Hydrogel based on piezoelectric hydroxyapatite heterostructure, preparation method and application thereof

Abstract

The application relates to a hydrogel based on a piezoelectric hydroxyapatite heterostructure, a preparation method and application, the hydrogel comprising a double-network structure formed by a skeleton and natural polyphenol crosslinking, the skeleton being loaded with a nano-heterostructure; the nano-heterostructure is loaded on the skeleton through microfluidic technology; the nano-heterostructure comprises reduced graphene oxide, gold nanoparticles and piezoelectric hydroxyapatite; the piezoelectric hydroxyapatite is prepared by depositing metal on the surface of hydroxyapatite in situ; the skeleton comprises 3-aminobenzene boronic acid and methyl methacrylated sodium hyaluronate; and the natural polyphenol comprises tannic acid. The microspheres prepared by the microfluidic technology ensure uniform distribution and spatial gradient delivery of the nano-heterostructure, and significant progress is achieved in self-driving, response accuracy, mechanical durability, multi-modal synergy and growth plate injury regeneration specificity, thereby providing a new efficient, safe and minimally invasive regeneration and repair way for growth plate injury.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to hydrogels based on piezoelectric hydroxyapatite heterostructures, their preparation methods, and applications. Background Technology

[0002] Growth plate (epithelial plate) injury is a common orthopedic condition in children and adolescents, primarily caused by trauma, infection, tumors, or iatrogenic factors. It often leads to bone bridge formation, skeletal growth imbalance, limb length discrepancies, or angular deformities, severely impacting children's growth, development, and quality of life. Traditional clinical treatment often involves bone bridge resection combined with fat or polymeric material filling to prevent recurrence, but the success rate is low, and later osteotomy or limb lengthening surgery is frequently required. With the development of tissue engineering and regenerative medicine, injectable hydrogels have shown potential as minimally invasive implant materials in the repair of growth plate injuries. This material can mimic the extracellular matrix (ECM), providing a three-dimensional scaffold to support cell adhesion, proliferation, and differentiation, and possesses good biocompatibility, biodegradability, and injectability. Currently, related technologies are mostly based on natural polymers (such as sodium alginate, hyaluronic acid, or recombinant collagen) to construct hydrogels, often loaded with growth factors, inhibitors, or stem cells to achieve local drug release or cell delivery, promoting cartilage regeneration and inhibiting bone bridge formation.

[0003] Existing technology discloses a bilayer drug-loaded sodium alginate (SA) microsphere hydrogel prepared using flow focusing and microfluidic control methods. The outer layer is loaded with anti-osteobridging factor (Enbrel® + Bevacizumab), and the inner layer is loaded with chondrogenic factor-1 (IGF-1), which is then mixed with bone marrow mesenchymal stem cells (BMSCs) to form a bio-ink. This hydrogel, when injected into the epiphyseal plate injury site, achieves a sequential release that initially inhibits inflammation and angiogenesis, followed by promotion of cartilage regeneration. This technology provides local, timed, and quantitative drug delivery for pediatric epiphyseal plate injuries involving bone bridging, exhibiting minimal invasiveness and bioactivity. Other existing technologies describe a hydrogel drug delivery system based on chitosan and / or alginate, loaded with anti-VEGF compounds, stem cell chemokines, and transforming growth factor-β cytokines. This gel, applied via injection to the growth plate injury site, aims to inhibit bone bridging and promote cartilage regeneration, exhibiting good biocompatibility and injectability, making it suitable for the treatment of pediatric growth plate defects. Existing technologies also disclose a hydrogel system for bone-interface tissue regeneration, particularly suitable for epiphyseal plate repair, including an injectable hydrogel form. This system inhibits osteogenic formation by promoting chondrogenic differentiation of mesenchymal stem cells (MSCs) through controlled morphogenetic diffusion and gradient formation, making it suitable for repairing large epiphyseal plate defects. Its potential to inhibit bone bridge formation and restore growth has been validated in a goat model. This technology emphasizes gradient design and photopolymerization for preparing bulk structures suitable for dynamic bone-interface regeneration.

[0004] While the aforementioned existing technologies have made progress in injectability and drug delivery, several drawbacks remain: First, these systems largely rely on passive drug release or static scaffolds, lacking self-driven mechanisms (such as generating electrical signals using physiological movement). They cannot effectively mimic the dynamic electrophysiological environment of the growth plate to promote directed cell differentiation and calcification, resulting in low regeneration efficiency in areas of high mechanical load. Second, the hydrogels have limited mechanical strength, interfacial stability, and microenvironment responsiveness, making them susceptible to uneven gelation or excessively rapid degradation due to pH, temperature, or inflammation. This makes them unable to precisely match the degradation needs of rapidly growing children and can easily lead to component separation or recurrence of inflammation. Third, while existing technologies can inhibit bone bridging, they are mostly limited to single or dual-factor release, lacking multimodal functions (such as conductive, photothermal, or antibacterial synergy), resulting in insufficient comprehensive therapeutic effects in complex injuries (such as those accompanied by inflammation or infection). Finally, although these hydrogels promote cartilage regeneration in growth plate repair, they often fail to achieve enhanced piezoelectric effects or intelligent responses, leaving room for improvement in overall regeneration specificity and efficiency. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide an injectable hydrogel system based on a piezoelectric hydroxyapatite heterostructure and its preparation method, so as to overcome the shortcomings of existing growth plate damage repair materials in several key aspects. Specifically, it includes: (1) lack of self-driven electrical stimulation mechanism: existing hydrogel systems (such as delivery systems based on sodium alginate microspheres or chitosan / alginate) mostly rely on passive drug release or static scaffold support, and cannot utilize physiological movement (such as mechanical stress generated by joint movement) to generate continuous electrical signals, making it difficult to effectively simulate the natural electrophysiological environment of the growth plate (epithelial plate), thereby limiting the promotion of cell-directed differentiation, calcium ion channel activation and cartilage mineralization process, resulting in low regeneration efficiency in high mechanical load dynamic areas (such as children's growth plates). (2) Insufficient mechanical strength, interfacial stability and biocompatibility: In existing technologies, hydrogels (such as recombinant collagen or bilayer drug-loaded microsphere systems) often have high cross-linking density to improve strength, but this will reduce porosity and hinder the deep infiltration and vascularization of host cells (such as mesenchymal stem cells or chondrocytes); at the same time, the interfacial bonding is mostly physical mixing or simple embedding, which is prone to component separation, delamination or mechanical failure, especially in the inflammatory microenvironment or rapid growth stage, the material is prone to uneven gelation, excessive degradation or local inflammation recurrence, and cannot maintain structural integrity and mechanical matching for a long time. (3) Weak microenvironment responsiveness and intelligent release ability: Existing hydrogels lack intelligent response characteristics to the microenvironment of the damaged site (such as pH decrease, ROS accumulation or glucose concentration increase), which makes it difficult to achieve precise spatiotemporal control of drug / growth factor release (such as sequential release or inflammation-induced release), and it is difficult to effectively inhibit bone bridge formation or promote cartilage regeneration in complex injuries (such as those accompanied by inflammation, infection or abnormal vascular proliferation), thus limiting the overall treatment specificity and efficiency. (4) Lack of multimodal functional synergy: Existing systems are mostly limited to single or dual factor delivery (such as anti-VEGF + chondroitin factor), lacking multimodal synergistic mechanisms such as conductivity, photothermal, antibacterial, and mineralization promotion, and cannot comprehensively address the various pathological processes of growth plate damage (such as inflammation, vascular invasion, ossification tendency and apoptosis). Especially in the highly sensitive environment of pediatrics, it is difficult to achieve efficient antibacterial, immune regulation and tissue integration. (5) Mismatch between degradation rate and children's growth and poor long-term stability: Existing hydrogels often degrade too quickly or too slowly, which is not coordinated with the rapid bone growth and tissue remodeling cycle of children, and is prone to scar tissue hyperplasia, bone bridge recurrence and increased risk of limb deformity. At the same time, the long-term stability of regenerated cartilage (such as anti-ossification ability and durable bioactivity) is insufficient, and problems such as immune rejection, material residue or functional decline are faced in clinical translation.

