Collagen-based composite piezoelectric hydrogel, preparation method and application

Abstract

The application belongs to the technical field of biomedical materials, functional polymer materials and regenerative medicine, and particularly relates to a collagen-based composite piezoelectric hydrogel based on non-solvent induced phase separation and extrusion type 3D printing, a preparation method and application. The preparation method comprises the following steps: mixing collagen solution, polyvinylidene fluoride and barium titanate to prepare a composite bio-printing ink; the printing ink is deposited and formed layer by layer through extrusion type 3D printing, and a non-solvent is introduced during the printing process, so that the system undergoes non-solvent induced phase separation and realizes in-situ curing, and the collagen-based composite piezoelectric hydrogel is obtained. The prepared composite piezoelectric hydrogel has good printing formability, structural stability, biocompatibility and piezoelectric response performance, can form a stable porous composite network structure, and can generate electrical signal output under the condition of external mechanical stimulation or ultrasonic stimulation. The collagen-based composite piezoelectric hydrogel can be used for wound repair, and is particularly suitable for diabetic chronic wound repair.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical materials, functional polymer materials and regenerative medicine, specifically relating to a collagen-based composite piezoelectric hydrogel, its preparation method and application. Background Technology

[0002] Diabetic chronic wounds are common, complex, and difficult-to-heal wounds in clinical practice. They are exposed to an abnormal pathological microenvironment characterized by high glucose, low oxygen, persistent inflammation, and susceptibility to infection. This often results in impaired angiogenesis, decreased cell migration and proliferation, and weakened tissue regeneration, leading to prolonged healing periods. In severe cases, it can even cause complications such as infection spread, tissue necrosis, and amputation, placing a heavy burden on patients' quality of life and the healthcare system. Currently, treatment methods for diabetic chronic wounds mainly include debridement, anti-infection therapy, negative pressure wound therapy, and various wound dressings. Among these, hydrogel dressings are widely used in chronic wound repair due to their good moisturizing properties, soft adherence, and certain biocompatibility. However, most existing hydrogel dressings primarily provide physical coverage and a basic moist healing environment, lacking the ability to actively regulate the complex pathological microenvironment. Furthermore, they often suffer from insufficient structural stability, limited functional continuity, and weak multifunctional synergistic effects during long-term use, making it difficult to meet the clinical needs for long-term repair of diabetic chronic wounds.

[0003] Collagen, as an important component of the extracellular matrix, possesses excellent biocompatibility, biodegradability, and bioactivity that promotes cell adhesion, migration, and proliferation, thus it is widely used in wound repair materials. However, natural collagen materials typically suffer from low mechanical strength, insufficient structural stability, and susceptibility to degradation or structural collapse in complex pathological environments, making it difficult to maintain stable wound support over the long term, thereby limiting their application in chronic wound repair.

[0004] In recent years, functional materials with electrostimulation capabilities have received widespread attention in the field of tissue repair. Appropriate electrostimulation helps mimic the body's natural bioelectrical signals, promoting cell migration, tissue repair, and angiogenesis to some extent. Traditional electrostimulation therapy typically relies on external power sources, electrodes, or wire devices, which suffer from problems such as complex equipment, poor flexibility, and inconvenience in long-term use, limiting its application in wound repair materials. Piezoelectric materials can convert mechanical energy into electrical signals under external mechanical stimulation, providing a new technical path for constructing self-powered electrostimulated wound repair materials. Among them, polyvinylidene fluoride (PVDF), as a typical polymeric piezoelectric material, possesses excellent piezoelectric properties and processing performance; barium titanate (BTO), as a typical inorganic piezoelectric material, exhibits high piezoelectric response performance. Introducing piezoelectric functional components into biomedical material systems is expected to endow materials with electrostimulation capabilities, thereby expanding their application value in wound repair.

[0005] However, existing technologies still face several technical challenges. On the one hand, PVDF itself is highly hydrophobic and has weak biocompatibility, limiting its use alone in wound repair. On the other hand, BTO, as an inorganic particulate material, still presents challenges in achieving uniform dispersion and interfacial compatibility within flexible biomaterial systems, potentially affecting the structural stability and functional consistency of composite materials. Therefore, achieving stable composite construction of bioactive matrix materials and piezoelectric functional materials remains a pressing technical problem to be solved in this field. Furthermore, 3D printing technology can construct tissue engineering materials with controllable morphologies layer by layer according to a pre-defined structure. Among these, extrusion 3D printing has attracted widespread attention due to its good adaptability to natural polymer materials and relatively simple equipment structure. However, for collagen-based materials, relying solely on printing is often insufficient to obtain a three-dimensional network structure that combines structural stability and functionality. Existing stabilization methods typically rely on chemical cross-linking or post-treatment curing steps, which not only increase process complexity but may also introduce potential biosafety risks.

