy, researchers have introduced electrolytes exogenously, such as ion-loaded conductive hydrogels. In short, current studies have mainly focused on "exogenous supplementation" and ignored physiological maintenance, which can hardly serve as an effective guide for clinical intervention strategies.

[0005] To replenish the missing electric field in the wound area, researchers have attempted to utilize exogenous electrical stimulation therapy and conductive or piezoelectric biomaterials (such as biobatteries, conductive polymers, and piezoelectric nanomaterials). For example, piezoelectric materials are widely used in tissue engineering due to their excellent biocompatibility, superior self-generating properties, multifunctionality, and adjustability. However, the formation and maintenance of bioelectricity is a complex physiological process, involving the synergistic interaction of multiple factors such as cells, molecules, and the external environment. A single material often fails to fully recreate the complex bioelectric field. To replenish the missing electric field caused by excessive electrolyte loss, ion-conductive hydrogels, previously used in flexible electronic devices such as capacitors, have garnered widespread attention due to their excellent flexibility and high conductivity. However, these technologies still face many challenges, primarily the difficulty in controlling the release rate, carrier instability, and low release efficiency. Particularly during wound healing, single exogenous electrolyte replenishment methods struggle to effectively restore local electrolyte balance and may fail to meet the complex electric field requirements of wound healing. In summary, even if the electric field supplemented by exogenous biomaterials can numerically make up for the missing electric field, the efficient conduction of bioelectricity depends on sufficient conductive media. Without sufficient electrolytes and ion gradients, the piezoelectric signal is difficult to effectively transmit to the wound repair site, resulting in delayed tissue healing. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention provides an electroactive dressing and a production method for negative pressure therapy for promoting bone cell differentiation and bone regeneration, which solves the problem that the efficient conduction of bioelectricity in the existing negative pressure therapy process depends on sufficient conductive medium. Without sufficient electrolytes and ion gradients, the piezoelectric signal is difficult to effectively transmit to the wound repair site, resulting in delayed tissue healing.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: an electroactive dressing for negative pressure therapy to promote bone cell differentiation and bone regeneration and a method for making the electroactive dressing, the method for making the electroactive dressing comprising:

[0008] S1: Preparation of PLLA / BG spinning solution;

[0009] S2: Preparation of PLLA / BG nanofiber membrane by electrospinning;

[0010] S3: vacuum drying;

[0011] S4: PAMAM-NH2 surface modification.

[0012] Preferably, the PLLA / BG spinning solution is prepared as follows: 1.2 g of PLLA powder is weighed, 10 mL of hexafluoroisopropanol is added, and the mixture is stirred at room temperature for 6 h using a magnetic stirrer until the PLLA is completely dissolved to obtain a 12 wt% PLLA solution, 0 mg, 60 mg, 120 mg, and 180 mg of BG particles are weighed respectively, corresponding to BG mass fractions of 0 wt%, 5 wt%, 10 wt%, and 15 wt%, and the BG particles are added to the PLLA solution. Ultrasonic dispersion is performed at a power of 300 W for 30 min to ensure that the BG is evenly dispersed to obtain a PLLA / BG mixed solution.

[0013] Preferably, the electrospinning method for preparing PLLA / BG nanofiber membrane is as follows: the PLLA / BG mixed solution is loaded into a 10 mL syringe, installed in an electrospinning device, and the parameters are set as voltage 18 KV, propulsion rate 1.5 mL / h, collection distance 18 cm, ambient temperature 25 ° C, relative humidity 40%, and the spun fibers are collected on aluminum foil. The spinning time is 2 h to obtain a uniform PLLA / BG nanofiber membrane.

[0014] Preferably, the vacuum drying is as follows: placing the PLLA / BG nanofiber membrane in a vacuum drying oven, setting the temperature to 40° C., the vacuum degree to <10 mbar, and drying for 24 hours to remove the residual solvent.

[0015] Preferably, the PAMAM-NH2 surface modification is as follows: 19.2 mg EDC and 11.5 mg NHS are weighed and dissolved in 1 mL PBS solution, the PLLA / BG nanofiber membrane is immersed in the EDC / NHS solution, reacted at room temperature for 2 hours to activate the surface carboxyl groups of the PLLA / BG nanofiber membrane, a 0.5 mg / mL-1 mg / mL PAMAM-NH2 PBS solution is prepared, the activated PLLA / BG nanofiber membrane is immersed in the PAMAM-NH2 solution, reacted at room temperature for 12 hours to graft PAMAM-NH2, after the reaction is completed, the PLLA / BG nanofiber membrane is washed with deionized water 3 times, each time for 10 minutes, and the PLLA / BG nanofiber membrane is placed in a vacuum drying oven and dried at 40°C for 24 hours to obtain a PLLA / BG / PAMAM-NH2 composite spinning membrane.

[0016] Beneficial effects

[0017] The present invention provides an electroactive dressing for negative pressure therapy to promote osteoblast differentiation and bone regeneration and a method for making the same, which has the following beneficial effects:

[0018] 1. When negative pressure creates mechanical deformation, the piezoelectric biofilm generates continuous electrical stimulation, which has a positive impact on the physiological processes of osteoblasts and bone tissue. This electrical effect continuously recruits functional electrolytes from the surrounding tissue, which are sequestered within the wound by the dendritic macromolecule PAMAM-NH2, effectively compensating for the excessive exudation caused by negative pressure therapy. Simultaneously, the flow of electrolytes further strengthens local electrical signals, effectively promoting cell migration and osteoblast proliferation and differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the preparation process of the PLLA / BG / PAMAM-NH2 electroactive dressing of the present invention.

[0020] Figure 2 The SEM, CLSM, and EDS images of the electroactive dressing of the present invention and a schematic diagram of the element composition and content of each group.

[0021] Figure 3 The figure shows the contact angle images of water and diiodomethane and the schematic diagram of the quantitative analysis of the contact angles of water and diiodomethane and the comparative analysis of surface energy.

[0022] Figure 4 Comparison of the extensibility of each group of electroactive dressings of the present invention.

[0023] Figure 5 The diagram shows the relationship between stress and deformation of each group of materials in the present invention and a statistical comparison of Young's modulus of each group of dressings.

[0024] Figure 6 The diagram shows the voltage intensity generated by the electroactive dressing of the present invention after mechanical stimulation, as well as a comparison of the voltage intensity generated by the electroactive dressing under different power mechanical stimulation intensities; the diagram shows the current intensity generated by the electroactive dressing after mechanical stimulation, as well as a comparison of the current intensity generated by the electroactive dressing under different power mechanical stimulation intensities.