[0006] To address the shortcomings of existing technologies, this invention provides an injectable hydrogel based on a piezoelectric hydroxyapatite heterostructure. By introducing a multimodal piezoelectric nanostructure and a dynamic response network, it achieves synergistic self-driven electrical stimulation and intelligent drug / factor release, promoting efficient repair and regeneration of growth plate damage. This invention uses an rGO-Au modified piezoelectric hydroxyapatite nanostructure as its functional core, combined with a HAMA-PBA dynamic response network, microfluidic microsphere loading, and TA polyphenol crosslinking, to form a multimodal self-driven injectable hydrogel system. This system achieves continuous electrical stimulation induced by physiological movement, intelligent microenvironment-responsive release, multifunctional synergistic regulation, and precise matching with the growth plate regeneration rate, thereby significantly improving the repair efficiency and long-term regeneration effect of growth plate damage in children.

[0007] The technical solution of the present invention is as follows: A hydrogel based on a piezoelectric hydroxyapatite heterostructure, characterized in that the hydrogel comprises a dual network structure formed by cross-linking a framework with natural polyphenols, wherein the framework is loaded with a nano-heterostructure; the nano-heterostructure is loaded onto the framework using microfluidic technology. The nano-heterostructure (rGO-Au-PHA) comprises reduced graphene oxide (rGO), gold nanoparticles, and piezoelectric hydroxyapatite (PHA); the piezoelectric hydroxyapatite is prepared by in-situ deposition of metal on the surface of hydroxyapatite. The skeleton (HAMA-PBA) comprises 3-aminophenylboronic acid (3-APBA) and sodium methacryloyl hyaluronic acid (HAMA). The natural polyphenols include tannic acid (TA).

[0008] In one embodiment, the concentration ratio of the nanoheterostructure:the skeleton:the natural polyphenol is (1~3) mg / mL:(2~4) wt%:3 wt%.

[0009] In one embodiment, the hydrogel further includes a chondrogenic factor loaded on the framework; the chondrogenic factor content in the piezoelectric hydroxyapatite heterostructure-based hydrogel is 100 μg / mL. The chondrogenic factors include KGN or BMP-7.

[0010] A second aspect of the present invention also provides a method for preparing the above-mentioned hydrogel based on a piezoelectric hydroxyapatite heterostructure, characterized by comprising the following steps: Preparation of piezoelectric hydroxyapatite: Dissolve anhydrous magnesium chloride (MgCl2) in water, add hydroxyapatite (HA), stir, add alkaline solution, adjust pH, continue stirring, wash, collect precipitate, and dry to obtain piezoelectric hydroxyapatite (PHA). Preparation of nano-heterostructures: Graphene oxide (GO) was dispersed in water, sonicated, and mixed with piezoelectric hydroxyapatite. The mixture was heated to react, cooled, centrifuged, washed, and dried to obtain a reduced graphene oxide-piezoelectric hydroxyapatite (rGO / PHA) composite. The reduced graphene oxide-piezoelectric hydroxyapatite composite was dispersed in water, sonicated, and tetrachloroauric acid (HAuCl4) was added. The mixture was stirred, sodium citrate was added, and the mixture was reacted, centrifuged, washed, and dried to obtain the nano-heterostructures. Preparation of the skeleton: Sodium hyaluronate (HA) was dissolved in water, the pH was adjusted, methacrylic anhydride (MA) was added, the reaction was carried out in an ice bath, the reaction was terminated, dialyzed, and freeze-dried to obtain methacrylamide hyaluronic acid; methacrylamide hyaluronic acid was dissolved in water, 3-aminophenylboronic acid (3-APBA) was added, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added, the pH was adjusted, the mixture was stirred in the dark, the pH was adjusted again, dialyzed, and freeze-dried to obtain the skeleton; Preparation of hydrogels: Dissolve photoinitiator in water, add to the skeleton and dissolve, add nano-heterogeneous structure and mix evenly to obtain a mixed solution. Place the mixed solution in a microfluidic device to prepare microdroplets, photocur, wash, and centrifuge to obtain skeleton microgel microspheres loaded with nano-heterogeneous structure; dissolve natural polyphenols to obtain a natural polyphenol solution, add to the skeleton microgel microspheres loaded with nano-heterogeneous structure, mix evenly to obtain hydrogel based on piezoelectric hydroxyapatite heterostructure.

[0011] In one embodiment, the method for preparing the hydroxyapatite includes the following steps: Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and diammonium hydrogen phosphate ((NH4)2HPO4) were dissolved in water and acid-adjusted to form a colloidal solution as the aqueous phase; cyclohexane was added as an emulsifier as the oil phase; the aqueous phase was added dropwise to the oil phase, the mixture was stirred and reacted, washed, subjected to hydrothermal reaction, cooled, centrifuged, washed, and dried to obtain the final product. According to the mass ratio, the calcium content of the calcium nitrate tetrahydrate to the phosphorus content of the diammonium hydrogen phosphate is (1~10):1; The hydrothermal reaction is carried out at a temperature of 100-120°C for 4-10 hours.

[0012] In one embodiment, the emulsifier is a mixed emulsifier comprising Tween 80 and Span 80, wherein the mass ratio of Tween 80 to Span 80 is 1:(1~2.5).

[0013] In one embodiment, in the preparation of piezoelectric hydroxyapatite, the anhydrous magnesium chloride to the hydroxyapatite is in a mass ratio of (0.1~1):1; In the preparation of the nano-heterostructure, the mass ratio of graphene oxide to piezoelectric hydroxyapatite is 1:(2~10); the mass ratio of the reduced graphene oxide-piezoelectric hydroxyapatite composite to tetrachloroauric acid is 1:(0.01~0.05).

[0014] In one embodiment, the stirring time in the preparation of piezoelectric hydroxyapatite is 20-40 min.