[0006] Non-solvent phase separation (NIPS) technology can induce phase separation in polymer systems and form porous structures through solvent-non-solvent substitution, showing potential applications in the preparation of functional materials. However, existing NIPS methods mostly employ overall immersion or post-printing processing, which are difficult to effectively coordinate with extrusion 3D printing processes. This can easily lead to deformation, collapse, or unstable distribution of functional components in the printed structure, limiting its application in the construction of biomedical materials with high structural stability.

[0007] Therefore, there is an urgent need to develop a new strategy for constructing collagen-based composite piezoelectric hydrogels that can achieve structural stability and reinforcement of collagen-based materials while maintaining good biocompatibility, and effectively integrate piezoelectric functional components, so that the materials can simultaneously possess good printability, structural stability and piezoelectric response performance, in order to meet the needs of biomedical applications such as chronic wound repair for diabetes. Summary of the Invention

[0008] The purpose of this invention is to address the technical problems of existing diabetic chronic wound repair materials, such as insufficient structural stability, limited functional continuity, and difficulty in simultaneously achieving good biocompatibility and electrostimulation function. This invention provides a collagen-based composite piezoelectric hydrogel, its preparation method, and its applications. The preparation method synergistically couples non-solvent-induced phase separation technology with an extrusion 3D printing process. During the layer-by-layer deposition and molding of the material, phase separation is simultaneously induced in the system, achieving in-situ solidification. This results in a collagen-based composite piezoelectric hydrogel that possesses good printability, structural stability, biocompatibility, and piezoelectric response properties.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a collagen-based composite piezoelectric hydrogel, comprising the following steps: (1) Preparation of composite bioprinting ink: Collagen solution, polyvinylidene fluoride and barium titanate particles are mixed and uniformly dispersed to obtain composite bioprinting ink; (2) Extrusion 3D printing and non-solvent phase separation coupling molding: The composite bioprinting ink obtained in step (1) is loaded into an extrusion 3D printing device and deposited layer by layer through the nozzle according to a preset path to form a three-dimensional structure; during the printing process, the composite bioprinting ink is brought into contact with a non-solvent, and solvent and non-solvent replacement occurs, inducing non-solvent phase separation of the system and achieving in-situ solidification, thereby obtaining the collagen-based composite piezoelectric hydrogel.

[0010] Preferably, the composite bioprinting ink comprises the following components: Collagen solution 0.5%–2% (m / v); Polyvinylidene fluoride 10%–20% (m / v); Barium titanate 0.05%~1% (m / v).

[0011] Preferably, the composite bioprinting ink comprises the following components: Collagen solution 2% (m / v); Polyvinylidene fluoride 10%–15% (m / v); Barium titanate 0.5% (m / v).

[0012] Preferably, the collagen is bovine collagen.

[0013] Preferably, the bovine collagen is selected from one or at least two of type I bovine collagen, type II bovine collagen, and type III bovine collagen.

[0014] Preferably, the polyvinylidene fluoride comprises a β-phase.

[0015] Preferably, the barium titanate is in the form of nano-sized particles.

[0016] Preferably, the non-solvent is water vapor.

[0017] Preferably, in step (2), the water vapor acts synchronously on the printing area during the printing process to induce the composite bioprinting ink to undergo in-situ phase separation and solidification.

[0018] In a second aspect, the present invention provides a collagen-based composite piezoelectric hydrogel, wherein the collagen-based composite piezoelectric hydrogel is prepared by the above-described preparation method.

[0019] A third aspect of the present invention provides the application of the collagen-based composite piezoelectric hydrogel in the preparation of wound repair dressings.

[0020] Preferably, the wound is a diabetic chronic wound.

[0021] A fourth aspect of the present invention provides a method for applying the collagen-based composite piezoelectric hydrogel, wherein the collagen-based composite piezoelectric hydrogel is applied to a wound and ultrasonic stimulation is applied.

[0022] Preferably, the ultrasonic stimulation is used to induce a piezoelectric response in the collagen-based composite piezoelectric hydrogel.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention combines non-solvent phase separation technology with extrusion 3D printing process to enable composite bioprinting ink to undergo phase separation and in-situ solidification during the layer-by-layer deposition process, thereby avoiding structural deformation or collapse that may be caused by traditional post-printing processing, and improving the printing accuracy and structural stability of the material.

[0024] (2) This invention uses collagen as a bioactive matrix material and introduces polyvinylidene fluoride and barium titanate piezoelectric functional components to construct a collagen-based composite material system with good biocompatibility, structural stability and piezoelectric response performance, which is conducive to expanding the application of piezoelectric functional materials in the biomedical field.

[0025] (3) The collagen-based composite piezoelectric hydrogel prepared by the present invention has good structural integrity and porous characteristics, which can meet the application requirements of wound covering materials for flexible adhesion and structural stability.