[0025] Figure 7 The XPS spectra of each group of dressings of the present invention are shown, and the PBS-added group verifies the ion adsorption capacity of the PLLA / BG / PAMAM-NH2 electroactive dressing, as well as the XRD patterns and infrared spectra of each group of dressings.

[0026] Figure 8 Schematic diagram of the in vitro Transwell co-culture system of the present invention, a CCK-8 experiment evaluating the effects of different concentrations of bioactive glass on cell proliferation, and a schematic diagram of the experimental evaluation of the differences in cell proliferation in different groups after the addition of PLLA.

[0027] Figure 9Schematic diagram of live / dead cell staining performed on days 1, 3, and 5 in the present invention and quantitative analysis of the number of live cells in each group using ImageJ software, as well as cytoskeleton and nuclear staining.

[0028] Figure 10 Schematic diagram showing the comparison of alkaline phosphatase staining at 7 days, alizarin red staining at 21 days, and statistical analysis of the average optical density of alkaline phosphatase and alizarin red staining in each group of the electroactive dressing experiment of the present invention.

[0029] Figure 11 Schematic diagram of qRT-PCR analysis of the mRNA expression levels of osteogenesis-related genes such as Alp, Runx2, Ocn, Opn, and Osx in the present invention.

[0030] Figure 12 This is a schematic diagram of Runx2 immunofluorescence staining (green represents the cytoskeleton, blue represents the cell nucleus, and red represents Runx2 fluorescence) of each experimental group after 14 days of osteogenic differentiation in the present invention and statistical analysis comparison of Runx2 fluorescence intensity of each experimental group.

[0031] Figure 13 Schematic diagram of Western blotting analysis of the osteogenesis-related genes Runx2 and Ocn protein expression levels and statistical analysis of the grayscale values ​​of the corresponding experimental groups.

[0032] Figure 14 The invention provides a rat skull critical bone defect model construction, treatment mode and time arrangement.

[0033] Figure 15 This is a statistical analysis and comparison of the electrolyte concentrations (Na+, K+, Ca2+) in the skull defect site of rats in the present invention.

[0034] Figure 16 This is a statistical analysis comparison of wound potentials during conventional negative pressure therapy and conventional treatment within 24 hours of the present invention, and a statistical analysis of changes in wound potential when conventional negative pressure therapy is added and removed with electroactive dressings.

[0035] Figure 17 The present invention is used to treat the electroactive dressing for 4 and 8 weeks. The growth status of the defect site after Micro-CT three-dimensional reconstruction of the skull model and the statistical analysis and comparison of Micro-CT bone volume fraction (BV / TV) and bone mineralization density (BMD) are compared.

[0036] Figure 18 H&E staining and Masson staining of skull model tissue sections after 4 and 8 weeks of treatment with the electroactive dressing of the present invention (black arrows represent inflammatory cells, blue and green represent host bone, and yellow represents new bone).

[0037] Figure 19The Runx2 and Ocn immunohistochemical staining of skull bone model tissue sections after 4 and 8 weeks of treatment with the electroactive dressing of the present invention, as well as the statistical analysis of the immunohistochemical activation areas were compared. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] See also Figure 1-2 The present invention provides a technical solution: an electroactive dressing for negative pressure therapy to promote bone cell differentiation and bone regeneration and a preparation method thereof. The preparation method of the electroactive dressing comprises:

[0040] S1: Preparation of PLLA / BG spinning solution;

[0041] S2: Preparation of PLLA / BG nanofiber membrane by electrospinning;

[0042] S3: vacuum drying;

[0043] S4: PAMAM-NH2 surface modification.

[0044] Furthermore, the PLLA / BG spinning solution was prepared as follows: 1.2 g of PLLA powder was weighed, 10 mL of hexafluoroisopropanol was added, and the mixture was stirred at room temperature for 6 h using a magnetic stirrer until the PLLA was completely dissolved to obtain a 12 wt% PLLA solution. 0 mg, 60 mg, 120 mg, and 180 mg of BG particles were weighed, corresponding to BG mass fractions of 0 wt%, 5 wt%, 10 wt%, and 15 wt%. The BG particles were added to the PLLA solution, and ultrasonic dispersion was performed at a power of 300 W using an ultrasonic disperser for 30 min to ensure that the BG was evenly dispersed to obtain a PLLA / BG mixed solution.

[0045] Furthermore, the electrospinning method for preparing PLLA / BG nanofiber membrane is as follows: the PLLA / BG mixed solution is loaded into a 10 mL syringe, installed in an electrospinning device, and the parameters are set as voltage 18 KV, propulsion rate 1.5 mL / h, collection distance 18 cm, ambient temperature 25°C, relative humidity 40%, and the spun fibers are collected on aluminum foil. The spinning time is 2 h to obtain a uniform PLLA / BG nanofiber membrane.

[0046] Furthermore, the vacuum drying is as follows: placing the PLLA / BG nanofiber membrane in a vacuum drying oven, setting the temperature to 40° C., the vacuum degree to <10 mbar, and drying for 24 hours to remove the residual solvent.

[0047] Furthermore, the PAMAM-NH2 surface modification is as follows: 19.2 mg EDC and 11.5 mg NHS are weighed and dissolved in 1 mL PBS solution, and then the PLLA / BG nanofiber membrane is immersed in the EDC / NHS solution, reacted at room temperature for 2 hours to activate the surface carboxyl groups of the PLLA / BG nanofiber membrane, and a 0.5 mg / mL-1 mg / mL PAMAM-NH2 PBS solution is prepared, and the activated PLLA / BG nanofiber membrane is immersed in the PAMAM-NH2 solution, reacted at room temperature for 12 hours to graft PAMAM-NH2. After the reaction is completed, the PLLA / BG nanofiber membrane is washed with deionized water 3 times, each time for 10 minutes, and then the PLLA / BG nanofiber membrane is placed in a vacuum drying oven and dried at 40°C for 24 hours to obtain a PLLA / BG / PAMAM-NH2 composite spinning membrane.

[0048] Characterization and performance verification of electroactive dressings:

[0049] ①SEM observation

[0050] The PLLA / BG / PAMAM-NH2 film was cut into 5 mm × 5 mm samples and gold-sprayed (thickness 5 nm).

[0051]

[0052] The surface morphology was observed using SEM with an accelerating voltage of 10 kV and a working distance of 10 mm.