[0015] In one embodiment, in the preparation of piezoelectric hydroxyapatite, the alkaline solution is a sodium hydroxide (NaOH) solution, and the stirring time is 1-3 hours.

[0016] In one embodiment, during the preparation of the nano-heterostructure, the heating reaction is carried out at a temperature of 150-200°C for 2-4 hours.

[0017] In one embodiment, during the preparation of the nano-heterostructure, the concentration of tetrachloroauric acid is 1-5 mM, and the stirring time is 0.5-2 h.

[0018] In one embodiment, the overall size of the nanoheterostructure is 100~300 nm in the preparation of the nanoheterostructure.

[0019] In one embodiment, during the preparation of the skeleton, the ratio of hyaluronic acid to methacrylic anhydride is 1 g: (1~5) mL. The mass ratio of the methacryloyl hyaluronic acid to the 3-aminophenylboronic acid is 0.5:(0.1~0.3).

[0020] In one embodiment, the light-protected stirring time during the preparation of the skeleton is 24-48 hours.

[0021] In one embodiment, during the preparation of the hydrogel, the contents of the nano-heterostructure, the framework, and the natural polyphenols in the piezoelectric hydroxyapatite heterostructure-based hydrogel are (1~3) mg / mL, (2~4) wt%, and 3 wt%, respectively. The volume ratio of the loaded nanostructured framework microgel microspheres to the natural polyphenol solution is 1:1.

[0022] In one embodiment, the preparation of the hydrogel further includes loading chondrogenic factors, and the preparation of the hydrogel includes the following steps: A photoinitiator was dissolved in water, added to the framework for dissolution, and then mixed with a nano-heterostructure and chondrogenic factor to obtain a homogeneous solution. The homogeneous solution was placed in a microfluidic device to prepare microdroplets, which were then photocured, washed, and centrifuged to obtain framework microgel microspheres loaded with nano-heterostructure and chondrogenic factor. Natural polyphenols were dissolved to obtain a natural polyphenol solution, which was then added to the framework microgel microspheres loaded with nano-heterostructure and chondrogenic factor and mixed to obtain a hydrogel based on a piezoelectric hydroxyapatite heterostructure. In the hydrogel based on the piezoelectric hydroxyapatite heterostructure, the contents of the nano-heterostructure, the framework, the natural polyphenols, and the chondroitin inducing factor are (1~3) mg / mL, (2~4) wt%, 3 wt%, and 100 μg / mL, respectively.

[0023] The volume ratio of the skeletal microgel microspheres loaded with nano-heterogeneous structures and chondrogenic factors to the natural polyphenol solution is 1:1.

[0024] A third aspect of the present invention also provides the application of the above-described hydrogel based on a piezoelectric hydroxyapatite heterostructure or the hydrogel based on a piezoelectric hydroxyapatite heterostructure obtained by the above preparation method in the preparation of growth plate damage repair drugs.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) Self-driven multimodal electrophysiological stimulation was achieved: Through the rGO-Au modified piezoelectric hydroxyapatite heterostructure, a continuous microcurrent (1~10mV) was generated under physiological mechanical load. Without the need for external power supply or auxiliary stimulation such as ultrasound, calcium ion channels, Wnt / β-catenin and PI3K / AKT signaling pathways were activated, promoting the efficient and directed differentiation of mesenchymal stem cells into chondrocytes (SOX9, Col2A1 and Aggrecan expression were significantly upregulated), effectively inhibiting hypertrophy and bone bridge formation, which is significantly better than the regeneration efficiency of existing passive drug release or static scaffold systems in dynamic growth plate environment.

[0026] (2) It has intelligent microenvironment response and precise release capabilities: The HAMA-PBA dynamic borate ester network is highly sensitive to pH increase (7.5~8.5), ROS accumulation and high sugar environment at the injury site. It can achieve inflammation-induced self-healing, local expansion and gradient, multi-stage precise release of cartilage-inducing factors (such as KGN, BMP-7), avoiding the problem of local toxicity or inflammation aggravation caused by sudden drug release in the existing technology, and improving the specificity and bioavailability of the treatment.

[0027] (3) Significantly improves mechanical strength, tissue adhesion and long-term stability: The dual network structure formed by TA polyphenol cross-linking endows the hydrogel with excellent compressive modulus (20~80kPa, matching the growth plate cartilage), fatigue resistance and strong wet adhesion (lasting adhesion at the dynamic bone interface), while the free radical scavenging rate is >85%, significantly inhibiting the expression of inflammatory factors such as TNF-α, IL-1β and IL-6, reducing the risk of fibrosis and inflammation recurrence; the degradation cycle matches the rapid bone remodeling, overcoming the defects of existing hydrogels such as uncoordinated degradation rate, easy residue or premature failure.

[0028] (4) More comprehensive multi-functional synergistic effect: It integrates multiple mechanisms such as piezoelectric stimulation, conduction enhancement (rGO), photothermal / antibacterial (AuNPs), and antioxidant / anti-inflammatory (TA) to achieve electro-biological-immune multimodal synergistic regulation. It shows stronger comprehensive therapeutic effect in complex injury environments (such as those accompanied by inflammation or mild infection). In vitro / in vivo experiments show that the differentiation efficiency of MSCs is increased by 20-40%, and the bone bridge inhibition rate is significantly higher than that of single factor delivery systems.

[0029] (5) The advantages of minimally invasive and personalized application are prominent: the loaded microspheres prepared by microfluidic technology ensure the uniform distribution of nano-heterogeneous structures and spatial gradient delivery. The hydrogel has shear-thinning properties and can be injected into irregular damaged areas through a 22~25G injection needle. It can quickly form gel by short-time room temperature self-crosslinking (10~30s), realizing in-situ molding and personalized filling. The operation is simple and the trauma is small. It is especially suitable for pediatric clinical application and has higher safety and translational potential.

[0030] In summary, this invention has made significant progress in terms of self-driving ability, precise response, mechanical durability, multimodal synergy, and specificity for regeneration of pediatric growth plate injuries, providing a new, efficient, safe, and minimally invasive regenerative repair approach for growth plate injuries. Attached Figure Description

[0031] Figure 1 SEM images of HAMA-PBA@rGO-Au-PHA@BMP-7 microspheres; Figure 2 SEM image of HAMA-PBA@rGO-Au-PHA@BMP-7 / TA hydrogel; Figure 3 Representative images of intracellular ROS levels in each group were obtained using the DCFH-DA fluorescent probe. Figure 4 Images of Safranin O / Fast Green stained at 8 weeks post-surgery for each group. Detailed Implementation