[0026] (4) The collagen-based composite piezoelectric hydrogel of the present invention can generate a piezoelectric response under external mechanical or ultrasonic stimulation, and has self-powered electrical stimulation characteristics. It has good application value in wound repair, especially in the field of chronic diabetic wound repair. Attached Figure Description

[0027] Figure 1 The image shows the preparation and structural characterization of a collagen-based composite piezoelectric hydrogel; where: Figure 1 a is a top view of a grid structure formed by extrusion 3D printing of composite bioprinting ink; Figure 1 b is a physical image of the printed mesh structure; Figure 1 c is the circular dichroism chromatogram of the collagen solution; Figure 1 d shows the scanning electron microscope image and EDS elemental distribution map of the collagen-based composite piezoelectric hydrogel; Figure 1 e is the rheological stress scanning result of the collagen-based composite piezoelectric hydrogel; Figure 1 f represents the tensile stress-strain curves of different samples; Figure 1 g is a graph showing the swelling performance test results of different samples.

[0028] Figure 2 The piezoelectric properties characterization diagram is shown for collagen-based composite piezoelectric hydrogels; where: Figure 2 a is a graph showing the voltage output curves of different component samples under cyclic mechanical load; Figure 2 b is the voltage output curve of the collagen-based composite piezoelectric hydrogel under different loading conditions; Figure 2 c is the voltage output curve of the collagen-based composite piezoelectric hydrogel under different loading frequencies; Figure 2 d is the voltage output curve of the collagen-based composite piezoelectric hydrogel under ultrasonic stimulation.

[0029] Figure 3 The diagram shows the in vitro biocompatibility evaluation of the collagen-based composite piezoelectric hydrogel; where: Figure 3 Figure a shows the cytotoxicity test results for different treatment groups; Figure 3 b shows the cell viability detection results of different treatment groups at different culture times; Figure 3 c shows the fluorescence images of live / dead cells in different treatment groups.

[0030] Figure 4 The graph shows the in vitro antibacterial properties evaluation of collagen-based composite piezoelectric hydrogels; where: Figure 4 a shows plate colony images of Escherichia coli and Staphylococcus aureus from different treatment groups; Figure 4 b is a statistical graph showing the antibacterial rates of different treatment groups against Escherichia coli; Figure 4 c is a statistical graph showing the antibacterial rates of different treatment groups against Staphylococcus aureus.

[0031] Figure 5 The image shows the immunofluorescence detection of macrophage phenotype-related markers using a collagen-based composite piezoelectric hydrogel; where: Figure 5 a shows CD163 immunofluorescence staining images in different treatment groups; Figure 5 b shows iNOS immunofluorescence staining images in different treatment groups.

[0032] Figure 6 The image shows the in vivo evaluation of collagen-based piezoelectric hydrogels in the repair of chronic wounds in diabetes; where: Figure 6 a shows wound photographs of different treatment groups at different time points; Figure 6 b is a statistical graph showing the changes in wound area among different treatment groups. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Equivalent substitutions or modifications made by those skilled in the art under the guidance of the present invention without departing from the spirit and substance of the present invention should all fall within the scope of protection of the present invention.

[0034] Unless otherwise specified, the methods used in this invention are all conventional methods in the art; the reagents, raw materials and instruments used are all commercially available.

[0035] The collagen used in this invention is preferably bovine collagen, which may be selected from one or at least two of type I, type II, and type III bovine collagen. This embodiment uses yak type I collagen as an example, but the invention is not limited to this specific source.

[0036] In this invention, the collagen-based composite piezoelectric hydrogel is composed of collagen, polyvinylidene fluoride (PVDF), and barium titanate (BTO), wherein the PVDF preferably contains a β-crystalline phase, and the BTO is preferably nano-sized particles.

[0037] In this invention, non-solvent-induced phase separation (NIPS) refers to the process in which the solvent in the composite bioprinting ink is replaced by an external non-solvent, thereby inducing phase separation in the system and achieving structural solidification.

[0038] Example 1: Preparation of Collagen-Based Composite Bioprinting Ink Freeze-dried collagen was added to dimethyl sulfoxide (DMSO) and stirred continuously at low temperature until completely dissolved to obtain a homogeneous collagen solution.

[0039] In this embodiment, the molding properties of the system were investigated at collagen concentrations of 0.5% (m / v), 1.0% (m / v), and 2.0% (m / v). The results showed that when the collagen concentration was low, the structural support of the resulting composite structure was weak; when the collagen concentration was 2.0% (m / v), better system stability could be obtained. Therefore, a collagen concentration of 2.0% (m / v) was preferred.

[0040] Subsequently, polyvinylidene fluoride was added to the collagen solution and thoroughly mixed to form a homogeneous system. Barium titanate particles were then added to the system and mechanically stirred and dispersed to achieve uniform dispersion, yielding the composite bioprinting ink.