[0053] ②CLSM observation

[0054] The sample was immersed in a 0.1 mg / mL FITC-labeled PAMAM-NH2 solution for 30 min, washed, and dried. Fluorescence distribution was observed using CLSM with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0055] ③EDS analysis

[0056] The elemental composition was analyzed by EDS with an accelerating voltage of 15 kV and an acquisition time of 60 s.

[0057] ④Contact angle and surface energy test

[0058] The samples were fixed to the contact angle measuring instrument platform. 2 μL of deionized water and diiodomethane were added, respectively. The contact angles were recorded, with five measurements per sample group taken as the average. Surface energy was calculated using the Owens-Wendt equation, where γL is the liquid surface tension, θ is the contact angle, and γSd and γSp are the dispersive and polar components of the material's surface energy.

[0059] The Owens-Wendt equation calculates the surface energy:

[0060] ⑤ Mechanical properties test

[0061] The samples were cut into 10 mm × 50 mm strips with a thickness of 0.1 mm. Tensile tests were performed using a universal material testing machine (Instron, 5967) at a tensile rate of 10 mm / min, and stress-strain curves were recorded.

[0062] The elastic modulus is calculated from the linear portion of the stress-strain curve, where E is the elastic modulus, Δσ is the change in stress, and Δ is the change in strain:

[0063] ⑥Material discharge performance under ultrasonic stimulation

[0064] The sample was immersed in deionized water and placed in an ultrasonic stimulation device with an ultrasonic power of 50 W, a frequency of 20 kHz, and a stimulation time of 10 minutes. A digital potentiometer was used to measure the short-circuit voltage and open-circuit current of the electroactive dressing under ultrasonic mechanical loading.

[0065] ⑦XPS analysis

[0066] The sample was cut into 5 mm × 5 mm and the surface chemical composition was analyzed by XPS with the following parameters: AlKα radiation (1486.6 eV), energy resolution 0.5 eV, and scanning range 0–1200 eV.

[0067] ⑧XRD analysis

[0068] Use XRD to analyze the crystal structure, setting parameters: CuKα radiation The scanning range is 10°–80°, with a step size of 0.02°.

[0069] ⑨FTIR analysis

[0070] Analyze chemical structure using FTIR with the following parameters: wave number range 4000–400 cm -1 , resolution 4cm

[0071]

[0072] -1, scan times 32 times.

[0073] Surface properties of electroactive dressings:

[0074] The results of the electroactive dressing showed that PLLA and BG were smooth and randomly arranged, with uneven distribution, forming a spider web structure, which is beneficial for cell attachment and growth. Inhomogeneous granular substances were seen on the surface of the electrospun fibers of the PLLA / BG / PAMAM-NH2 group, indicating that the dendritic macromolecules were effectively distributed on the surface of the spinning membrane. At the same time, confocal microscopy showed that the surface of the material was uneven, which helped to enhance cell adhesion and proliferation. The uneven surface and color differences of the material together reflected the diversity of the microstructure of the composite material and the heterogeneity of its component distribution. Energy dispersive X-ray spectroscopy showed the spatial distribution of different elements on the surface of the material, including oxygen (O), carbon (C), calcium (Ca), silicon (Si), nitrogen (N), etc. Quantitative analysis of each group of elements more intuitively displayed the content of each element.

[0075] The water contact angles of PLLA, PLLA / BG, and PLLA / BG / PAMAM-NH2 were 132°±2.5°, 114°±1.9°, and 79.8°±3.1°, respectively. The diiodomethane contact angles were 44.9°±0.9°, 36.6°±0.6°, and 23.4°±1°, respectively. The contact angles of PLLA / BG / PAMAM-NH2 were lower than those of pristine PLLA and PLLA / BG, indicating that the dendrimer coating enhanced the surface hydrophilicity of the pristine spun membrane. Furthermore, the surface energy of the electroactive dressing was significantly increased with the addition of the dendrimers. Higher surface energy indicates stronger wettability and interaction with the surrounding environment. Therefore, the electroactive dressing has a positive effect on cell adhesion.

[0076] Mechanical properties, piezoelectric properties and structural characteristics of electroactive dressings:

[0077] The mechanical ductility of piezoelectric materials is a key factor influencing their stress-electric coupling efficiency. When subjected to mechanical tension, the material must exhibit both good toughness and ductility while also generating enhanced discharge efficiency under minimal mechanical force. First, we conducted mechanical property tests on electroactive dressing samples. As shown in the figure, PLLA exhibits significant brittleness, while PLLA / BG exhibits semi-ductility. After incorporating the dendrimer PAMAM-NH2 coating, the sample exhibited improved ductility.

[0078] During the material strain process, PLLA showed a high initial stress (approximately 200 MPa) but a low strain, indicating that it has high rigidity and lacks significant ductility during stretching. In the presence of bioglass, the tensile stress decreased compared to pure PLLA (maximum approximately 50 MPa), and the curve showed a larger strain value, indicating that it has greater toughness and elongation ability than pure PLLA during stretching. After the introduction of PAMAM-NH2, the electroactive dressing exhibited a lower tensile stress, indicating that the addition of PAMAM-NH2 enhanced the plasticity of the material. These results indicate that the dendrimer effectively improved the mechanical properties of the electroactive dressing. Quantitative analysis of Young's modulus showed that the modulus of the dressing decreased significantly after the addition of the dendrimer PAMAM-NH2, indicating that its rigidity decreased and the material was more easily deformed under external force, resulting in a higher electrical energy conversion rate.

[0079] When testing the electroactive dressing PLLA / BG / PAMAM-NH2, the open-circuit voltage was 0.549 V and the short-circuit current was 2.386 μA. Polyrotaxane can effectively regulate the charge accumulation and release process during piezoelectric material discharge, improving the stability of charge transfer. Furthermore, during the piezoelectric material discharge process, polyrotaxane can adjust the charge storage and release rates, achieving more efficient energy conversion. After the addition of polyrotaxane, the electroactive dressing exhibited an open-circuit voltage of 0.574 V and a short-circuit current of 2.875 μA, demonstrating high discharge efficiency. When the ultrasonic power was gradually increased to 2.0 W, the open-circuit voltage and short-circuit current reached 0.858 V and 3.516 μA, respectively, representing increases of 1.56 and 1.49 times, respectively. This indicates that under the action of a periodic constant force, the open-circuit voltage and short-circuit current increase with increasing mechanical force.