[0032] The present invention provides a multimodal self-driven injectable hydrogel system based on a piezoelectric hydroxyapatite heterostructure and its preparation method, for the repair and regeneration of growth plate damage. The system uses rGO-Au modified piezoelectric hydroxyapatite (PHA) nanostructures (100-300 nm in size) as the core functional unit, generating a continuous microcurrent (1-10 mV) under physiological mechanical stress. This activates calcium ion channels, Wnt / β-catenin, and PI3K / AKT pathways, promoting mesenchymal stem cell differentiation into chondrocytes and inhibiting osteobridging. Using HAMA-PBA as the main network framework, the system leverages the sensitivity of phenylboronic acid ester bonds to pH, glucose, and ROS to achieve network expansion, self-healing, and loading of chondrogenic factors triggered by the damage microenvironment. Precise gradient release of glycoproteins (such as KGN or BMP-7); preparation of HAMA-PBA microspheres (100-400 μm in diameter) loaded with heterostructures and chondroitinogens using microfluidic technology to ensure uniform spatial distribution and multi-stage controlled release of nanoparticles; finally, a dual-network structure is formed by combining with tannic acid (TA). The polyphenol crosslinking of TA provides strong antioxidant (free radical scavenging rate >85%), anti-inflammatory (significantly inhibits TNF-α, IL-1β and IL-6) and mechanical reinforcement (compressive modulus 20-80 kPa), while also imparting excellent wet tissue adhesion and anti-fatigue properties. The preparation process includes: hydrothermal synthesis of PHA nanoparticles and in-situ loading of rGO-Au to form a heterostructure; dispersion of the heterostructure and chondroitinogens in HAMA-PBA precursor solution, and preparation of microspheres using a microfluidic device (UV photocrosslinking); mixing of microspheres with tannic acid (TA) solution, and rapid crosslinking at room temperature (10-30 s) to form a microgel. This hydrogel can be minimally invasively injected into the damaged site of the growth plate via a 22-25G injection needle, achieving in-situ shaping, dynamic electrical stimulation, intelligent response release, anti-inflammatory protection and degradation matching, thereby significantly improving regeneration efficiency, inhibiting bone bridge formation and supporting normal bone growth.

[0033] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0036] Example This invention provides a method for preparing a hydrogel based on a piezoelectric hydroxyapatite heterostructure, comprising the following steps: (1) Preparation of hydroxyapatite (HA) First, hydroxyapatite (HA) was synthesized using a reverse emulsification-hydrothermal method: Ca(NO3)2·4H2O and (NH4)2HPO4 were dissolved in an appropriate amount of deionized water at a Ca:P mass ratio of (1~10):1, and 0.1M HCl or H2SO4 was added to adjust the solution to form a colloidal solution as the aqueous phase; cyclohexane was used as the continuous phase in the oil phase, and a mixed emulsifier (Tween 80: Span 80 mass ratio of 1:1 to 1:2.5, HLB value controlled in the range of 5.5~9.0) was added to ensure a stable W / O emulsion system. The above colloid was slowly dripped into the oil phase and emulsified for 2 hours under magnetic stirring at 40℃ and 300~500rpm to form a uniform reverse microemulsion. After the reaction, the precipitate was washed three times with cyclohexane to remove residual emulsifier. The resulting product was then placed in a high-pressure reactor and hydrothermally treated at 100~120℃ for 4~10 hours to promote crystal growth and improve crystallinity. After cooling, the product was collected by centrifugation, washed, and dried to obtain nano-sized hydroxyapatite powder.

[0037] (2) Preparation of piezoelectric hydroxyapatite (PHA) To deposit magnesium in situ on the surface of hydroxyapatite (HA), 0.48 g of anhydrous magnesium chloride (MgCl2, 95.21 g / mol) was first weighed and dissolved in 50 mL of deionized water to obtain a 0.1 M MgCl2 solution. The solution was stirred for 5–10 minutes until completely dissolved. Next, 1.0 g of hydroxyapatite powder (HA) was added to the solution, and the mixture was stirred continuously for 30 minutes to ensure that HA was uniformly dispersed and stably suspended in the system. Subsequently, a 0.1 M sodium hydroxide (NaOH) solution was slowly added dropwise to the above mixture to adjust the pH of the system to 10.0. At this point, Mg... 2+ Begin to gradually interact with OH - The reaction generates an in-situ Mg(OH)2 deposit layer on the HA surface: Mg 2+ +2OH - →Mg(OH)₂↓. Stir continuously for 1 hour during the dropwise addition to avoid localized deposition or agglomeration. After the deposition reaction is complete, continue stirring the entire mixture for 2 hours, maintaining the temperature at room temperature to help with deposition homogeneity and nucleation control. After the reaction is complete, wash the deposit 3-4 times with deionized water to remove excess Na. + Cl -Byproducts, etc. The resulting sediments were collected by centrifugation or filtration and dried in a 60°C oven for 12 hours to ensure complete dehydration. To enhance the bonding force and structural stability between Mg(OH)2 and the HA substrate, the dried sample could be heat-treated (calcined at 400°C for 2 hours) to obtain PHA.

[0038] (3) Preparation of reduced graphene oxide-gold nanoparticle modified PHA (rGO-Au-PHA) Subsequently, the rGO / PHA composite was prepared: 0.1~0.5g of GO (prepared by modified Hummers method) was dispersed in 100mL of deionized water and sonicated for 1h (concentration 1~5mg / mL). 1g of the above PHA powder (GO:PHA weight ratio 1:10~1:2) was added and sonicated for 30min. The mixture was then transferred to a high-pressure reactor and hydrogen gas was injected (pressure 1~2MPa). The reactor was heated at 150~200°C for 2~4h. After cooling, the mixture was centrifuged (6000rpm, 15min), washed three times with water / ethanol, and dried under vacuum at 80°C to obtain the rGO / PHA composite. Finally, in-situ decoration of AuNPs: 1g of rGO / PHA complex was dispersed in 50mL of deionized water and sonicated for 30min. 0.01~0.05g of HAuCl4 (Au loading 1~5wt%, concentration 1~5mM) was added and stirred for 1h. 0.1g of sodium citrate (0.1M) was slowly added as a reducing agent. The mixture was stirred at room temperature for 24h or heated to 60°C to accelerate the reaction. After centrifugation (5000rpm, 20min), washing with deionized water 3~5 times, and vacuum drying at 60°C, rGO-Au-PHA heterostructures with an overall size of 100~300nm were obtained.

[0039] (4) Synthesis of 3-aminophenylboronic acid modified methacryloyl hyaluronic acid sodium hyaluronate (HAMA-PBA) In this invention, the synthesis method of HAMA-PBA (3-aminophenylboronic acid modified methacrylated hyaluronic acid) adopts a two-step method: first, hyaluronic acid (HA) is methacrylated to obtain HAMA, and then phenylboronic acid (PBA) groups are further grafted onto HAMA. Specifically, HAMA was first synthesized by dissolving 1.0 g of sodium hyaluronate (HA, molecular weight approximately 100-300 kDa) in 100 mL of deionized water and stirring until completely dissolved (concentration 1% w / v). The pH was then adjusted to 8-9 with 0.5 M NaOH. Subsequently, methacrylic anhydride (MA, 1-5 mL, typically HA repeating unit:MA molar ratio 1:2-1:5) was slowly added dropwise. The reaction was carried out in an ice bath (0-5℃) for 24 h, during which the pH was maintained at 8-9 with 0.5 M NaOH every 4-6 h. After the reaction was completed, the pH was adjusted to 7.0 with 0.1 M HCl to terminate the reaction. The solution was then transferred to a dialysis bag (molecular weight cutoff 3.5-14 kDa) and dialyzed against deionized water for 3-5 days (water changed every 12 h) to remove unreacted MAA and small molecule byproducts. The dialysis solution was then freeze-dried (-80℃, 24-48 h) to obtain a white, fluffy powder of methacryloyl hyaluronic acid (HAMA).

