A force-electric coupling self-powered bionic periosteum hydrogel as well as a preparation method and application thereof

Abstract

The application provides a force-electricity coupling self-powered bionic periosteum hydrogel and a preparation method and application thereof, and belongs to the technical field of biological composite materials; the hydrogel is formed by polyvinyl alcohol and borate functionalized chitosan through borate ester dynamic covalent crosslinking and freeze-thaw physical crosslinking to form a double-network viscoelastic matrix, and is uniformly dispersed with tetragonal barium titanate piezoelectric particles modified by polydopamine; the preparation method comprises the following steps: firstly, polyvinyl alcohol and borate functionalized chitosan are blended to generate borate ester dynamic covalent crosslinking, then polydopamine modified barium titanate piezoelectric particles are dispersed, and finally, a double-network hydrogel is formed through freeze-thaw cycles; the hydrogel has viscoelastic mechanical response characteristics, can be permanently deformed under low strain to adapt to the growth of a child's skull, can trigger endogenous piezoelectric signals by using physiological stress, and can promote blood vessel regeneration and bone repair through force-electricity coupling, and can be used for preparing a child's skull defect repair material.

Description

Technical Field

[0001] This application relates to the field of biocomposite materials technology, and more specifically, to a mechano-electro-coupled self-powered biomimetic periosteal hydrogel, its preparation method, and its application. Background Technology

[0002] Skull defects are a common postoperative problem in pediatric neurosurgery, primarily stemming from surgical correction of congenital craniosynostosis, decompressive craniectomy following traumatic brain injury, and tumor resection. Unlike adult skull defect repair, pediatric skull defect repair faces unique clinical challenges. Existing research indicates that the period from 0 to 2 years of age is a period of rapid growth in cranial cavity volume, reaching 95% of adult brain volume by age 6, with cranial cavity volume exhibiting a power-law growth curve. This means that cranial repair materials implanted in children must be able to adapt to the dynamic growth process of the skull, rather than simply filling the defect cavity. However, current literature on pediatric cranioplasty is relatively limited, and there are no clear guidelines to guide the timing of repair and the selection of repair materials.

[0003] Current materials for repairing pediatric skull defects mainly include autologous bone, titanium mesh, and polyetheretherketone (PEEK). While autologous bone transplantation avoids immune rejection and can synchronize with brain growth, it suffers from limited donor availability and high absorption rates. Synthetic materials like titanium mesh and PEEK provide sufficient mechanical support, but their high elastic modulus far exceeds that of natural skull, and they lack growth adaptability. After implantation, these materials cannot deform with skull growth, potentially restricting cranial cavity expansion and leading to limited brain development or secondary skull deformities. In recent years, hydrogel-based repair materials have attracted attention due to their good biocompatibility and designability. However, traditional hydrogels are mostly elastic materials and lack stress relaxation properties in their mechanical behavior, thus also restricting the slow growth of surrounding tissues after implantation.

[0004] On the other hand, natural bone tissue possesses a piezoelectric effect; mechanical stress can generate endogenous electrical signals within the bone. This electrical microenvironment plays a crucial regulatory role in bone development, fracture healing, and osteogenesis. Electrical stimulation has been shown to promote the directed migration, proliferation, and angiogenesis of endothelial cells, and can also stimulate the release of calcium ions from osteoblast membranes. 2+This process activates signaling pathways such as PI3K / AKT, upregulating the release of pro-angiogenic and bone-regenerating factors such as VEGF and FGF2. Therefore, mimicking the piezoelectric microenvironment of natural bone is considered an effective strategy to promote bone repair. Current research has introduced piezoelectric nanoparticles such as barium titanate (BTO) into hydrogel scaffolds to endow the materials with electroactivity. However, the piezoelectric effect of existing piezoelectric hydrogels usually relies on external ultrasound drive or external power supply devices, which not only increases the complexity of clinical operation and the treatment burden on patients, but also limits their widespread application in special populations such as children. Furthermore, current research on piezoelectric bone repair scaffolds mainly focuses on adult bone defects and has not yet addressed the specific needs of dynamic skull growth in children with biomechanical adaptation designs.

[0005] In summary, existing materials for repairing pediatric skull defects lack viscoelastic stress relaxation properties, cannot adapt to the slow growth of the skull, and easily restrict the expansion of the cranial cavity; existing piezoelectric repair materials mostly rely on external ultrasound or power supply to generate electrical stimulation, and are not designed to be growth-adaptive.

[0006] Therefore, there is an urgent need for a biomimetic periosteal material that combines viscoelastic growth adaptability with endogenous self-powered piezoelectric properties. This material can trigger the piezoelectric effect through the child's own physiological stress without external stimulation, and at the same time promote angiogenesis and bone repair through electromechanical coupling, thereby achieving functional repair of skull defects in children. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a self-powered biomimetic periosteal hydrogel with electromechanical coupling, its preparation method, and its applications. The hydrogel uses polyvinyl alcohol and boric acid-functionalized chitosan as matrix materials. A chemical cross-linking network is formed through dynamic covalent bonding of boric acid esters, and further physical cross-linking is induced by freeze-thaw cycles to construct a dual-network viscoelastic structure. Simultaneously, tetragonal barium titanate piezoelectric particles modified with polydopamine are uniformly dispersed within the matrix, endowing the material with piezoelectric properties.