[0041] In this embodiment, the preferred composition of the composite bioprinting ink is as follows: Collagen solution: 2.0% (m / v); Polyvinylidene fluoride: 15% (m / v); Barium titanate: 0.5% (m / v).

[0042] In other embodiments of the present invention, the concentrations of each component in the composite bioprinting ink can be adjusted within the following ranges: Collagen solution: 0.5%~2.0% (m / v); Polyvinylidene fluoride: 10%~20% (m / v); Barium titanate: 0.05%~1.0% (m / v).

[0043] The resulting composite bioprinting ink is defoamed and then used for later use.

[0044] Example 2: Preparation of collagen-based composite piezoelectric hydrogel The composite bioprinting ink obtained in Example 1 was loaded into an extrusion 3D printing device.

[0045] During printing, the nozzle extrudes and deposits layer by layer according to a preset printing path to form a predetermined three-dimensional structure.

[0046] During the printing process, non-solvents are introduced into the printing area simultaneously, causing the solvent in the composite bioprinting ink to be replaced by the non-solvent, thereby inducing non-solvent phase separation and achieving in-situ solidification, ultimately obtaining collagen-based composite piezoelectric hydrogel.

[0047] In this embodiment, the non-solvent is preferably water vapor.

[0048] Water vapor continuously acts on the nozzle extrusion area during the printing process, causing the composite bioprinting ink to undergo phase separation and solidification simultaneously during deposition, thereby maintaining the integrity of the printed structure.

[0049] After printing, the resulting collagen-based composite piezoelectric hydrogel is washed in deionized water or buffer solution to remove residual solvent and unbound components, and then set aside for use.

[0050] In a preferred embodiment of the present invention, the printing parameters may include: Nozzle inner diameter: 200–600 μm; Printing speed: 1~20 mm / s; Extrusion pressure: 20–200 kPa; Floor height: 100~500 μm.

[0051] The above parameters can be adjusted according to the rheological properties of the composite bioprinting ink.

[0052] Example 3: Structural and physicochemical characterization of collagen-based composite piezoelectric hydrogels Characterization of Extrusion 3D Printing Effect: A mesh model was designed and printed, and the printed model was photographed for documentation. The optimal ink formula was determined after screening different proportions, and printing experiments were conducted on different models. The mesh model was clearly distinguishable, demonstrating high printing precision. Figure 1 As shown in Figure a, the printed model can stand stably when held by tweezers, indicating that the printed gel has sufficient support. Figure 1 As shown in b.

[0053] Circular dichroism (CD) characterization: The collagen solution was characterized by circular dichroism. The sample was equilibrated at 4°C for at least 24 hours prior to CD measurement. A cuvette with a path length of 1 mm was used, the wavelength scan range was 180–260 nm, the step size was 1.0 nm, and the averaging time was 1.0 second. The results are as follows: Figure 1 As shown in Figure c, collagen dissolved in DMSO exhibits a negative peak near 200 nm and a positive peak near 222 nm, representing a typical triple helix structure. This indicates that DMSO, as a solvent, did not disrupt the triple helix structure of collagen. Figure 1 As shown in c.

[0054] Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) characterization: The CPB composite piezoelectric printing gel was freeze-dried and then sputter-coated with gold. The surface and cross-sectional morphology of the gel were observed using scanning electron microscopy. SEM images showed that collagen constituted a continuous three-dimensional network structure in the composite gel, serving as the main supporting phase of the overall structure. Polyvinylidene fluoride (PVDF) precipitated and distributed within the continuous collagen network during the non-solvent-induced phase separation and curing process, spatially interpenetrating with collagen to form a physically interpenetrating network structure. Barium titanate particles were fixed within the collagen / PVDF composite network structure during phase separation and curing, and the three components together constituted a structurally stable composite system. Further EDS elemental mapping analysis results were consistent with the SEM observations, confirming the coexistence of collagen, PVDF, and barium titanate in the composite dressing and their spatial distribution characteristics within the collagen-dominated network, such as... Figure 1 As shown in d.

[0055] Rheological analysis: The rheological properties of CPB hydrogel were tested using a rheometer in oscillation mode. The storage modulus (G′) and loss modulus (G″) were determined by stress scanning within a shear stress range of 0–2500 Pa. During the test, the angular frequency was set to 1 rad / s, and the strain was controlled at 1% to characterize the in-situ gelation behavior of the printing ink during the molding process. The test results show that within the tested stress range, the storage modulus (G′) of the CPB hydrogel is consistently greater than the loss modulus (G″), indicating that the formed hydrogel system exhibits predominantly elastic behavior and good structural stability. Figure 1 As shown in e.