[0040] Next, PBA grafting was performed to obtain HAMA-PBA: 0.5g of the above HAMA powder was dissolved in 50mL of deionized water (concentration 1% w / v) and stirred evenly; 3-aminophenylboronic acid (3-APBA, 0.1~0.3g, HA repeating unit: 3-APBA molar ratio 1:0.1~1:0.3) was added; then, coupling agents EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.2~0.5g) and NHS (N-hydroxysuccinimide, 0.15~0.4g) were added as the activation system (EDC:NHS molar ratio 1:1), the pH was adjusted to 4.5~5.5 with 0.5M NaOH, and stirred in the dark at room temperature (25℃) for 24~48h to promote the amide bonding of carboxyl and amino groups to form HAMA-PBA; after the reaction was completed, 0.1M NaOH was used to adjust the pH to 4.5~5.5. Adjust the pH to neutral with HCl; transfer the solution to a dialysis bag (molecular weight cutoff 3.5~14kDa), and dialyze against deionized water for 3~5 days to remove unreacted substances and small molecules; freeze-dry the solution after dialysis to obtain a light yellow, fluffy HAMA-PBA powder.

[0041] Examples and comparative examples of injectable hydrogels based on piezoelectric hydroxyapatite heterostructures of the present invention, wherein the components contained are as follows: 2~4wt% HAMA-PBA, 0.2wt% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), 3wt% TA, 1~3mg / mL rGO-Au-PHA, and 100μg / mL BMP-7.

[0042] Table 1. Formulation Mass Fraction Table

[0043] In the embodiments, HAMA-PBA is preferably 3 wt%; TA is preferably 3 wt%; rGO-Au-PHA is preferably 2 mg / mL; and BMP-7 is preferably 100 μg / mL.

[0044] Examples 1-6 Weigh 10 mg of LAP (photoinitiator) and dissolve it completely in 5 mL of distilled water (0.2%). Then, weigh the corresponding mass fraction of HAMA-PBA solid material and place it in the EP tube. Dissolve it in a 50°C water bath for 2 hours, resulting in a homogeneous, transparent liquid, thus obtaining a known concentration of HAMA-PBA aqueous solution (containing LAP). Subsequently, add the predetermined concentrations of rGO-Au-PHA and BMP-7, vortex for 3 minutes to mix thoroughly, obtaining a HAMA-PBA@rGO-Au-PHA@BMP-7 solution. This solution is then placed in a 2.5 mL syringe as the dispersed phase and placed in a microfluidic device. The continuous phase is 2% droplet-generating oil (fluorinated oil). The flow rate of the dispersed phase was controlled at 0.2 mL / h, and the flow rate of the continuous phase was 2.4 mL / h. The generated microdroplets were collected and cured under ultraviolet light to form microgel microspheres. After adding a demulsifier to remove the fluorinated oil on the surface of the microspheres, they were washed with deionized water and centrifuged to obtain clean HAMA-PBA@rGO-Au-PHA@BMP-7 microspheres. Next, a TA solution (3%) was placed on a vortex mixer, and HAMA-PBA@rGO-Au-PHA@BMP-7 microspheres were added while vortexing, with a volume ratio of HAMA-PBA@rGO-Au-PHA@BMP-7 microspheres to TA solution of 1:1. After mixing, the mixture was vortexed for 30 s to form a uniform hydrogel.

[0045] Comparative Example 1 10 mg of LAP (photoinitiator) was weighed and completely dissolved in 5 mL of distilled water (0.2%). Then, the corresponding mass fraction of HAMA-PBA solid material was weighed and placed in the aforementioned EP tube. The solution was dissolved in a 50°C water bath for 2 hours, resulting in a homogeneous, transparent liquid, yielding a known concentration of HAMA-PBA aqueous solution (containing LAP). Subsequently, BMP-7 of a predetermined concentration was added, and the mixture was vortexed for 3 minutes to obtain a homogeneous HAMA-PBA@BMP-7 solution. This solution was then placed in a 2.5 mL syringe as the dispersed phase and placed in a microfluidic device. A 2% droplet-generating oil (fluorinated oil) was used as the continuous phase. The flow rate of the dispersed phase was controlled at 0.2 mL / h, and the flow rate of the continuous phase was 2.4 mL / h. The generated microdroplets were collected and cured under UV light to form microgel microspheres. After removing the fluorinated oil from the surface of the microspheres with a demulsifier, the microspheres were washed with deionized water and centrifuged to obtain clean HAMA-PBA@BMP-7 microspheres. Next, the TA solution (3%) was placed on a vortex mixer, and HAMA-PBA@BMP-7 microspheres were added while vortexing. The volume ratio of HAMA-PBA@BMP-7 microspheres to TA solution was 1:1. After mixing, the mixture was vortexed for 30 seconds to form a uniform hydrogel.

[0046] Comparative Example 2 10 mg of LAP (photoinitiator) was weighed and completely dissolved in 5 mL of distilled water (0.2%). Then, the corresponding mass fraction of HAMA-PBA solid material was weighed and placed in the EP tube. The solution was dissolved in a 50°C water bath for 2 hours, resulting in a homogeneous, transparent liquid, yielding a known concentration of HAMA-PBA aqueous solution (containing LAP). Subsequently, a predetermined concentration of rGO-Au-PHA was added, and the mixture was vortexed for 3 minutes to obtain a HAMA-PBA@rGO-Au-PHA solution. This solution was placed in a 2.5 mL syringe as the dispersion phase and placed in a microfluidic device. A 2% droplet-generating oil (fluorinated oil) was used as the continuous phase. The flow rate of the dispersed phase was controlled at 0.2 mL / h, and the flow rate of the continuous phase was 2.4 mL / h. The generated microdroplets were collected and cured under UV light to form microgel microspheres. After removing the fluorinated oil from the surface of the microspheres with a demulsifier, the microspheres were washed with deionized water and centrifuged to obtain clean HAMA-PBA@rGO-Au-PHA microspheres. Next, the TA solution (3%) was placed on a vortex mixer, and HAMA-PBA@rGO-Au-PHA microspheres were added while vortexing. The volume ratio of HAMA-PBA@rGO-Au-PHA microspheres to TA solution was 1:1. After mixing, the mixture was vortexed for 30 seconds to form a uniform hydrogel.