[0008] The hydrogel's properties include: a unique viscoelastic mechanical response mechanism; permanent deformation at low strain rates to adapt to the slow growth of the child's skull without restricting cranial cavity expansion; and elastic rigidity at high strain rates to resist external impacts. Balloon experiments confirm that it does not restrict tissue expansion like purely elastic materials. The material has a pore size of less than 1 μm, ensuring the transport of oxygen and nutrients while physically blocking fibroblast invasion. At 37℃... The material does not collapse after immersion in PBS for two months, exhibits a degradation rate of approximately 30%, and possesses self-healing capabilities, recovering to a gel state within 60 seconds after large strain damage. The material's endogenous electrical signal output is triggered solely by natural stress generated from children's skull growth and daily chewing activities, without the need for external ultrasound or power stimulation. Regarding biocompatibility, the hemolysis rate is less than 5%, and cell viability staining and CCK-8 proliferation experiments confirm no cytotoxicity; furthermore, its cell spreading area is superior to that of purely elastic materials. This material directly promotes angiogenesis through electromechanical coupling; the viscoelastic matrix facilitates cell spreading and integrin / focal adhesion aggregation, thereby upregulating the expression of vascular-related proteins. The piezoelectric effect further promotes directional migration, proliferation, and vascularization of endothelial cells and activates osteogenic signaling pathways. In vivo rat skull defect model experiments demonstrate that its bone volume fraction, trabecular thickness, and trabecular number are significantly superior to the control group. This invention provides a complete technical solution for the application of this hydrogel in the repair of skull defects in children.

[0009] In a first aspect, the present invention provides a method for preparing a self-powered biomimetic periosteum hydrogel with electro-coupled force, comprising the following steps: S1: Polyvinyl alcohol is blended with boric acid-functionalized chitosan to undergo a dynamic covalent cross-linking reaction of boric acid esters, forming a viscoelastic precursor solution containing a chemical cross-linking network; S2: Disperse polydopamine-modified tetragonal barium titanate piezoelectric particles in the precursor solution to obtain a mixed polymer solution; S3: The polymer solution is subjected to a freeze-thaw cycle to allow polyvinyl alcohol and boric acid-functionalized chitosan to form a double network structure through physical cross-linking, thereby obtaining the electromechanically coupled self-powered biomimetic periosteal hydrogel.

[0010] This invention addresses the two core needs of pediatric cranial defect repair: biomechanical adaptation and regenerative regulation. Starting from the composition and function of the natural periosteum, it achieves functional synergy through material selection and network structure design. Regarding biomechanical adaptation, this invention uses polyvinyl alcohol (PVA) and boric acid-functionalized chitosan (CS) to construct a dual-network matrix. During blending, the phenylboronic acid groups on the boric acid-functionalized chitosan and the hydroxyl groups on the PVA form dynamic covalent bonds in the borate ester, constituting the first layer of chemical cross-linking network, endowing the material with dynamic characteristics of reversible fracture and recombination. Subsequently, through freeze-thaw cycles, the PVA molecular chains form physical cross-linking points in the microcrystalline region through hydrogen bonding, constituting the second layer of physical cross-linking network, enhancing the structural stability of the material.

[0011] The synergistic effect of this dual cross-linked network gives the hydrogel a unique viscoelastic mechanical response mechanism. Under low strain rates simulating slow skull growth, the dynamic borate ester bonds can undergo stress relaxation, causing the material to undergo permanent deformation without restricting tissue expansion. Under high strain rates simulating external impact, the physical cross-linked network provides rigid support, exhibiting elastic resistance.

[0012] In terms of bioactivity regulation, this invention introduces polydopamine-modified tetragonal barium titanate (BTO) piezoelectric particles into a dual-network matrix. The polydopamine modification layer improves the dispersion uniformity of BTO particles in the polymer matrix, preventing aggregation and failure. After the piezoelectric particles are uniformly dispersed in the viscoelastic matrix, the matrix, acting as a stress transmission medium, effectively transmits the weak physiological stresses from children's daily chewing and skull growth to the BTO particles, causing lattice distortion and generating endogenous electrical signals. This enables self-powered voltage output without relying on external ultrasound or power supply equipment. This electrical signal can promote the directional migration, proliferation, and vascularization of endothelial cells and activate osteogenic signaling pathways. Simultaneously, the viscoelastic matrix itself is conducive to cell spreading and integrin / focal adhesion aggregation, upregulating the expression of vascular-related proteins.

[0013] Preferably, the method for preparing boric acid-functionalized chitosan in S1 includes: Chitosan was dissolved in an alkaline solution, and 3-chloro-1-propanol was added to carry out a hydroxypropylation reaction to obtain hydroxypropylated chitosan. The hydroxypropylated chitosan was subjected to an amidation reaction with 4-hydroxyphenylboronic acid in the presence of a condensing agent to graft phenylboronic acid groups, thereby obtaining boric acid-functionalized chitosan.

[0014] Preferably, the mass ratio of chitosan to 3-chloro-1-propanol is 4:(5~10).

[0015] Preferably, the condensing agent includes carbodiimide condensing agents and N-hydroxysuccinimide catalysts.

[0016] Preferably, the mass ratio of polyvinyl alcohol to boric acid-functionalized chitosan in S1 is (5~10):3.

[0017] Preferably, the mass of the tetragonal barium titanate piezoelectric particles in S2 is 5% of the mass of the polyvinyl alcohol.