[0056] Tensile property analysis: The tensile properties of CPB piezoelectric hydrogel were tested using a universal testing machine, and the results are shown in Figure 1. The CPB piezoelectric support exhibits good mechanical properties, with a tensile strength of approximately 60 kPa (range 50–500 kPa) and a fracture strain of approximately 55% (range 30–70%). These mechanical parameters indicate that the material meets the basic mechanical property requirements for dressings used on skin surfaces in terms of flexibility and strength. Figure 1 As shown in f.

[0057] Swelling performance analysis: The CPB piezoelectric scaffold was immersed in phosphate-buffered saline (PBS), and its swelling ratio (SR) was measured. The results showed that the CPB hydrogel maintained a stable swelling ratio of approximately 93% in PBS and reached swelling equilibrium within about 2 hours. This result indicates that the prepared hydrogel exhibits relatively stable swelling behavior in an aqueous environment, which is beneficial for its structural maintenance and application stability in wound environments. Figure 1 As shown in g.

[0058] Example 4: Piezoelectric property testing of collagen-based composite piezoelectric hydrogel The CPB piezoelectric hydrogel prepared in Example 2 was used to fabricate a sensor structure, and its piezoelectric output performance was tested using a Keithley 6514 electrometer. During the test, external mechanical stimuli of different intensities were applied to the composite piezoelectric hydrogel, and the corresponding changes in its electrical signal output were recorded.

[0059] Comparison of intrinsic piezoelectric output results of different samples under the same mechanical load conditions ( Figure 2a) It can be seen that the pure collagen sample only produces a weak signal close to the baseline, indicating that its contribution to the overall electrical output is small. After the introduction of PVDF, the sample can produce a stable periodic voltage signal, indicating that the piezoelectric response mainly comes from the electroactive phase of PVDF. Further addition of BTO further enhances the output signal. Notably, the output of the Col I+PVDF+BTO sample is significantly higher than that of the Col I+PVDF+BTO(No) composite system without NIPS-3D structural reconstruction, indicating that the improvement in piezoelectric performance depends not only on the introduction of functional components but also on their organization in the reconstructed network. Subsequently, the force response output performance of the CPB piezoelectric hydrogel was investigated under cyclic loads of 5–35 N. Figure 2 b). A stable electrical signal synchronized with the loading rhythm was recorded throughout the test, and the output amplitude gradually increased with the increase of the applied force, indicating that the hydrogel has stable and adjustable electromechanical conversion capability over a wide load range. With other loading conditions basically the same, its frequency response behavior in the 1–4 Hz frequency range was further investigated. Figure 2 c). The CPB piezoelectric hydrogel maintains a stable periodic output across the entire frequency range, and the voltage signal increases with increasing frequency, indicating that it can respond rapidly and continuously to dynamic mechanical stimulation and has good frequency adaptability. Furthermore, the composite piezoelectric hydrogel was placed under ultrasonic stimulation conditions, and its electrical response behavior under applied ultrasound was recorded. Figure 2 d). Under ultrasonic stimulation, the composite piezoelectric hydrogel can generate a detectable electrical signal output and exhibits a significant response change with ultrasonic stimulation, indicating that the material can effectively generate a piezoelectric response under ultrasonic-induced mechanical disturbance and has good ultrasonic response characteristics. The CPB piezoelectric hydrogel exhibits good piezoelectric response performance under force stimulation, frequency change, and ultrasonic stimulation, and can achieve stable and adjustable electrical signal output, demonstrating good application potential. The above results show that the collagen / polyvinylidene fluoride / barium titanate physical interpenetrating network structure synergistically constructed by extrusion printing and non-solvent-induced phase separation not only ensures the structural stability of the composite hydrogel, but also endows it with the ability to achieve stable piezoelectric output under mechanical and ultrasonic stimulation conditions.

[0060] Example 5: Biocompatibility Evaluation of Collagen-Based Composite Piezoelectric Hydrogel The CPB piezoelectric hydrogel prepared in Example 2 was washed and sterilized before being used in cell culture experiments to evaluate its biocompatibility. Human foreskin fibroblasts (HFF-1) were purchased from Pronosei Biotechnology Co., Ltd. (China) and cultured in DMEM complete medium containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. Cells were routinely cultured in a 37 °C, 5% CO2 cell culture incubator. Experimental groups included a blank control group (Control), a collagen hydrogel group (ColI), a CPB biopiezoelectric hydrogel group (CPB), and a CPB combined with ultrasound stimulation group (CPB+US). In the ultrasound safety verification experiment, an additional ultrasound-only treatment group (Blank+US) was set up. Unless otherwise specified, non-contact ultrasound stimulation was used in cell experiments. According to the previous experimental settings, the ultrasound parameters were 1.0 W cm⁻¹. -2 (1 MHz), 50% duty cycle. After cell adhesion, sonication was initiated, four times daily for 60 seconds each time. All sonication groups were treated under the same conditions. The prepared CoL, CPB, and CPB+US hydrogels were pre-equilibrated in DMEM medium at 37 ℃ for subsequent cell co-culture experiments.