[0047] Comparative Example 3 Weigh 10 mg of LAP (photoinitiator) and dissolve it completely in 5 mL of distilled water (0.2%). Then, weigh the corresponding mass fraction of HAMA-PBA solid material and place it in the EP tube. Dissolve it in a 50°C water bath for 2 hours, resulting in a homogeneous, transparent liquid, thus obtaining a known concentration of HAMA-PBA aqueous solution (containing LAP). Subsequently, add the predetermined concentrations of rGO-Au-PHA and BMP-7, vortex for 3 minutes to mix thoroughly, obtaining a HAMA-PBA@rGO-Au-PHA@BMP-7 solution. This solution is then placed in a 2.5 mL syringe as the dispersed phase and placed in a microfluidic device. The continuous phase is 2% droplet-generating oil (fluorinated oil). The flow rate of the dispersed phase was controlled at 0.2 mL / h, and the flow rate of the continuous phase was controlled at 2.4 mL / h. The generated microdroplets were collected and cured by ultraviolet light to form microgel microspheres. After adding a demulsifier to remove the fluorinated oil on the surface of the microspheres, deionized water was added for washing, and the clean HAMA-PBA@rGO-Au-PHA@BMP-7 microspheres were obtained by centrifugation.

[0048] from Figure 1 The results show that the HAMA-PBA@rGO-Au-PHA@BMP-7 microspheres prepared in this invention exhibit a relatively regular spherical or near-spherical structure with good dispersibility and no obvious large-area aggregation, indicating that the microfluidic method can achieve stable preparation and relatively uniform shaping of microspheres. The microsphere particle size is within a suitable micrometer scale range, and the surface exhibits certain roughness and wrinkled micro / nano structures, which may be related to the loading of internal functional components and subsequent drying treatment. This rough surface morphology suggests that the microspheres have a relatively stable three-dimensional structure and also helps to increase the specific surface area, providing a structural basis for subsequent dynamic assembly with TA solution, interfacial interaction, and sustained release of active factors. Overall, the obtained microspheres have complete morphology and stable structure, indicating that the microfluidic preparation and photocuring process adopted in this invention has good feasibility and can provide a reliable material basis for the subsequent construction of injectable microsphere assembly-type dual-network hydrogel systems.

[0049] from Figure 2The results show that the HAMA-PBA@rGO-Au-PHA@BMP-7 / TA hydrogel exhibits a relatively continuous and interconnected three-dimensional porous network structure with relatively uniform pore wall distribution. Abundant pores and an interwoven network framework are visible internally, indicating that the microspheres and TA successfully formed a structurally complete assembled hydrogel system. This porous morphology suggests that the material has a large specific surface area and good connectivity, which is beneficial for water permeation, nutrient exchange, and the continuous release of active factors. It also provides a suitable microenvironment for cell adhesion, migration, and tissue ingrowth. The relatively dense local areas and network connection structure in the figure suggest that the participation of TA enhances the cross-linking degree and framework stability of the system, resulting in a tighter interfacial bond between microspheres, thus endowing the hydrogel with better integrity and mechanical support. Overall, the HAMA-PBA@rGO-Au-PHA@BMP-7 / TA hydrogel obtained in this invention has a typical porous network microstructure, proving that its dual-network assembly strategy is feasible and can provide a good structural basis for subsequent injectable filling, tissue adhesion, and growth plate damage repair.

[0050] from Figure 3 The results showed that oxidative stress induction (culturing in a simulated oxidative damage environment of 200 μM H2O2 for 24 hours) without any intervention was designated as the oxidative stress group; simultaneously, normal culture conditions without oxidative stimulation or intervention served as the control group. The control group showed weak intracellular fluorescence signals, indicating low intracellular ROS levels under basal conditions and a relatively stable redox equilibrium. In contrast, the oxidative stress group exhibited significantly enhanced green fluorescence with a wide distribution and high intensity, suggesting a large accumulation of reactive oxygen species and significant oxidative damage after oxidative stimulation. After treatment in Example 4 (oxidative stress + Example 4 treatment group), the intracellular fluorescence signal significantly decreased, approaching the level of the control group, indicating that this material system can effectively reduce intracellular ROS accumulation under oxidative stress and significantly alleviate oxidative damage. The above results demonstrate that the HAMA-PBA@rGO-Au-PHA@BMP-7 / TA hydrogel constructed in this invention has excellent antioxidant stress regulation capabilities. It can remove excess reactive oxygen species and improve the local microenvironment of cells, thus providing favorable conditions for subsequent cell survival, function maintenance, and growth plate damage repair.

[0051] from Figure 4The results showed that in the control group (normal growth plate), the growth plate structure was intact, chondrocytes were regularly arranged, columnar layers were clear, and Safranin-O staining showed obvious red staining, indicating that local proteoglycans and cartilage matrix were well preserved and the tissue structure was in a normal state. In contrast, in the growth plate injury model group, the tissue structure in the injured area was significantly disordered, the normal columnar arrangement was destroyed, and there was a tendency for bone tissue invasion and bone bridge formation. Safranin-O staining was significantly weakened, indicating loss of cartilage matrix and decreased proteoglycan content, suggesting abnormal growth plate repair after injury and a significant tendency for ossification. After treatment in Example 4 (growth plate injury model + Example 4 treatment group), the tissue continuity in the injured area was significantly improved, cartilage-like tissue increased, cell arrangement was more regular than in the model group, Safranin-O staining was enhanced, and the area of ​​bone invasion shown by Fast Green staining was reduced, indicating that the material system can effectively promote cartilage matrix deposition and growth plate-like structure reconstruction, while inhibiting abnormal ossification and bone bridge formation. The above results indicate that the HAMA-PBA@rGO-Au-PHA@BMP-7 / TA hydrogel constructed in this invention can significantly improve the histological repair effect after growth plate damage, providing favorable conditions for restoring normal growth plate structure and promoting regeneration and repair.

[0052] Implementation effect evaluation 1. Rheological characterization Test Methods: The rheological properties of each group of hydrogel samples were characterized using a rotational rheometer to evaluate their viscoelasticity and network structure stability. Specifically, after the samples prepared according to Examples 1-6 and Comparative Examples 1-3 were gelled at room temperature, an appropriate amount of sample was placed between the parallel plate clamps of the rheometer for measurement. The test mode was a small-amplitude oscillating shear mode. A parallel plate geometry with a plate diameter of 20 mm was recommended for the clamps, with a test gap of 1.0 mm. The test temperature was controlled at 25℃ or 37℃. To avoid the influence of water loss at the sample edges on the test results, a suitable amount of low-viscosity silicone oil could be applied to the sample edges for protection, or a solvent trapping device could be used. First, the linear viscoelastic range of the samples was determined by strain scanning. Based on this, 1% strain was selected as a fixed test condition, and oscillation tests were performed at a fixed frequency of 1 Hz. The storage modulus (G') and loss modulus (G”) of each group of samples were recorded. Among them, the storage modulus G' is used to characterize the elastic response and network carrying capacity of the hydrogel system, and the loss modulus G” is used to characterize the viscous dissipation behavior of the system.