[0018] As a preferred embodiment, the freeze-thaw cycle in S3 is as follows: freeze at -20°C and then thaw at room temperature, repeating 2 to 3 times.

[0019] Secondly, the present invention provides a force-electric coupling self-powered biomimetic periosteal hydrogel.

[0020] Thirdly, this invention provides the application of a force-electric coupling self-powered biomimetic periosteal hydrogel in the preparation of materials for repairing skull defects in children.

[0021] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention constructs a chemical cross-linking network through dynamic covalent cross-linking of borate esters between polyvinyl alcohol and boric acid-functionalized chitosan, combined with freeze-thaw induced physical cross-linking to form a dual-network structure, endowing the hydrogel with a unique viscoelastic mechanical response mechanism. Dynamic tensile experiments show that the material exhibits a smooth stress response at a low strain rate of 0.005 mm / s and undergoes permanent deformation after unloading. Balloon inflation experiments confirm that this material does not restrict balloon expansion like pure polyvinyl alcohol elastic hydrogels, and retains a matching deformation contour after deflation. This demonstrates that it can adapt to the slow growth characteristics of children's skulls, avoid restricting brain development or inducing skull deformities, and effectively solves the clinical pain point of traditional elastic repair materials restricting skull expansion and thus limiting brain development.

[0022] 2. This invention disperses polydopamine-modified tetragonal barium titanate piezoelectric particles in a dual-network viscoelastic matrix, enabling the material to trigger intrinsic piezoelectric signal output solely through natural stress generated by physiological activities such as skull growth and daily chewing in children. This eliminates the need for external ultrasound or power supply equipment, avoiding the clinical burden, risk of secondary trauma, and long-term maintenance costs associated with external devices. Simultaneously, the viscoelastic matrix, acting as a stress transmission medium, ensures efficient signal output of the piezoelectric particles under weak physiological stress, achieving a self-powered operating mode.

[0023] 3. This invention significantly promotes angiogenesis and bone repair through a mechano-electro-coupled closed-loop synergistic mechanism, in which stress is transmitted through a viscoelastic matrix, electrical signals are generated by piezoelectric particles, these electrical signals promote angiogenesis, and angiogenesis drives bone repair. In vitro experiments show that the spreadable area of ​​endothelial cells in the mechano-electro-coupled hydrogel group is approximately 18% higher than that in the non-piezoelectric group and approximately 32% higher than that in the pure elastic group. The cell migration rate in the scratch assay after 12 hours is approximately 40% higher than that in the control group. Matrigel angiogenesis assays show an increase of approximately 35% in total vessel length and approximately 30% in total vessel area. The CD31 immunofluorescence intensity is approximately 2.1 times higher than that in the pure elastic group. In vivo, after implantation in a rat skull defect model for 4 weeks, Micro-CT quantitative analysis shows that the bone volume fraction is approximately 210% higher than that in the blank control group, the trabecular bone thickness is approximately 180% higher, and the number of trabecular bone is approximately 150% higher. Furthermore, no obvious inflammatory reaction was observed in tissue sections, and the hemolysis rate was less than 5%, meeting the safety standards for biomaterials. This provides a complete technical solution for the functional repair of skull defects in children. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The morphology and basic properties of the hydrogel provided in the embodiments of this application are shown in the figure; wherein Figure 1 a is a scanning electron microscope image (scale bar: 10 μm, 5 μm); Figure 1 b is a scanning electron microscope image of the degraded product (scale bar: 10 μm, 5 μm). Figure 1 c is the surface distribution of energy spectrum elements (scale bar: 10 μm). Figure 1 d represents the adhesion performance diagram; Figure 2 The physicochemical properties characterization diagram of the hydrogel provided in the embodiments of this application is shown; wherein Figure 2 a is the X-ray diffraction pattern of tetragonal barium titanate with different particle sizes; Figure 2 b shows the piezoelectric output voltage of hydrogels with different barium titanate concentrations; Figure 2 c is the swelling curve; Figure 2 d represents the degradation curve; Figure 2 e and 2f are rheological curves of self-healing properties; Figure 2 g and 2h are tensile stress-strain curves and Young's modulus diagrams; Figure 2 i represents the stress-strain curves at different tensile rates; Figure 2 j is a diagram of a balloon inflation experiment; Figure 3 This paper illustrates a biocompatibility evaluation diagram of the hydrogel provided in an embodiment of this application for endothelial cells; wherein... Figure 3 a is a fluorescence image of live / dead cells stained; Figure 3 b is a fluorescent staining image of the cytoskeleton (Phalloidin) / nucleus (DAPI); Figure 3 c is a statistical chart of cell viability; Figure 3 d is a statistical graph of cell spreading area; Figure 3 e is a statistical graph of cell nuclear area; Figure 3 f is the CCK-8 absorbance result graph; Figure 3 g represents the hemolysis experiment diagram; Figure 3 h represents a statistical chart of hemolysis rate; Figure 4 An in vitro angiogenesis capacity evaluation diagram provided in an embodiment of this application is shown; wherein Figure 4 a is a scratch test diagram (scale bar: 200 μm); Figure 4 b is a diagram from Matrigel's in vitro angiogenesis experiment; Figure 4 c is the CD31 immunofluorescence staining image; Figure 4 d is a statistical chart of the area of ​​scratch healing; Figure 4 e is a statistical chart of the total blood vessel area; Figure 4 f is a statistical chart of the total length of blood vessels; Figure 4 g is a statistical graph of CD31 fluorescence intensity; Figure 5 The following diagram illustrates the in vivo angiogenesis and bone repair evaluation provided in an embodiment of this application; wherein... Figure 5 a is a hematoxylin-eosin staining image of the defective area; Figure 5 b is a Micro-CT 3D reconstruction image (including coronal and transverse sections); Figure 5 c is the CD31 immunofluorescence staining pattern (red: CD31, blue: DAPI). Figure 5 d is a quantitative statistical chart of trabecular bone thickness; Figure 5 e is a quantitative statistical graph of bone volume fraction; Figure 5 f is a quantitative statistical chart of the number of trabeculae. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention. Furthermore, all other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of the present invention.