[0080] XPS (Extensive Photonic Scanning) was used to identify the relative contents of the major elements on the surface of the electroactive dressing. The results showed that the piezoelectric PLLA element primarily exhibited C1s and O1s peaks, including C–C (~284.8 eV), C–O (~286.5 eV), and O–C=O (~288.5 eV). In contrast, the addition of bioglass to the spun membrane revealed Ca2p and Si2p peaks. When the spun membrane coated with PAMAM-NH2 dendrimers was immersed in PBS buffer, the scan revealed Na1s and K2p peaks, indicating that Na and K ions in the buffer were adsorbed and accumulated on the membrane surface, confirming the electroactive dressing's ability to capture ions and prevent excessive electrolyte loss.

[0081] XRD analysis of the electroactive dressing's crystal structure revealed that PLLA, PLLA / BG, and PLLA / BG / PAMAM-NH2 all exhibited irregular, jagged curves rather than sharp, smooth diffraction peaks, indicating that the electroactive dressings were semicrystalline and exhibited a degree of disorder in the crystal structure. Low crystallinity allows for easier deformation and flow during processing, which is beneficial for the fabrication of electrospun fibers. Furthermore, low-crystallinity materials may exhibit improved solubility, flexibility, or biocompatibility. The diffraction peaks of PLLA and PLLA / BG were similar, with a broad peak in the 15°-20° region, but the overall crystallinity remained low. The addition of PAMAM-NH2 resulted in multiple sharp peaks, particularly near 15° and 30°, suggesting that the PAMAM-NH2 dendrimers promoted crystallization, resulting in a more ordered crystal arrangement. These changes in the peak spectrum indicate improved material stability, as well as enhanced toughness and durability.

[0082] FTIR results showed that the PLLA / BG spun membrane showed a peak at 1750 cm -1 The C=O stretching vibration peak and 1180cm -1 The C–O stretching vibration peak is a characteristic of PLLA, indicating that the material contains a large number of ester groups. The Si-O stretching vibration peak in bioactive glass is usually located at about 1000-1200 cm -1 and 500-600cm -1 In addition, at 3400cm -1 N–H stretching peak, 1600 cm -1 The N–H bending peak and 1100–1200 cm -1 The C–N stretching peaks of PAMAM-NH2 demonstrate that PAMAM-NH2 has been attached to the surface of the PLLA / BG composite.

[0083] Verification of biosafety and proliferation performance of electroactive dressings:

[0084] 1. Methods for verifying the biosafety and value-added performance of electroactive dressings

[0085] ① Bioglass concentration detection

[0086] BMSCs were seeded in the lower chamber of a Transwell chamber, and a spinning membrane containing varying concentrations of bioglass (0 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL) was added to the upper chamber. Mechanical energy was imparted to the Transwell chamber using an ultrasound probe at an intensity of 1.0 W / cm² for 3 minutes twice daily. After 24 hours of culture, 10 μL of CCK-8 reagent was added to each well, and the cells were cultured for an additional 2 hours. The absorbance was measured at 450 nm using a microplate reader.

[0087] ②Cell proliferation assay

[0088] BMSCs were seeded in the lower chamber of a Transwell chamber. The upper chamber was filled with the following spun membranes: blank control (no material), PLLA / BG group, PLLA / BG / PAMAM-NH2 group, PLLA / BG + US group, and PLLA / BG / PAMAM-NH2 + US group. After 24 hours of stimulation with the same ultrasound intensity and duration, 10 μL of CCK-8 reagent was added to each well and cultured for an additional 2 hours. The absorbance was measured at 450 nm using a microplate reader.

[0089] ③ Live and dead cell staining

[0090] BMSCs were seeded in the lower chamber of a Transwell chamber, and the corresponding grouped spunbond membranes were added to the upper chamber for culture for 1, 3, and 5 days. Live-dead staining solution was prepared with 2 μM calcein-AM and 4 μM EthD-1 in PBS. The culture medium was aspirated, and 200 μL of the staining solution was added to each well. The cells were incubated at 37°C in the dark for 30 minutes. Observation was performed using a fluorescence microscope.

[0091] ④Cytoskeleton / DAPI staining

[0092] BMSCs were seeded in the lower chamber of a Transwell chamber, and the corresponding grouped spun membrane was added to the upper chamber and cultured for 5 days. The culture medium was aspirated, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, and permeabilized with 1 ml of immunostaining permeabilization solution for 10 minutes, and washed three times with PBS. Actin-Tracker Green was diluted 1:40-200 with PBS, 2 ml per well, incubated in the dark for 30 minutes, and washed three times with PBS. 500 μL of DAPI was added to each well, incubated in the dark for 5 minutes, and washed three times with PBS. Observed using a fluorescence microscope.

[0093] 2. Experimental Results

[0094] ①Cell proliferation assay

[0095] We conducted in vitro experiments to determine the effects of the optimal bioglass concentration and electroactive dressing on cell proliferation in order to evaluate the biocompatibility of the biomaterial. We co-cultured the material with bone marrow mesenchymal stem cells (BMSCs) using a Transwell system and stimulated them with ultrasound. Studies have shown that the ultrasound intensity that can trigger the piezoelectric material to produce an electrical effect is 1.0 W / cm-2, 3 minutes each time, twice a day

[16] . First, the effects of different concentrations of bioglass (0.5, 1.0, 1.5 mg / mL) on BMSC proliferation were tested by CCK-8 experiments to determine the optimal bioactive concentration of bioactive glass in the electrospinning state. The results showed that 1.0 mg / mL of bioglass significantly promoted the proliferation of BMSCs, which was significantly better than other concentrations. Therefore, we used 1.0 mg / mL of bioactive glass as the basis and composited it with L-polylactic acid (PLLA) to produce an electrospun film, and then made an amine-terminated dendrimer (PAMAM-NH2) coating to form an electroactive dressing. We further evaluated the effect of electroactive dressing combined with ultrasound stimulation on BMSCs proliferation. CCK-8 test results showed that the electroactive dressing combined with ultrasound stimulation group significantly stimulated BMSCs proliferation compared with other experimental groups.

[0096] Live-dead staining experiments on days 1, 3, and 5 further demonstrated that the BMSCs in the electroactive dressing combined with ultrasound stimulation group exhibited the best growth trend, with virtually no dead cells observed. Live cell counts also yielded the same conclusion. Furthermore, cytoskeleton and nuclear staining of cells co-cultured for 5 days revealed that the BMSCs in the electroactive dressing combined with ultrasound stimulation group exhibited favorable morphology, with significantly enhanced cell expansion and alignment.