[0053] Table 2 Rheological properties characterization of hydrogels

[0054] Table 2 shows the results: As shown in Table 2, the hydrogel systems obtained in each embodiment all exhibited a relatively obvious elastic-dominant characteristic, specifically, the storage modulus (G') was generally higher than the loss modulus (G"). This indicates that after the addition of TA, the HAMA-PBA functional microspheres can further assemble into a stable three-dimensional network structure, giving the system good structural integrity and mechanical support capabilities. As the concentration of HAMA-PBA increased from 2% to 4%, the G' and G" of the hydrogel generally showed an increasing trend, indicating that the increase in polymer backbone concentration can effectively improve the network crosslinking density, thereby enhancing the viscoelasticity of the system. At the same time, when the concentration of rGO-Au-PHA increased from 1 mg / mL to 3 mg / mL, the hydrogel modulus also showed an increasing trend, indicating that this heterostructure filler played a significant reinforcing role in the network, helping to improve the overall stability and deformation resistance of the system. Comparative Example 1, lacking the addition of rGO-Au-PHA, exhibited lower G' and G” values ​​compared to the corresponding intact system, indicating that the introduction of rGO-Au-PHA not only endowed the material with functionality but also positively enhanced the network's mechanical properties. Comparative Example 2, without BMP-7 loading, showed relatively small differences in rheological properties compared to the corresponding intact system, suggesting that BMP-7's primary role in this system was more focused on bioactivity regulation rather than direct network enhancement. Comparative Example 3, lacking TA, failed to form a stable dual-network assembly structure, resulting in significantly insufficient overall viscoelasticity, thus making it difficult to obtain stable rheological data comparable to the other groups. These results demonstrate that TA is crucial for constructing a complete hydrogel network, while the concentration of HAMA-PBA and the amount of rGO-Au-PHA added are key factors in regulating the hydrogel's rheological properties and structural stability.

[0055] 2. Adhesion performance characterization Test Method: The wet tissue adhesion properties of each group of hydrogels were characterized using the overlap shear method. Specifically, fresh pig skin was selected as the adhesion substrate material. The substrate was cut into rectangular samples of uniform size, and the surface was kept moist to simulate the in vivo tissue interface environment. A certain volume of the hydrogel sample was uniformly coated onto the overlapping area of ​​two substrates, with the overlap area controlled at a fixed value (10mm × 10mm). The samples were then allowed to stand at room temperature for a certain time (15min) to allow for full adhesion and formation. For the embodiment group of this invention, the microsphere-assembled hydrogel was allowed to stand and form a gel after contact with the tissue surface; the control group was treated in the same way. After adhesion was completed, the samples were fixed on the clamps at both ends of a universal testing machine, and a shear tensile test was performed at a constant tensile rate (5mm / min). The maximum load at which the samples separated was recorded, and the adhesion strength was calculated by dividing the maximum load by the overlapping adhesion area, in kPa.

[0056] Table 3. Wet tissue adhesion strength of hydrogels

[0057] Table 3 shows the results: As shown in Table 3, all hydrogels containing TA exhibited good wet tissue adhesion ability, and were significantly better than Comparative Example 3 without TA, indicating that TA played a key role in imparting tissue adhesion to the system. As the HAMA-PBA concentration increased from 2% to 4%, the adhesion strength of the hydrogels generally showed an upward trend, indicating that increasing the polymer backbone concentration is beneficial to improving network cohesion and interfacial film-forming ability. Meanwhile, the appropriate introduction of rGO-Au-PHA can further enhance the structural stability of the system, thereby improving adhesion performance. Example 4 showed the highest adhesion strength, suggesting a better synergistic balance between 3% HAMA-PBA, 3% TA, and 2 mg / mL rGO-Au-PHA. When the rGO-Au-PHA content continued to increase to 3 mg / mL, the adhesion strength decreased slightly, presumably related to the decrease in interfacial spreadability and wettability caused by excessive filler content. Comparative Example 1, lacking rGO-Au-PHA, exhibited lower adhesion strength than the corresponding intact system, indicating that this heterostructure plays a positive role in enhancing the overall cohesive stability of the hydrogel. Comparative Example 2 showed little difference from the intact system, suggesting that BMP-7's direct contribution to adhesion performance is limited, with its main function leaning more towards bioactivity regulation. Overall, the results demonstrate that the microsphere-assembled dual-network hydrogel system constructed in this invention possesses excellent wet tissue adhesion properties, meeting the application requirements for in-situ fixation of damaged growth plate sites and interfacial adhesion.

[0058] 3. Piezoelectric performance evaluation Test Method: The piezoelectric constants of each group of samples were characterized using a quasi-static piezoelectric constant meter to evaluate the electrical response of different hydrogel systems under external force. Specifically, samples prepared according to Examples 1-6 and Comparative Examples 1-3 were injected into pre-set molds to form uniformly sized circular or cylindrical test samples, which were then allowed to stand at room temperature or 37°C until the system structure stabilized. Before testing, free liquid on the sample surface was appropriately removed to reduce the influence of free water on measurement stability. Subsequently, the samples were placed on the piezoelectric constant meter platform, and their longitudinal piezoelectric constant d was measured under a constant, small periodic normal force. 33 The unit is pC / N.

[0059] Table 4. Piezoelectric constants of hydrogels

[0060] Table 4 Results: As shown in Table 4, all samples in the rGO-Au-PHA group exhibited measurable piezoelectric responses, while the comparative example 1 without rGO-Au-PHA showed only extremely low d... 33The values ​​indicate that the rGO-Au-PHA heterostructure is the main source of the piezoelectric properties of this system. As the concentration of rGO-Au-PHA increased from 1 mg / mL to 3 mg / mL, the piezoelectric constant of the hydrogel system generally showed an upward trend, indicating that increasing the content of the piezoelectric functional phase is beneficial to improving the system's charge response capability under stress. Meanwhile, under the condition of fixed rGO-Au-PHA content, the concentration of HAMA-PBA also had a significant impact on the piezoelectric properties. A moderate concentration of HAMA-PBA was more conducive to achieving a balance between network integrity and stress transfer efficiency; therefore, Examples 2 and 4 exhibited better piezoelectric response levels. When the polymer concentration further increased, the increased proportion of the continuous polymer phase may have a certain dilution effect on the stress transfer of the functional filler, causing a slight decrease in the piezoelectric constant. Although Comparative Example 2 did not contain BMP-7, its piezoelectric constant was only slightly different from the corresponding intact system, indicating that BMP-7 has a limited direct contribution to the piezoelectric properties of the system; its main role is in subsequent bioactivity regulation. Comparative Example 3, without the addition of TA, still contained rGO-Au-PHA, but due to the lack of a complete and stable dual-network assembly structure, the overall continuity and stress coupling ability of the sample were weakened, and its d 33 The value is significantly lower than that of the complete system, further illustrating the important role of TA in improving the structural integrity of the system and promoting the transmission of mechanical and electrical signals. In summary, the microsphere-assembled dual-network hydrogel system constructed in this invention exhibits excellent apparent piezoelectric response performance, providing a basis for continuous self-driven micro-electrical stimulation at damaged sites in the growth plate.