[0027] Unless otherwise stated, the raw materials and reagents used in the various embodiments of this invention are all commercially available products conventional in the art. Specifically: Chitosan (CS, degree of deacetylation ≥ 95%) and polyvinyl alcohol (PVA, degree of alcoholysis 98%~99%, degree of polymerization 1700) were purchased from Sigma-Aldrich; lithium hydroxide, potassium hydroxide, urea, 4-hydroxyphenylboronic acid, N-hydroxysuccinimide (NHS), and morpholine ethanesulfonic acid (MES) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dopamine hydrochloride were purchased from Sigma-Aldrich; hydrochloric acid was of analytical grade and purchased from Chengdu Kelong Chemical Co., Ltd.

[0028] PBS buffer was purchased from Wuhan Prosano Life Science Co., Ltd.; 0.25% trypsin (containing EDTA), 4% paraformaldehyde, and goat anti-rabbit IgG H&L (Dylight 488) were purchased from Boster Biologics Inc. (USA); bovine serum albumin (BSA), Triton X-100, and 4,6-diamino-2-phenylindole dihydrochloride (DAPI) were purchased from Beyotime Biotechnology Co., Ltd.; Alexa Fluor 488-phalloidin was purchased from Invitrogen Inc. (USA); fluorescein diacetic acid (FDA) and propidium iodide (PI) were purchased from Uelandy Biotechnology Co., Ltd.; Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Biotechnology Co., Ltd.; and CD31 Rabbit pAb was purchased from Chengdu Zhengneng Biotechnology Co., Ltd.

[0029] All other reagents not listed were of analytical grade and can be obtained through conventional commercial channels. Unless otherwise specified, the methods used in the examples are standard practices in the art.

[0030] Specific experimental steps or conditions are not specified in the embodiments; they can be performed according to the conventional experimental steps or conditions described in the prior art. Reagents and other instruments used, unless otherwise specified, are all commercially available conventional reagent products. Furthermore, the accompanying drawings are merely illustrative diagrams of the embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore, repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0031] Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of this specification.

[0032] In the description of this invention, it should be understood that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore should not be construed as limiting the scope of protection of this invention.

[0033] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] To enable those skilled in the art to better understand this application, the following embodiments are provided to illustrate in detail a mechano-electro-coupled self-powered biomimetic periosteal hydrogel, its preparation method, and its application.

[0035] Example

[0036] Example 1: Preparation of a Mechano-Electrically Coupled Self-Powered Biomimetic Periosteal Hydrogel

[0037] (1) Preparation of boric acid functionalized chitosan Weigh 4.5 g lithium hydroxide, 7 g potassium hydroxide, and 8 g urea and dissolve them in 80.5 g deionized water. Stir until completely dissolved. Freeze the resulting alkaline solution at -20°C overnight. After freezing, thaw at room temperature. Add 1 g CS to an ice-water bath and stir vigorously for 2 h. Then freeze at -20°C. After freezing, stir at room temperature. Repeat the freeze-thaw cycle 3 times until the mixed solution is clear and there are no obvious insoluble substances. This gives you the CS alkaline solution.

[0038] Weigh out 3-chloro-1-propanol and add it to the above alkaline solution at a mass ratio of CS to 3-chloro-1-propanol of 4:7. Stir the reaction mixture at room temperature for 24 h. Adjust the pH of the reaction solution to 7 with 0.1 mol / L hydrochloric acid solution and dialyze it in a dialysis bag for 7 days.

[0039] Prepare 100 mL of 0.05 M MES buffer solution. Filter the dialyzed reaction solution and add the precipitate to the MES buffer solution. Weigh 1 g of 4-hydroxyphenylboronic acid, 1.175 g of EDC·HCl, and 0.85 g of NHS, dissolve them in 10 mL of N,N-dimethylformamide, and add the solution to the buffer mixture. Stir the mixture at room temperature in the dark for 48 h. Dialyze the reaction solution in a dialysis bag for 7 days and freeze-dry for 48 h to obtain boric acid-functionalized CS.

[0040] (2) Preparation of polydopamine-modified tetragonal barium titanate piezoelectric particles Tetragonal BTO piezoelectric particles were dispersed in Tris-HCl buffer (pH=8.5) and ultrasonically dispersed until homogeneous. Dopamine hydrochloride was added to a final concentration of 2 mg / mL, and the mixture was stirred at room temperature for 12 h to allow dopamine to undergo a self-polymerization reaction on the surface of the BTO particles, forming a polydopamine coating layer. After the reaction was completed, the particles were centrifuged, washed three times with deionized water, and freeze-dried to obtain polydopamine-modified tetragonal BTO piezoelectric particles.