[0097] Ultrasound-stimulated electroactive dressing promotes osteogenic differentiation in vitro:

[0098] 1. Experimental Methods for Promoting Osteogenic Differentiation with Electroactive Dressings in Vitro

[0099] ①ALP and ARS staining

[0100] BMSCs were seeded in the lower chamber of a Transwell microplate and cultured with α-MEM for 3 days. When the cell population reached 90%, the cells were digested with TRYPLE (500 μL / well) for 3 minutes, the culture medium was collected, and centrifuged (1000 rpm, 5 minutes) to remove cell debris. 2 mL of osteogenic induction medium was added and plated at 5 × 104 cells per well in a 6-well plate. After 14 days of culture, ALP staining was performed as follows: the culture medium was aspirated, the cells were washed three times (5 minutes each) with PBS, fixed with 4% paraformaldehyde solution for 30 minutes, and washed three times (5 minutes each) with PBS. The alkaline phosphatase working solution was prepared according to the instructions of the alkaline phosphatase staining kit, and 2 mL / well was incubated in the dark for 20 minutes. The cells were then rinsed three times (5 minutes each) with distilled water and observed under a microscope. After 14 days of culture, ARS staining was performed as follows: the culture medium was aspirated, the cells were washed three times with PBS, the staining working solution was prepared using the Alizarin Red staining kit, 200 μL of ARS staining solution was added to each well, the cells were incubated at room temperature for 30 min, and the cells were washed three times with PBS, 5 min each time, and observed under a microscope.

[0101] ②Osteogenic gene qPCR analysis

[0102] RNA was extracted using TRIzol reagent: The entire experiment was performed on ice. 1 mL of TRIzol was added to each well, and the cells were lysed for 5 minutes. 200 μL of chloroform was added, and the cells were shaken for 15 seconds, allowed to stand for 10 minutes, and centrifuged (12,000 rpm, 15 minutes, 4°C). The upper transparent liquid was removed, and an equal volume of isopropanol was added. The cells were shaken for 15 seconds, allowed to stand for 10 minutes, and centrifuged (12,000 rpm, 15 minutes, 4°C). The supernatant was discarded, and 1 mL of 75% ethanol was added. The cells were shaken for 15 seconds, allowed to stand for 10 minutes, and centrifuged (12,000 rpm, 15 minutes, 4°C). The supernatant was discarded, and the cell suspension was dissolved in 20 μL of DEPC water after air drying.

[0103] Detect RNA concentration using a microplate reader.

[0104] Use a reverse transcription kit to synthesize cDNA: prepare 20 μL of the system reaction solution according to the kit instructions and place it in a PCR instrument for reaction. The reaction program is: 50°C for 15 minutes, 85°C for 5 seconds.

[0105] Gene expression was detected using a qPCR kit: reaction system: 10 μL SYBR Green Master Mix, 0.5 μL cDNA, 1.2 μL primers, 8.5 μL DEPC water; reaction procedure: 95°C for 30 s, 95°C for 10 s, 60°C for 30 s, 40 cycles of 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. Real-time fluorescence quantitative PCR analysis was used.

[0106] Primer sequences of rat osteoblast differentiation-related genes

[0107]

[0108] ③ Runx2 immunofluorescence staining

[0109] BMSCs were seeded in the lower chamber of the Transwell chamber, and 2 mL of osteogenic induction medium was added. The medium was changed every 3 days. After 14 days of culture, the medium was discarded and the cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 30 minutes. The cells were washed with PBS 3 times, 5 minutes each time, and 1 ml of immunostaining permeabilization solution was added to the cells for 15 minutes at room temperature. After washing with PBS, the cells were quickly blocked with protein-free antibody for 30 minutes at room temperature. The Runx2 primary antibody was incubated with the cells overnight at 4°C. After washing, the secondary antibody was incubated at room temperature in the dark for 1 hour. After washing again, the cells were stained with DAPI solution at room temperature in the dark for 10 minutes to stain the cell nuclei. After sealing, the cells were observed under a fluorescence microscope. The Runx2 fluorescent signal was marked in red, and the DAPI-stained cell nuclei were marked in blue.

[0110] ④ Western Blot experiment of Runx2 and OCN protein expression

[0111] BMSCs were cultured at a rate of 1 × 10 4 Cells were seeded at a density of 10 cells / well in the lower chamber of a Transwell chamber and 2 mL of osteogenic induction medium was added. Runx2 and OCN protein expression was assessed by Western blotting on day 14. The specific steps were as follows: Protein extraction was performed using RIPA lysis buffer: 200 μL of RIPA lysis buffer was added to each well, lysed on ice for 10 minutes, centrifuged (12,000 rpm, 15 minutes, 4°C), and the supernatant was collected. One-quarter volume of instant protein loading buffer was added, and the proteins were denatured at 95°C for 5 minutes.

[0112] The SDS-PAGE gel was prepared using a one-step PAGE gel rapid preparation kit, and electrophoresis was performed using Tris-glycine-SDS electrophoresis buffer and DD water. The electrophoresis was performed at 80V for 25min, then switched to 120V for 40min. The eBlot TMThe L1 Rapid Wet Blotter transfers proteins to a PVDF membrane according to the program based on protein size. Cut designated bands based on the molecular weight of the protein being detected and block with protein-free rapid blocking buffer at room temperature for 40 minutes. Wash three times with 1×TBST wash buffer for 10 minutes. Then, prepare the primary antibody at a 1:1000 ratio using universal antibody diluent and incubate at 4°C overnight. The next day, wash the membrane three times with 1×TBST wash buffer and add the secondary antibody prepared at a 1:1000 ratio. Incubate at room temperature for 2 hours. Wash the membrane again three times with 1×TBST. Add the Omni-ECL™ Ultra-Sensitive Chemiluminescent Detection Kit for ECL chemiluminescence development, and then capture images for analysis.

[0113] 2. Experimental Results

[0114] ①ALP and ARS staining

[0115] We used a Transwell system to co-culture induced osteoblasts with various materials and stimulated them accordingly. Alkaline phosphatase (ALP) staining was performed 7 days later, and Alizarin red (ARS) staining and activity assays were performed 21 days later. The experimental groups stained darker than the control group, and the electroactive dressing combined with ultrasound stimulation exhibited the best cell morphology. This finding was confirmed by quantitative analysis of the optical density of ALP and ARS staining.