[0061] (1) Cytotoxicity test Cytotoxicity assays were performed using the CCK-8 assay. HFF-1 fibroblasts were cultured at a rate of 1 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured in complete medium (89% DMEM, 10% FBS, and 1% penicillin-streptomycin) at 37 °C and 5% CO2 for 24 h to ensure full cell adhesion. Subsequently, different pre-prepared hydrogel samples were added to the cell culture system for direct co-culture with the cells for another 24 h. After culture, the medium was discarded, the cells were washed three times with PBS, and 100 μL of DMEM-diluted CCK-8 working solution was added to each well. The cells were incubated at 37 °C in the dark for 1 h. The absorbance (OD) of each sample at 450 nm was then measured using a multi-mode microplate reader, and cell viability was calculated.

[0062] The results of the cytotoxicity experiment are shown in Figure 3a. Compared with the blank control group (Blank, 100%), the cell viability of CoL, CPB and CPB+US groups was significantly higher than 100%, indicating that the above hydrogels had no obvious cytotoxicity to fibroblasts under direct co-culture conditions.

[0063] (2) Cell proliferation experiment Cell proliferation behavior was evaluated using the CCK-8 assay. The cell seeding method was consistent with the cytotoxicity assay; different hydrogel samples were added to three independent 96-well plates and co-cultured directly with HFF-1 fibroblasts. Cell viability was measured using a multi-mode microplate reader on days 1, 3, and 5 of culture. The blank control group (containing only DMEM) on day 1 of culture was used as the baseline for calculations.

[0064] As shown in Figure 3b, the cell proliferation rates of the CPB and CPB+US hydrogel groups were significantly higher than those of the Blank and CoL groups after 1, 3, and 5 days of culture. Specifically, the CPB+US group achieved cell proliferation rates of 303.3% and 409.6% on Day 3 and Day 5, respectively, significantly higher than the other groups. This indicates that the CPB piezoelectric hydrogel exhibits good cell compatibility under direct co-culture conditions and further promotes fibroblast proliferation under ultrasonic stimulation.

[0065] (3) Cell biocompatibility experiment HFF-1 fibroblasts were qualitatively analyzed using the Calcein-AM / PI live / dead cell staining kit to further evaluate the biocompatibility of the hydrogel. Following the kit instructions, samples from each group were stained on days 1, 3, and 5 after direct co-culture with the hydrogel, and then observed using a laser confocal microscope.

[0066] Staining results as follows Figure 3 As shown in c, after 5 days of culture, each group still showed a large amount of green fluorescence (live cells) and very little red fluorescence (dead cells), indicating that the prepared CPB piezoelectric hydrogel has good biocompatibility in the direct co-culture system and has not had an adverse effect on cell survival.

[0067] Example 6: Evaluation of the antibacterial properties of collagen-based composite piezoelectric hydrogels The CPB piezoelectric hydrogel prepared in Example 2 was used in an in vitro antibacterial experiment, and its antibacterial properties were evaluated under conditions with and without ultrasonic stimulation. Gram-negative bacteria *Escherichia coli* (E. coli) were selected. Escherichia coli E. coli) and Gram-positive bacteria Staphylococcus aureus ( Staphylococcus aureus *S. aureus* was used as a representative strain to evaluate the broad-spectrum antibacterial properties of the Col / PVDF / BTO piezoelectric composite hydrogel. 100 μL of a 1 × 10⁻⁶ concentration was used. 7 CFU·mL -1Bacterial suspensions were inoculated onto the surfaces of sterilized samples. Experimental groups included a blank control group (Blank), a sonication-only group (US), a collagen hydrogel group (COL), a CPB piezoelectric hydrogel group (CPB), and a CPB piezoelectric hydrogel combined with sonication treatment group (CPB+US). All samples were placed in 6-well plates and incubated at 37 ℃ for 24 h. For the sonication groups (US and CPB+US), sonication was performed after the addition of an ultrasonic coupling agent, with ultrasonic parameters set to a frequency of 1.0 MHz and a sound intensity of 2.5 W·cm. -2 The duty cycle was 50%, and the treatment time was 8 min. During the ultrasonic treatment, the ultrasonic probe was kept in close contact with and fixed to the bottom of the 6-well culture plate to ensure effective transfer of ultrasonic energy. After incubation, bacteria on the surface of each group of samples were washed off and collected. The resulting bacterial suspension was serially diluted with physiological saline, and 100 μL was inoculated onto agar plates and cultured at 37 ℃ for 20 h. The antibacterial effect of each group of samples was analyzed by counting the number of colony forming units (CFU).