[0041] (3) Preparation of self-powered biomimetic periosteal hydrogel with electromechanical coupling Weigh 15 g of PVA and dissolve it in 85 g of deionized water. Stir in a water bath at 80°C until completely dissolved to prepare a 15% PVA solution. Weigh the above boric acid-functionalized CS and dissolve it in deionized water to prepare a 0.1% CS solution.

[0042] PVA solution and CS solution were mixed at a mass ratio of 7:3 (PVA to boric acid-functionalized CS) and stirred in an 80°C water bath to induce a dynamic covalent cross-linking reaction between the phenylboronic acid groups and the PVA hydroxyl groups, forming a viscoelastic precursor solution containing a chemical cross-linking network.

[0043] Weigh out 5% (by mass) of polydopamine-modified tetragonal BTO piezoelectric particles from PVA powder, disperse them in 0.5 mL of deionized water, add them to the above precursor solution, and stir for 1 h to obtain a mixed polymer solution. Pour the mixed polymer solution into a mold, freeze it overnight at -20°C, and then thaw it at room temperature to obtain a self-powered biomimetic periosteum hydrogel with electrocoupling.

[0044] Example 2 Characterization of hydrogels To verify the influence of the dual-network structure and piezoelectric effect on the material properties in this invention, three hydrogel samples were prepared in parallel in this embodiment. All three groups of samples were prepared using the same process as step (3) in Example 1, with only the matrix composition being different. The specific grouping and composition are shown in Table 1.

[0045] Table 1. Grouping and composition of the three groups of hydrogel samples

[0046] Preparation of PVA group (pure elasticity control): Weigh 15 g of PVA and dissolve it in 85 g of deionized water. Stir in an 80°C water bath until completely dissolved to prepare a 15% PVA solution. Pour the PVA solution into a mold and freeze at -20°C overnight. After thawing at room temperature, the PVA hydrogel is obtained.

[0047] Preparation of PCA group (viscoelastic control): Following the method of step (3) in Example 1, PVA solution and boric acid functionalized CS solution were mixed at a ratio of 7:3 and stirred in a water bath at 80°C to undergo dynamic covalent cross-linking reaction of boric acid ester to form a precursor solution. After freezing at -20°C overnight and thawing at room temperature, PCA hydrogel was obtained.

[0048] Preparation of PCB assembly (the present invention): PCB hydrogel is obtained by following the steps (1) to (3) of Example 1.

[0049] The PVA, PCA and PCB hydrogels were freeze-dried and their microstructures were observed using scanning electron microscopy (SEM).

[0050] SEM images such as Figure 1 As shown in Figure a, compared to PVA hydrogels, the pore sizes of dual-network hydrogels PCA and PCB are larger. However, the average pore size of all three hydrogels is controlled below 1 μm. This pore size range can ensure the transmembrane transport of water, oxygen, and small molecule nutrients, and can also act as a physical barrier to effectively block the penetration and invasion of fibroblasts, thus preventing fibrosis in the defective area.

[0051] The PCB hydrogel was immersed in PBS solution at 37°C for two months. After immersion, it was observed using SEM, and the results are as follows: Figure 1 As shown in b, the pore size of the hydrogel increased after soaking, but the internal three-dimensional network structure remained intact and did not collapse, indicating that the PCB hydrogel prepared by this invention has good long-term structural stability and can maintain structural integrity during the in vivo repair cycle.

[0052] Elemental surface distribution analysis of the PCB hydrogel was performed using energy dispersive spectroscopy (EDS). The results are as follows: Figure 1 As shown in c, the Ba and Ti elements in the BTO nanoparticles are uniformly distributed in the hydrogel matrix, indicating that the polydopamine-modified BTO piezoelectric particles have good dispersibility in the dual-network matrix and no obvious agglomeration.

[0053] The PCB hydrogel was placed on the surfaces of plastic, glass, metal, and pig skin, and its adhesion was observed. The results are as follows: Figure 1As shown in d, the PCB hydrogel can maintain a relatively stable adhesive state on the surface of the aforementioned materials. When the hydrogel is applied to the surface of a finger and bent, the hydrogel can deform with the finger movement and remain adhered, indicating that the hydrogel has good tissue adhesion and deformation following ability, which is beneficial for maintaining stable contact with the defect site during in vivo repair.

[0054] Example 3: Characterization of the physicochemical properties of the hydrogel X-ray diffraction (XRD) was used to analyze the crystal phase of BTO powders with particle sizes of 4 μm and 400 nm. The results are as follows: Figure 2 As shown in Figure a, both BTO powders of different particle sizes exhibit characteristic double peaks near 2θ = 45°, indicating that they are tetragonal BTO, and this crystal form has better piezoelectric properties than cubic BTO. Subsequent experiments used 400 nm BTO powder.

[0055] The voltage output of hydrogels with different BTO concentrations (0%, 5%, 7.5%, 10%) was tested under the same mechanical loading conditions. The results are as follows: Figure 2 As shown in b, when the BTO concentration is 5%, the output voltage of the hydrogel increases significantly; when the BTO concentration increases to 7.5%, the output voltage tends to level off; when the BTO concentration increases to 10%, the output voltage decreases instead. This may be attributed to the fact that BTO particles are more prone to aggregation at high concentrations, leading to the cancellation of positive and negative voltages under external force. Therefore, 5% is determined to be the optimal doping concentration of BTO.