[0116] Quantitative analysis of osteogenic gene expression by qRT-PCR

[0117] We used qRT-PCR to measure the expression of Alp, Runx2, Osx, Ocn, and Opn genes during each stage of osteogenic differentiation under mechanical stimulation of the electroactive dressing. The results showed that the expression of Alp, Runx2, Osx, Ocn, and Opn was significantly higher in the electroactive dressing combined with ultrasound stimulation group than in the other groups. This suggests that mechanical stimulation of the electroactive dressing enhanced the osteogenic differentiation capacity of BMSCs at both the early and late stages of osteogenic differentiation.

[0118] Immunofluorescence staining of osteogenic gene Runx2

[0119] We co-cultured osteoblasts in a Transwell system for 7 days and performed Runx2 immunofluorescence staining. Osteoblasts in the different compositions grew well and exhibited differential osteogenic differentiation. The electroactive dressing group exhibited stronger fluorescence under ultrasound stimulation, and analysis of fluorescence intensity further confirmed its osteogenesis-promoting effect.

[0120] Western blotting analysis of osteogenic protein expression.

[0121] To further validate the expression of osteoblastic proteins, osteoblast proteins were extracted and analyzed by western blotting (WB) after 7 and 14 days of co-culture of BMSCs and the materials. The expression of Runx2 and Ocn was measured and their grayscale values ​​were quantitatively analyzed. Compared with the control group, the expression of osteoblast proteins in all experimental groups was significantly increased. Grayscale analysis of the target bands showed that the expression of osteoblast proteins was highest in the electroactive dressing combined with ultrasound stimulation group. These results further confirmed the electroactive dressing's ability to stimulate osteogenesis.

[0122] Negative pressure therapy restores the endogenous electric field in vivo, thereby promoting osteogenic differentiation and bone regeneration:

[0123] 1. Detection of the restoration of the endogenous electric field in vivo by electroactive dressings under negative pressure therapy

[0124] ① Establishment of a critical bone defect model in rat skull

[0125] Male SD rats weighing 250-300 g were selected as experimental animals. Rats were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital at 1 mL / 100 g body weight. After successful anesthesia, the rats were placed on a 37°C constant temperature operating table, the hair on the top of the skull was removed, and the top skin was disinfected with iodine. A surgical incision of approximately 1.5 cm was made along the midsagittal suture, and the skin and subcutaneous tissue were cut in turn, and the skull was exposed by blunt separation. Critical bone defects were made on both sides of the midcranial suture with a 5 mm diameter trephine drill, taking care to avoid injuring the meninges. The defect was covered with an electroactive dressing, and then covered with a polyurethane foam dressing on the surface of the dressing, fixed with a film, and an opening was reserved at the top of the film for connecting a negative pressure therapy device. The dressing and foam were disinfected and replaced every week after surgery.

[0126] ②Analysis of electrolyte concentration in exudate

[0127] The blank control group, NPWT group, PLLA / BG+NPWT group, and PLLA / BG / PAMAM-NH2+NPWT group were designed to evaluate the dressing's control effect on electrolyte ions. The blank group was also covered with polyurethane foam, but no negative pressure suction was performed. After one day of treatment, the polyurethane foam of each group was collected and placed in 10 ml of phosphate buffer for 3 days. After 3 days, it was placed on an oscillator and shaken at 150 rpm for 48 hours. The supernatant was taken and centrifuged at 3000 rpm for 10 minutes to remove particulate matter. A standard electrolyte solution (Orion940907 (Na + )、940908(K + )、940909(Ca 2+ )) After that, take 1ml of the soaked supernatant and add it to the sample pool, select Na + , K + , Ca 2+ Mg 2+In the detection mode, the concentration of each ion was recorded, and each group of samples was tested three times and the average value was taken.

[0128] ③Measurement of wound potential changes

[0129] Twenty-four hours after surgery, the polyurethane foam was removed, and an Ag / AgCl reference electrode was placed in the normal tissue near the skull. A glass microelectrode was used to touch the center of the defect. The potential was measured and the baseline value was recorded. Data was collected for at least 5 minutes to obtain a stable reading. The wound potential values ​​were measured and recorded at different times, 1, 2, 3, 6, 12, and 24 hours. Similarly, the wound potential values ​​were measured and recorded at the same time points under NPWT alone using the same method.

[0130] Under conventional NPWT treatment, the intra-wound potential value was measured using the above method. An electroactive dressing was placed on the defect site, covered with negative pressure foam and suctioned. Changes in the intra-wound potential were detected and recorded every 0.25 hours according to the above method. After 1 hour of treatment, the electroactive dressing was removed, and the intra-wound potential value of conventional negative pressure treatment was continued to be measured.

[0131] 2. Experimental Methods for Promoting Osteogenic Differentiation and Bone Regeneration in Vivo with Electroactive Dressings Under Negative Pressure Therapy

[0132] ① Imaging evaluation of bone regeneration

[0133] Skull specimens were obtained from rats at 4 and 8 weeks of age, and the skin and tissue covering the skulls were removed. Micro-CT scans (scanning resolution: 10 μm, voltage: 70 kVp, current: 114 μA, exposure time: 300 ms) were performed. 3D reconstruction and bone mass calculations, as well as bone volume fraction and bone density measurements, were performed using Scanco Analysis software. Furthermore, the area of ​​newly formed skull bone at the defect site was compared between groups at each time period.

[0134] ②H&E staining of skull specimens

[0135] Rat skull slice specimens reserved at 4 and 8 weeks were dewaxed according to the above method. Stain with hematoxylin for 5 minutes, rinse with tap water for 5 minutes to remove unbound dye, differentiate with 1% hydrochloric acid alcohol (1% HCl in 70% ethanol) for 3-5 seconds, rinse with distilled water for 30 seconds, return to blue with 0.2% ammonia water for 1 minute, and rinse with tap water for 5 minutes. Stain with 1% eosin Y for 3 minutes, rinse with tap water for 1 minute. Dehydrate again with gradient ethanol solution (80%, 95%, 100% ethanol), and clear with xylene twice, each for 2 minutes. Add neutral gum mounting medium, cover with a coverslip, and dry at room temperature. Observe tissue morphology using an optical microscope.