[0068] Experimental results are as follows Figure 4 As shown in a~c, under ultrasonic stimulation, CPB piezoelectric hydrogel showed significant inhibitory effects on the growth of Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus, demonstrating good antibacterial properties. Compared with the group without ultrasonic treatment, the antibacterial effect of the CPB+US group was more significant, indicating that ultrasonic stimulation can enhance the antibacterial effect of CPB piezoelectric hydrogel.

[0069] Example 7: Evaluation of Immunomodulatory Effects of Collagen-Based Composite Piezoelectric Hydrogels Add 1×10 to the laser confocal dish 5 RAW 264.7 cell suspensions were cultured at 100 cells / mL for 24 hours. The culture medium was discarded, and the cells were washed three times with PBS for 5 minutes each time. Then, 0.1 mg / mL LPS diluted in complete culture medium was added and the cells were cultured for 12 hours to polarize M0 macrophages to the M1 phenotype. The extracts from each group were then added to small dishes and cultured for 24 hours for immunofluorescence staining.

[0070] Antibody staining was performed on M0 cells from each group after 24 hours of culture. First, the samples were washed three times with PBS for five minutes each time, followed by permeabilization with 0.5% Triton diluted in PBS for ten minutes. After three washes with PBS for five minutes each time, 5% goat serum was added for blocking for one hour. Then, the samples were washed for five minutes, and CD163 and iNOS antibody markers diluted 250-fold were added, respectively, and incubated at 4°C for 16 hours. The same procedure was repeated, washing with PBS and incubating with diluted secondary antibody for two hours. After two hours, DAPI staining was performed for ten minutes according to the manufacturer's instructions. Fluorescence imaging of each group was performed using laser confocal microscopy at the same parameters.

[0071] Immunofluorescence staining results as follows Figure 5 As shown, after 12 h of stimulation with lipopolysaccharide (LPS), macrophages exhibited significant iNOS positive expression, indicating that the cells were in a typical M1 inflammatory phenotype. During the subsequent 3 days of culture, the iNOS expression levels in the Blank group and the sonication-only group (US group) remained essentially unchanged, showing no significant difference compared to the initial state (0 h), indicating that M1 macrophages are unlikely to spontaneously transform into an anti-inflammatory phenotype under conditions without functional material intervention.

[0072] In contrast, regarding the expression of the M2 phenotypic marker CD163, the results from each group showed an opposite trend to that of iNOS. CD163, a typical marker of anti-inflammatory (M2) macrophages, exhibited significantly higher fluorescence intensity in the CPB+US treatment group than in the Blank and US groups, and also higher than in the CPB group without ultrasound. This indicates that, while maintaining good biocompatibility, the CPB composite hydrogel, under ultrasound stimulation, can more effectively promote macrophage polarization towards the M2 phenotype through piezoelectric effects. Figure 5 As shown in Figure a, in the treatment groups in contact with the hydrogel, the iNOS fluorescence signal area and single-cell fluorescence intensity both showed varying degrees of decrease, indicating that the prepared hydrogel material itself has a certain regulatory effect on the state of inflammatory macrophages. Among them, the iNOS fluorescence intensity of the CPB+US group was significantly lower than that of the CPB group without ultrasound, indicating that under ultrasound stimulation, the piezoelectric response generated by the composite hydrogel further enhanced the inhibitory effect on the M1 phenotype. Figure 5 As shown in b.

[0073] The above results demonstrate that the CPB composite piezoelectric hydrogel not only possesses good stability and biocompatibility at the material level, but also generates a piezoelectric response under external ultrasound stimulation, thereby synergistically regulating the inflammatory phenotypic transformation of macrophages, manifested as a decrease in M1 phenotype-related iNOS expression and an increase in M2 phenotype-related CD163 expression. This property helps alleviate chronic inflammation and provides a more favorable repair microenvironment for wounds. Considering that diabetic wounds are usually located in a pathological environment rich in high glucose and advanced glycation end products (AGEs), which easily leads to prolonged inflammation and inhibits the transformation of M1 to M2 phenotypes, the CPB+US piezoelectric hydrogel of this invention provides an effective material regulation strategy for promoting inflammation resolution and accelerating wound healing through its material structure characteristics and ultrasound-induced piezoelectric effect.

[0074] Example 8: Evaluation of the application of collagen-based composite piezoelectric hydrogel in the repair of chronic diabetic wounds An artificial skin model was designed using 3D modeling software. Samples were prepared using extrusion 3D printing combined with a solvent-free phase separation method for subsequent animal experiments and in vitro culture studies. This molding method enables simultaneous phase separation and curing of the material during the printing process, ensuring that the prepared artificial skin has a stable 3D structure and good adhesion performance.