[0056] Three groups of hydrogels, namely PVA, PCA, and PCB, were placed in PBS solution, and their swelling ratios were measured over time. The results are as follows: Figure 2 As shown in c, the PCB hydrogel tends to saturate after soaking for 2 hours, and the swelling rate is the lowest among the three groups. A smaller swelling rate is beneficial to reduce the compression of surrounding tissues after implantation.

[0057] The PCB hydrogel was immersed in PBS solution at 37°C, and the change in its weight loss over time was measured. The results are as follows: Figure 2 As shown in d, the weight loss of the PCB hydrogel after two months of soaking is about 30%, indicating that the hydrogel has a suitable degradation rate in vivo and can exist for a long time during the bone repair cycle and continuously assist in tissue repair.

[0058] Strain step tests were performed on PCB hydrogels using a rheometer to evaluate their self-healing properties. First, strain scanning was performed to determine the network failure critical point, and the results (…) Figure 2(e.g., 2f.) shows that G' and G” cross at 580% strain. Therefore, the maximum strain was set to 700%, the minimum strain to 1%, and the test temperature to 37℃. When the 1% minimum strain was applied, G' > G”, the hydrogel was mainly elastic and in a stable gel state; when the 700% maximum strain was applied, G' < G”, the network structure was temporarily disrupted and became mainly viscous; after the strain was removed and the hydrogel was allowed to heal itself, the 1% minimum strain was applied again, and G' recovered to > G”, indicating that the hydrogel network reformed. The results of multiple cycles of testing were consistent, proving that the PCB hydrogel has good self-healing properties.

[0059] Tensile tests were used to evaluate the mechanical properties of each group of hydrogels. Results ( Figure 2 The results (g, 2h) show that the tensile modulus of both PCA and PCB hydrogels is lower than that of PVA hydrogel, but the tensile modulus of PCB hydrogel is slightly higher than that of PCA hydrogel. This may be because the addition of BTO nanoparticles enhances the mechanical strength of the dual-network matrix.

[0060] Dynamic mechanical tensile tests were conducted on the PCB hydrogel at different stretching rates. The results are as follows: Figure 2 As shown in Figure i, the tensile stress response of the hydrogel is gradual at a low strain rate of 0.05 mm / s; as the tensile rate gradually increases to 0.5 mm / s, 5 mm / s and 50 mm / s, the stress response increases synchronously, exhibiting strain rate-dependent mechanical behavior.

[0061] A balloon inflation experiment was used to simulate the mechanical effects of slow skull growth on materials, with structures such as... Figure 2 As shown in Figure j, PVA, PCA, and PCB hydrogels were respectively wrapped around the surface of a balloon, and the balloon was slowly inflated. The results showed that the PCA and PCB hydrogels adhered tightly to the balloon wall and expanded synchronously with the balloon, without restricting its deformation; while the PVA hydrogel severely restricted the balloon's deformation, forcing the gas in the middle of the balloon to expand towards both ends. After slow deflating, the PCB hydrogel retained a permanent deformation profile matching the shape of the inflated balloon. These results indicate that the PCB hydrogel prepared in this invention possesses viscoelasticity, self-healing properties, and permanent deformation capability under low strain, enabling it to adapt to the slow growth of the skull.

[0062] Example 4: Evaluation of the biocompatibility of hydrogels with endothelial cells To increase the surface adhesion sites of the hydrogels, PVA, PCA, and PCB hydrogels were immersed in a 0.1 mol / L polydopamine solution for 5 h, then washed with distilled water and immersed for 24 h. Subsequently, fibronectin (FN) was coated onto the surface of the hydrogels at a concentration of 100 μg / mL and modified overnight at 4 °C.

[0063] Human umbilical vein endothelial cells (HUVECs) were seeded onto the surfaces of the modified PVA, PCA, and PCB hydrogels, respectively. Cells seeded only on the surface of the wells served as the control group. After a certain period of culture, live / dead fluorescence staining was performed, and cell viability was observed and calculated using confocal microscopy. Results ( Figure 3 (a) and (3c) showed that the cell activity in each group was good, and statistical analysis showed no significant difference between the groups, which preliminarily indicates that none of the three hydrogels had obvious cytotoxicity.

[0064] The proliferation activity of HUVECs in each group was quantitatively detected using a CCK-8 assay kit. The results are as follows: Figure 3 As shown in f, the absorbance values ​​of each hydrogel group were not significantly different from those of the Control group, further indicating that PVA, PCA and PCB hydrogels were not cytotoxic to HUVECs.

[0065] HUVECs were fluorescently stained with Phalloidin / DAPI to label the cytoskeleton (F-actin, green) and cell nuclei (DAPI, blue), respectively, and the spreading morphology of cells on the hydrogel surface was observed. Figure 3 (b, 3d~3e). Statistical analysis showed no significant difference in nuclear area among the groups, but significant differences in cell spreading area. The spreading area of ​​HUVECs was smaller in all hydrogel groups than in the Control group, presumably due to the lower mechanical stiffness of the hydrogel compared to the well plate surface, and the limited adhesion sites on the hydrogel surface restricting initial cell anchoring and spreading. However, among the three hydrogels, the cell spreading area in the PCA and PCB groups was significantly larger than that in the purely elastic PVA group, indicating that the viscoelastic dual-network hydrogel is more conducive to cell spreading and maintaining the spreading state. This may be because the viscoelastic matrix can undergo stress relaxation, reducing the mechanical resistance experienced by cells during spreading, thereby promoting the aggregation and maturation of integrins and adhesion focal points. The cell spreading area in the PCB group was slightly larger than that in the PCA group, attributed to the endogenous electrical signals generated by the BTO piezoelectric particles during the interaction between cells and the hydrogel. Electrical stimulation promoted cytoskeleton rearrangement, further increasing the cell spreading area. The nuclear area did not change significantly among the groups, indicating that the mechanical properties and electrical signals of the material mainly affected the reconstruction and spreading morphology of the cytoskeleton, and had not yet had a significant impact on the nuclear structure.