[0136] ③Masson staining of skull specimens

[0137] After dewaxing, the specimens were stained with hematoxylin for 5 minutes, rinsed with tap water for 5 minutes to remove unbound dye, differentiated with 1% hydrochloric acid alcohol for 10 seconds to remove background staining, rinsed with distilled water for 1 minute, blued with 0.2% ammonia solution for 1 minute, and rinsed with tap water for 5 minutes. The specimens were stained with 1% acid fuchsin for 5 minutes, rinsed with distilled water for 1 minute, soaked in 5% phosphomolybdic acid solution for 10 minutes to enhance collagen fiber staining, stained with 2% aniline blue for 5 minutes, and differentiated with 1% glacial acetic acid for 1 minute. The specimens were dehydrated with graded ethanol solutions (80%, 95%, and 100% ethanol) and cleared with xylene twice for 2 minutes each. A neutral gum mounting medium was added, and the specimens were covered with a coverslip and allowed to dry at room temperature. The tissue morphology was observed using an optical microscope.

[0138] ④ Runx2 and OCN immunohistochemical staining

[0139] Skull sections were removed and treated with xylene twice for 5 minutes each. Dehydrated with graded ethanol, the sections were washed with PBS for 1 minute to deparaffinize and rehydrate. Tris-EDTA (pH 9.0) was used as an antigen retrieval solution. The sections were heated in a 95°C waterbath for 15 minutes, cooled to room temperature, and washed three times with PBS for 5 minutes each. Endogenous peroxidase was blocked by incubation with 3% H₂O₂ for 10 minutes, followed by three washes with PBS for 5 minutes each. The sections were then blocked with 5% goat serum for 30 minutes at 37°C. Anti-Runx2 antibody was added and incubated overnight at 4°C. The sections were washed three times with PBS for 5 minutes each. HRP-conjugated secondary antibody was then added and incubated at 37°C for 30 minutes. The sections were washed again with PBS three times for 5 minutes each. DAB color development solution was added and incubated at room temperature for 5 minutes. The reaction was terminated with running water. Hematoxylin staining was performed for 30 seconds, followed by 5-minute washing with running water, followed by 10-second differentiation with 1% hydrochloric acid and ethanol, followed by 1-minute washing with PBS, followed by 1-minute blueing with 0.2% ammonia solution, followed by 5-minute washing with PBS, and counterstaining with hematoxylin. Dehydrate again with graded ethanol and xylene twice for 2 minutes each time to achieve transparency. Neutral gum mounting medium was added, and coverslips were placed. The sections were allowed to dry at room temperature. OCN immunohistochemical staining was performed in the same manner. After successful staining, the sections were observed under a fluorescence microscope.

[0140] Experimental results:

[0141] A 5mm critical bone defect model was created in rats' skulls. The defect was not completely closed, creating an open fracture model. Furthermore, a negative pressure-driven electrical stimulation device was constructed using polyurethane foam. This device covered the defect in the rat skull fracture and simulated negative pressure therapy. The dressing was removed weekly and replaced with a polyurethane foam dressing after wound disinfection.

[0142] One day after surgery, the wound exudate in the polyurethane foam was soaked in phosphate buffer for 3 days. The exudate was collected and placed in a serum electrolyte analyzer to detect the concentrations of K+, Na+, and Ca2+. Compared with the blank control group, the concentrations of various ions in the wound exudate in the simple negative pressure treatment group were significantly increased. Moreover, after the addition of ordinary piezoelectric dressing, the concentrations of the absorbed ions further increased, indicating that under ordinary negative pressure conditions, a large amount of functional ions are exuded from the open injury site during the acute injury period. When the simple piezoelectric dressing is added, the electrolytes recruited by electrical stimulation are also absorbed, which is extremely detrimental to wound and bone growth. When PAMAM-NH2 is added, the electrolytes at the wound site are adsorbed to the material site, effectively blocking the loss of electrolytes.

[0143] The changes in electrical potential around the wound were detected over 24 hours under conventional bandaging treatment and negative pressure suction treatment. The results showed that the wound potential was approximately 80mv within 24 hours of conventional bandaging treatment, while the wound potential continued to decrease after negative pressure treatment. This suggests that under negative pressure suction conditions, as electrolytes and other exudates are lost, the potential within the wound is significantly suppressed. After the negative pressure-driven electroactive dressing produces an electrical stimulation effect, the potential within the wound is significantly enhanced, and after the device is removed, the potential within the wound returns to its original state. The results suggest that the electroactive dressing is converted into electrical energy after being mechanically stimulated by negative pressure, which has a positive and beneficial effect on the recovery of the potential within the wound.

[0144] Imaging evaluation of electroactive dressing promoting bone regeneration in vivo under negative pressure therapy

[0145] To evaluate the therapeutic efficacy of a negative pressure-driven electrical stimulation device in a rat open skull fracture model, we performed micro-CT scans on skull defect specimens from each group, observing the defect site individually. Reconstructed images revealed minimal new bone growth at the defect site in the blank control group, while the electroactive dressing group exhibited optimal growth. Quantitative analysis of bone volume fraction and bone mineralization density revealed that the electroactive dressing group exhibited significantly higher values ​​than the other groups at the same time point. Analysis and comparison of newly formed bone area also yielded similar results. These results demonstrate that the combined stimulation of exogenous piezoelectric material electrical stimulation and endogenous bioelectricity generated by PAMAM-NH2-locked electrolyte ions significantly enhances bone regeneration.

[0146] Further histological analysis of the skull specimens showed that HE staining and Masson staining showed no significant new bone formation in the blank control group at 4 weeks, and only a small amount of new bone formation at 8 weeks.

[0147] Immunohistochemical staining for Runx2 and OCN, the osteogenic markers, was performed on skull specimens taken at 4 and 8 weeks to assess bone formation. The results showed a significant increase in the positive staining area in the electroactive dressing group compared with the other groups. Quantitative analysis of the positive area yielded the same results. Histological analysis confirmed that mechanical force triggering the electroactive dressing's electrical stimulation plays a more positive role in bone regeneration.

[0148] This solution can be applied to electroactive dressings with piezoelectric properties for negative pressure therapy. When the negative pressure produces mechanical deformation, the piezoelectric biofilm generates continuous electrical stimulation. We have confirmed that this electrical stimulation has a positive and beneficial effect on the physiological processes of osteoblasts and bone tissue. The electrical effect it produces continuously recruits functional electrolytes from the surrounding tissues and is isolated inside the wound by the dendritic macromolecule PAMAM-NH2, effectively compensating for the excessive exudation caused by negative pressure therapy. At the same time, the flow of electrolytes further strengthens the local electrical signals, effectively promoting cell migration and osteoblast proliferation and differentiation. Specifically as follows: 1. The effectiveness of the combination of negative pressure therapy and electroactive dressings was verified through experiments. The two complement each other, solving the problem of the lack of a self-powered system for conventional piezoelectric materials and the disadvantage of excessive suction during negative pressure therapy, thereby optimizing the clinical application of negative pressure devices.