[0075] Male SD rats weighing 150–200 g were selected and a drug-induced diabetes model was established by a single intraperitoneal injection of streptozotocin (STZ, 65 mg / kg). During the modeling period, rats were provided with ample water and food, and their bedding was changed regularly. Three weeks after modeling, a full-thickness skin defect model with a diameter of 9 mm was prepared on the rat's back. The diabetes control group received no material treatment; the CoL group and CPB group had prepared collagen hydrogel and CPB piezoelectric hydrogel applied to the wound surface, respectively; a wound model of healthy rats without diabetes was established as a control group. All experimental groups used 3M transparent dressings to fix the wounds, which naturally fell off during subsequent feeding without further replacement. To avoid interference from infection, all rats underwent systemic disinfection, and the wounds were locally treated with iodine-soaked cotton balls.

[0076] Wounds in each group were photographed on postoperative days 0, 4, 7, 10, and 14, and uniformly cropped to 2 cm × 2 cm for observing wound healing over time, as shown in Figure 6a. ImageJ software was used for quantitative analysis of wound area changes and wound closure rate. The results, as shown in Figure 6b, indicate that compared to the acute wound control group, the diabetes control group showed significant delayed wound healing on days 4, 7, 10, and 14, consistent with typical characteristics of impaired healing in chronic diabetic wounds. In contrast, the experimental groups receiving material coverage showed faster wound closure rates at different time points, indicating that the prepared hydrogel material itself can provide a favorable repair environment for diabetic wounds. The CoL group showed a certain degree of improved healing compared to the control group, demonstrating the good biocompatibility and tissue support of the collagen material. The CPB+US group showed significantly higher wound closure rates at all time points than the CoL group and the CPB group without ultrasound, indicating faster healing speed and better healing quality. The above results demonstrate that CPB piezoelectric hydrogels, based on their inherent good biocompatibility and structural stability, can generate a piezoelectric response under external ultrasonic stimulation, thereby further improving the wound microenvironment and promoting the repair process of diabetic wounds. This synergistic effect highlights the potential advantages of piezoelectric functional hydrogel-based material strategies in the treatment of chronic diabetic wounds.

[0077] In summary, this invention provides a collagen-based composite piezoelectric hydrogel and its preparation method. This method synergistically couples a non-solvent-induced phase separation technique with an extrusion 3D printing process, enabling simultaneous phase separation and in-situ solidification of the composite bioprinting ink during layer-by-layer deposition, thereby obtaining a collagen-based composite piezoelectric hydrogel with good structural stability, biocompatibility, and piezoelectric response properties. The collagen-based composite piezoelectric hydrogel can be used for wound repair, especially suitable for the repair of chronic diabetic wounds.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a collagen-based composite piezoelectric hydrogel, characterized in that, Includes the following steps: (1) Preparation of composite bioprinting ink: Collagen solution, polyvinylidene fluoride and barium titanate particles are mixed and uniformly dispersed to obtain composite bioprinting ink; (2) Extrusion 3D printing and non-solvent phase separation coupling molding: The composite bioprinting ink obtained in step (1) is loaded into an extrusion 3D printing device and deposited layer by layer through the nozzle according to a preset path to form a three-dimensional structure; during the printing process, the composite bioprinting ink is brought into contact with a non-solvent, and solvent and non-solvent replacement occurs, inducing non-solvent phase separation of the system and achieving in-situ solidification, thereby obtaining the collagen-based composite piezoelectric hydrogel.

2. The preparation method according to claim 1, characterized in that, The composite bioprinting ink described in step (1) comprises the following components: collagen solution 0.5%–2% m / v, polyvinylidene fluoride 10%–20% m / v and barium titanate 0.05%–1% m / v.

3. The preparation method according to claim 2, characterized in that, The composite bioprinting ink described in step (1) comprises the following components: 2% m / v collagen solution, 10%–15% m / v polyvinylidene fluoride and 0.5% m / v barium titanate.

4. The preparation method according to claim 1, characterized in that, The collagen mentioned in step (1) is bovine collagen, which is selected from one or at least two of type I bovine collagen, type II bovine collagen and type III bovine collagen.

5. The preparation method according to claim 1, characterized in that, The polyvinylidene fluoride mentioned in step (1) contains a β-crystal phase, and the barium titanate mentioned in step (1) is nano-sized particles.

6. The preparation method according to claim 1, characterized in that, The non-solvent mentioned in step (2) is water vapor.

7. A collagen-based composite piezoelectric hydrogel, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 6.

8. The application of the collagen-based composite piezoelectric hydrogel as described in claim 7 in the preparation of wound repair dressings.

9. The application as described in claim 8, characterized in that, The wound in question is a chronic diabetic wound.

10. The application method of the collagen-based composite piezoelectric hydrogel as described in claim 7, characterized in that, The collagen-based composite piezoelectric hydrogel was applied to the wound, and ultrasonic stimulation was then applied.

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