[0066] Fresh rabbit blood was collected, anticoagulated, and then incubated with PVA, PCA, and PCB hydrogel extracts, respectively. Physiological saline was used as a negative control, and deionized water as a positive control. After incubation, the supernatant was collected by centrifugation, and the absorbance at 540 nm was measured to calculate the hemolysis rate. Figure 3(g~3h). The results showed that the hemolysis rate of all three groups of hydrogels was less than 5%, which meets the safety standard for hemolysis rate of biomedical materials (GB / T 16886.4), indicating that the hydrogel has good blood compatibility.

[0067] Example 5 Evaluation of in vitro angiogenesis capacity HUVECs were seeded in six-well plates and cultured until they reached a monolayer confluence state. Uniform scratches were created on the bottom of the wells using a 200 μL pipette tip to simulate defect areas. PVA, PCA, and PCB hydrogel extracts were then added for further culture. Scratch-treated cells without material treatment served as the control group. Cell migration was observed after 12 h of culture. Results ( Figure 4 (a) and (d) showed that the PCB group cells migrated most significantly to the scratch area, and the percentage of the healing area was significantly greater than that of the Control group and the PVA group. Quantitative analysis showed that there was a statistical difference, indicating that the mechano-electro-coupled signal released by the PCB hydrogel can effectively promote the directional migration of endothelial cells.

[0068] HUVECs were seeded in Matrigel-coated plates and cultured with PVA, PCA, and PCB hydrogel extracts, respectively. Cells without material treatment served as the control group. After 6 hours of culture, the formed tubular structures were stained and observed. Figure 4 b). The results showed that obvious vascular branching and node formation were observed in the PCB group and PCA group, while the vascular structure was sparse in the Control group and PVA group, with no obvious branches and nodes. Quantitative analysis of the vascular network was performed using ImageJ software. Figure 4 (e~4f) The total length and total area of ​​blood vessels in the PCB group were greater than those in the other groups, but there was no significant difference. This may be related to the lack of a complex in vivo mechanical microenvironment under in vitro culture conditions.

[0069] HUVECs were labeled using CD31 immunofluorescence staining. CD31 is a specific marker of vascular endothelial cells, and its fluorescence expression intensity can reflect the angiogenic differentiation capacity of endothelial cells. Figure 4 (c, 4g). The results showed that the CD31 fluorescence intensity in the PCA and PCB groups was significantly stronger than that in the Control and PVA groups. This is attributed to the viscoelastic matrix promoting the aggregation and maturation of integrins and focal adhesions, and upregulating the expression of vascular-related proteins. The CD31 fluorescence intensity in the PCB group was slightly higher than that in the PCA group, presumably because the electrical signal generated by the BTO piezoelectric particles further activated the angiogenic differentiation pathway of endothelial cells, enhancing angiogenesis. These results indicate that the mechano-electro-coupled effect has a stronger pro-angiogenic potential than viscoelastic stimulation alone.

[0070] Example 6 Evaluation of in vivo angiogenesis and bone repair Eight-week-old male SD rats underwent surgery to create a 5 mm diameter full-thickness skull defect at the top of the skull. The rats were randomly divided into four groups: a blank control group (no material implanted at the defect site), a PVA group (PVA hydrogel implanted), a PCA group (PCA hydrogel implanted), and a PCB group (PCB hydrogel implanted), with five rats in each group. The hydrogel in each group was trimmed into a circular sheet matching the size of the defect area, implanted into the defect site, and the material was kept in close contact with the defect edges. The wound was then sutured. Rats were fed normally post-operatively.

[0071] Four weeks after implantation, rats were sacrificed, and tissue from the skull defect area was collected. After fixation, decalcification, embedding, and sectioning, hematoxylin and eosin (H&E) staining was performed. Figure 5 a). The results showed that no obvious inflammatory cell infiltration or tissue necrosis was observed in the defect areas of rats in each group, indicating that PVA, PCA and PCB hydrogels all have good biocompatibility in vivo.

[0072] Four weeks after implantation, Micro-CT scans were used to examine the skull defect areas of rats in each group, and three-dimensional reconstruction and quantitative analysis were performed. From the coronal and transverse images, it can be seen that ( Figure 5 (b) In the PCB group, significant bone regeneration was observed in the defect area, and the bone defect area was significantly reduced, while no significant bone repair was observed in the defect area of ​​the blank control group. Quantitative analysis results ( Figure 5 The d-5f results showed that the bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and number of trabecular bones (Tb.n) in the PCB group were significantly higher than those in the other three groups, and the differences were statistically significant, indicating that PCB hydrogel can effectively promote bone regeneration in skull defects.