[0149] 2. It was confirmed that the electroactive dressing generates an electric effect through the piezoelectric element PLLA through negative pressure. This exogenous piezoelectric field mobilizes and transports electrolyte ions with electrotaxis, and through the "sealing" effect of PAMAM-NH2, it achieves the coupling of endogenous and exogenous electric fields under the guidance of the exogenous electric field, thereby jointly restoring the endogenous electric field under physiological conditions.

[0150] 3. Electroactive dressings can promote osteoblast differentiation and bone regeneration after restoring the local endogenous electric field.

[0151] By those skilled in the art, the scheme in this case is operated in sequence. The specific operation sequence should refer to the following working principle. The detailed connection means are well-known technologies in the field. The following mainly introduces the working principle and process.

[0152] Example: First, 1.2 g of PLLA powder was weighed, 10 mL of hexafluoroisopropanol was added, and the mixture was stirred at room temperature for 6 h using a magnetic stirrer until the PLLA was completely dissolved to obtain a 12 wt% PLLA solution. 0 mg, 60 mg, 120 mg, and 180 mg of BG particles were weighed, corresponding to BG mass fractions of 0 wt%, 5 wt%, 10 wt%, and 15 wt%. The BG particles were added to the PLLA solution, and ultrasonic dispersion was performed at a power of 300 W for 30 min to ensure that BG was uniformly dispersed to obtain a PLLA / BG mixed solution. The PLLA / BG mixed solution was then loaded into a 10 mL syringe and installed in an electrospinning device. The parameters were set as a voltage of 18 KV, a propulsion rate of 1.5 mL / h, a collection distance of 18 cm, an ambient temperature of 25 ° C, and a relative humidity of 40%. The spun fibers were collected on aluminum foil for 2 h to obtain a uniform PLLA / BG nanofiber membrane. Then, PLLA was added to a 10 mL syringe and the syringe was placed in an electrospinning device. The PLLA / BG nanofiber membrane was placed in a vacuum drying oven, set to 40°C and a vacuum degree of <10mbar, and dried for 24 hours to remove the residual solvent. Finally, 19.2mg EDC and 11.5mg NHS were weighed and dissolved in 1mL PBS solution. The PLLA / BG nanofiber membrane was then immersed in the EDC / NHS solution and reacted at room temperature for 2 hours to activate the carboxyl groups on the surface of the PLLA / BG nanofiber membrane. A 0.5mg / mL-1mg / mL PAMAM-NH2 PBS solution was prepared, and the activated PLLA / BG nanofiber membrane was immersed in the PAMAM-NH2 solution and reacted at room temperature for 12 hours to graft PAMAM-NH2. After the reaction, the PLLA / BG nanofiber membrane was washed with deionized water 3 times for 10 minutes each time. The PLLA / BG nanofiber membrane was placed in a vacuum drying oven and dried at 40°C for 24 hours to obtain a PLLA / BG / PAMAM-NH2 composite spinning membrane.

[0153] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include," "comprise," or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations. The phrase "includes an element defined by..." does not exclude the presence of other identical elements in the process, method, article, or device that includes the element.

[0154] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An electroactive dressing for negative pressure therapy to promote bone cell differentiation and bone regeneration and a method for making the same, characterized in that: The method for making the electroactive dressing includes: S1: Preparation of PLLA / BG spinning solution; S2: Preparation of PLLA / BG nanofiber membrane by electrospinning; S3: vacuum drying; S4: PAMAM-NH2 surface modification.

2. The electroactive dressing for negative pressure therapy for promoting bone cell differentiation and bone regeneration and the method for making the same according to claim 1, characterized in that: The PLLA / BG spinning solution was prepared by weighing 1.2 g of PLLA powder, adding 10 mL of hexafluoroisopropanol, and stirring at room temperature for 6 hours using a magnetic stirrer until the PLLA was completely dissolved to obtain a 12 wt% PLLA solution. 0 mg, 60 mg, 120 mg, and 180 mg of BG particles were weighed, corresponding to BG mass fractions of 0 wt%, 5 wt%, 10 wt%, and 15 wt%. The BG particles were added to the PLLA solution, and ultrasonic dispersion was performed at a power of 300 W using an ultrasonic disperser for 30 minutes to ensure that the BG was evenly dispersed to obtain a PLLA / BG mixed solution.

3. The electroactive dressing for negative pressure therapy for promoting bone cell differentiation and bone regeneration and the method for making the same according to claim 2, characterized in that: The electrospinning method for preparing the PLLA / BG nanofiber membrane is as follows: the PLLA / BG mixed solution is loaded into a 10 mL syringe, installed in an electrospinning device, and the parameters are set as a voltage of 18 KV, a propulsion rate of 1.5 mL / h, a collection distance of 18 cm, an ambient temperature of 25°C, and a relative humidity of 40%. The spun fibers are collected on aluminum foil and the spinning time is 2 h to obtain a uniform PLLA / BG nanofiber membrane.

4. The electroactive dressing for negative pressure therapy for promoting bone cell differentiation and bone regeneration and the method for making the same according to claim 3, characterized in that: The vacuum drying step is as follows: placing the PLLA / BG nanofiber membrane into a vacuum drying oven, setting the temperature to 40° C. and the vacuum degree to <10 mbar, and drying for 24 hours to remove the residual solvent.

5. The electroactive dressing for negative pressure therapy for promoting bone cell differentiation and bone regeneration and the method for making the same according to claim 4, characterized in that: The PAMAM-NH2 surface modification is as follows: 19.2 mg EDC and 11.5 mg NHS are weighed and dissolved in 1 mL PBS solution, the PLLA / BG nanofiber membrane is immersed in the EDC / NHS solution, reacted at room temperature for 2 hours to activate the surface carboxyl groups of the PLLA / BG nanofiber membrane, a 0.5 mg / mL-1 mg / mL PAMAM-NH2 PBS solution is prepared, the activated PLLA / BG nanofiber membrane is immersed in the PAMAM-NH2 solution, reacted at room temperature for 12 hours to graft PAMAM-NH2, after the reaction is completed, the PLLA / BG nanofiber membrane is washed with deionized water 3 times, each time for 10 minutes, and the PLLA / BG nanofiber membrane is placed in a vacuum drying oven and dried at 40°C for 24 hours to obtain a PLLA / BG / PAMAM-NH2 composite spinning membrane.

Images