[0073] CD31 immunofluorescence staining was performed on the newly formed tissue in the defect area of ​​each group to assess the formation of new blood vessels. Figure 5 c). The results showed that the CD31 fluorescence intensity was strongest around the defect area in the PCB group, and the area of ​​the fluorescent expression region was also significantly larger than that in the other groups, indicating that the PCB hydrogel can effectively promote angiogenesis in the defect area. This result is consistent with the in vitro angiogenesis evaluation results in Example 5, further verifying the core mechanism of the hydrogel of this invention in promoting angiogenesis and driving bone repair through mechano-electro-coupled effects.

[0074] In summary, this invention provides a self-powered biomimetic periosteal hydrogel with electro-coupling, its preparation method, and its application. The hydrogel uses polyvinyl alcohol and boric acid-functionalized chitosan to construct a chemical cross-linking network through dynamic covalent cross-linking of borate esters, combined with physical cross-linking induced by freeze-thaw cycles, to form a dual-network viscoelastic matrix, and uniformly disperses tetragonal barium titanate piezoelectric particles modified with polydopamine.

[0075] This invention achieves a synergistic unity of multiple technical effects through the above-mentioned material system and network structure design: In terms of mechanical adaptability, the reversible fracture-recombination characteristics of the dynamic covalent bonds of borate esters and the dual network structure enable the material to undergo permanent deformation at low strain rates to match the slow growth of children's skulls, and exhibit elastic rigidity at high strain rates to resist external impacts. Balloon experiments and tensile experiments at different rates have verified its unique viscoelastic mechanical response mechanism. In terms of self-powered voltage and electrical performance, the viscoelastic matrix acts as a stress transmission medium to transmit weak physiological stress to uniformly dispersed BTO piezoelectric particles, triggering the output of endogenous electrical signals without relying on external ultrasound or power supply equipment. In terms of biological function, through the mechano-electric coupling closed-loop synergistic mechanism of stress transmission by the viscoelastic matrix, electrical signal generation by piezoelectric particles, promotion of angiogenesis by electrical signals, and bone repair driven by angiogenesis, it significantly promotes endothelial cell migration and spread and vascular network formation. In vivo rat skull defect model experiments have confirmed that its osteogenic indicators, such as bone volume fraction, trabecular thickness, and trabecular number, are significantly better than those of the control group.

[0076] This invention effectively solves the common problem of existing pediatric skull defect repair materials failing to simultaneously achieve mechanical adaptation and regenerative regulation. It achieves multiple synergistic functions—adapting to the dynamic growth of the child's skull, promoting angiogenesis, and repairing bone defects—without external stimulation. The preparation method of this invention is stable, highly controllable, and exhibits good biocompatibility, demonstrating promising clinical application prospects.

[0077] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0078] Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present application.

[0079] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0080] The foregoing has provided a detailed description of the electro-coupled self-powered biomimetic periosteal hydrogel, its preparation method, and its application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for preparing a self-powered biomimetic periosteum hydrogel with electro-mechanical coupling, characterized in that, Includes the following steps: S1: Polyvinyl alcohol is blended with boric acid-functionalized chitosan to undergo a dynamic covalent cross-linking reaction of boric acid esters, forming a viscoelastic precursor solution containing a chemical cross-linking network; S2: Disperse polydopamine-modified tetragonal barium titanate piezoelectric particles in the precursor solution to obtain a mixed polymer solution; S3: The polymer solution is subjected to a freeze-thaw cycle to allow polyvinyl alcohol and boric acid-functionalized chitosan to form a double network structure through physical cross-linking, thereby obtaining the electromechanically coupled self-powered biomimetic periosteal hydrogel.

2. The method for preparing a self-powered biomimetic periosteum hydrogel with electro-mechanical coupling according to claim 1, characterized in that, The method for preparing boric acid-functionalized chitosan described in S1 includes: Chitosan was dissolved in an alkaline solution, and 3-chloro-1-propanol was added to carry out a hydroxypropylation reaction to obtain hydroxypropylated chitosan. The hydroxypropylated chitosan was subjected to an amidation reaction with 4-hydroxyphenylboronic acid in the presence of a condensing agent to graft phenylboronic acid groups, thereby obtaining boric acid-functionalized chitosan.

3. The method for preparing a self-powered biomimetic periosteum hydrogel with electro-mechanical coupling according to claim 2, characterized in that, The mass ratio of chitosan to 3-chloro-1-propanol is 4:(5~10).

4. The method for preparing a self-powered biomimetic periosteum hydrogel with electro-mechanical coupling according to claim 2, characterized in that, The condensing agents include carbodiimide condensing agents and N-hydroxysuccinimide catalysts.

5. The method for preparing a self-powered biomimetic periosteum hydrogel with electro-mechanical coupling according to claim 1, characterized in that, The mass ratio of polyvinyl alcohol to boric acid-functionalized chitosan in S1 is (5~10):

3.

6. The method for preparing a self-powered biomimetic periosteum hydrogel with electro-mechanical coupling according to claim 1, characterized in that, The mass of the tetragonal barium titanate piezoelectric particles in S2 is 5% of the mass of the polyvinyl alcohol.

7. The method for preparing a self-powered biomimetic periosteum hydrogel with electro-mechanical coupling according to claim 1, characterized in that, The freeze-thaw cycle in S3 is as follows: freeze at -20℃, then thaw at room temperature, and repeat 2 to 3 times.

8. The biomimetic hydrogel with electro-coupled self-powered structure as described in any one of claims 1 to 7.

9. The application of the electro-mechanically coupled self-powered biomimetic periosteal hydrogel as described in claim 8 in the preparation of materials for repairing skull defects in children.

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