[0061] 4. PCR detection of inflammatory factors Testing Methods: Real-time quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expression levels of inflammation-related factors TNF-α, IL-1β, and IL-6 in cells after treatment of each group of samples, in order to evaluate the regulatory ability of different hydrogel systems on the inflammatory microenvironment. Specifically, BMSCs were seeded in 6-well plates. After the cells adhered and grew to an appropriate density, an inflammatory stimulation model was established, and the corresponding treatment solutions or extraction solutions from each example and comparative example were added for intervention culture. After treatment, the culture medium was discarded, and the cells were gently washed 2-3 times with pre-cooled PBS. Trizol reagent was added to lyse the cells and extract total RNA. The RNA concentration and purity were measured using a spectrophotometer, and an equal amount of RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. Subsequently, using the cDNA as a template, SYBR Green quantitative PCR was used for amplification to detect the expression levels of TNF-α, IL-1β, IL-6, and the internal reference gene GAPDH. The relative expression levels of each gene were calculated using the 2^-ΔΔCt method, with the inflammatory model group serving as the calibration group. In in vitro experiments, low-intensity pulsed ultrasound (LIPUS) was used to mechanically stimulate cells. Ultrasound stimulation was provided by an ultrasound therapy device (Sonicator 740, Mettler Electronics Corp., USA). The ultrasound frequency was set to 1.5 MHz, and the spatially averaged temporally averaged sound intensity (SATA) was 30 mW / cm². 2 The duty cycle was 20% (pulse duration 200 μs, pulse repetition frequency 1 kHz). Cell culture plates were placed above a LIPUS transducer in a 37°C incubator. A sterile coupling agent was used between the transducer and the bottom of the culture plate to ensure uniform ultrasonic energy delivery to the cell layer. Each stimulation lasted 20 minutes, once daily, for the designated experimental time point.

[0062] Table 5. Relative expression levels of inflammation-related factors by qPCR

[0063] The expression levels of TNF-α, IL-1β, and IL-6 in the Control group were 0.21±0.03, 0.18±0.02, and 0.24±0.03, respectively; while the expression levels of TNF-α, IL-1β, and IL-6 in the oxidative stress model group (inflammation model group) were 1.00±0.08, 1.00±0.09, and 1.00±0.10, respectively.

[0064] Table 5 shows the results: qPCR results indicate that, compared with the inflammation model group, all hydrogel systems containing TA and functional components could upregulate the mRNA expression levels of TNF-α, IL-1β, and IL-6 to varying degrees, demonstrating that the microsphere-assembled dual-network hydrogel constructed in this invention has a good anti-inflammatory regulatory effect. Overall, the complete system group showed the most significant anti-inflammatory effect, with the lowest expression levels of the three inflammatory factors in Examples 4 and 5, suggesting that appropriate HAMA-PBA concentration, dynamic TA assembly network, and the synergistic effect of rGO-Au-PHA and BMP-7 can help to more effectively alleviate the inflammatory response. As the HAMA-PBA concentration increased from 2% to 4%, the expression of inflammatory factors in each example generally showed a decreasing trend, indicating that moderately increasing the network backbone concentration is beneficial to improving system stability and achieving more effective local microenvironment regulation; while as the rGO-Au-PHA content increased from 1 mg / mL to 2 mg / mL, the anti-inflammatory effect was further enhanced, but when it continued to increase to 3 mg / mL, the expression of inflammatory factors slightly rebounded, suggesting that excessively high filler content may have a certain impact on the system interface adaptability or biological regulatory balance. Comparative Example 1, lacking rGO-Au-PHA, exhibited a weaker anti-inflammatory effect than the intact system, indicating that this heterostructure plays a positive role in regulating the inflammatory microenvironment. Comparative Example 2, despite lacking BMP-7, still demonstrated a certain degree of anti-inflammatory ability, suggesting that the synergistic effect of TA and rGO-Au-PHA can still exert an anti-inflammatory effect. Comparative Example 3, without the addition of TA, showed significantly higher expression levels of TNF-α, IL-1β, and IL-6 than the intact system, further demonstrating that TA plays a crucial role in anti-oxidation, anti-inflammation, and microenvironment improvement. Overall, the results indicate that the injectable microsphere-assembled dual-network hydrogel constructed in this invention can effectively inhibit the expression of inflammation-related factors, providing favorable conditions for the regulation of the inflammatory microenvironment and tissue regeneration and repair after growth plate injury.

[0065] 5. Bone microstructural parameters in the growth plate injury area Test Methods: Healthy SPF-grade male SD rats (120-150g) aged 4 weeks were randomly divided into the Sham group, Model group, various example groups, and comparative group. After anesthesia, the groove at the distal femur was exposed under sterile conditions. A central defect of approximately 2.0mm was created at the distal femur by drilling through the articular cartilage using a 2.0mm drill. In the Sham group, only incision and exposure were performed without causing growth plate damage; in the Model group, no material intervention was performed after establishing growth plate damage; in the example groups and comparative group, injectable microsphere-assembled hydrogels of the corresponding formulation were injected into the damaged area immediately after modeling, allowing the material to fill the defect in situ and form a local treatment system. Postoperatively, the incision was sutured layer by layer, and the animals were fed and observed according to the requirements for experimental animal management. All animals were sacrificed 8 weeks postoperatively for tissue sampling for subsequent Micro-CT scanning. Specific test steps for Micro-CT scanning: After tissue sampling 8 weeks postoperatively, the bone specimen containing the growth plate damage area was completely separated, the surrounding soft tissue was removed, and the specimen was fixed in 4% paraformaldehyde. After fixation, high-resolution Micro-CT systems were used to scan each group of specimens, obtaining continuous tomographic images of the damaged area. Three-dimensional reconstruction analysis was then performed using accompanying three-dimensional reconstruction software. During the scanning process, parameters such as voltage, current, resolution, and exposure time were uniformly set to ensure consistent image acquisition conditions across different groups. After three-dimensional reconstruction, a region of interest (ROI) was selected within a uniform range at the growth plate injury site. The ROI was primarily limited to the damaged area and potential bone bridge formation zone to assess the degree of local abnormal ossification. Subsequently, bone tissue and non-bone tissue were segmented using a uniform threshold, and quantitative analysis of bone microstructural parameters within this region was performed, including bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), trabecular bone separation (Tb.Sp), and bone mineral density (BMD). BV / TV, Tb.N, Tb.Th, and BMD primarily reflect the degree of bony tissue filling and bone bridge formation in the damaged area, while Tb.Sp reflects changes in local trabecular bone spacing. All samples underwent ROI selection and threshold analysis using the same criteria to improve the comparability and accuracy of the results. In animal experiments, low-intensity pulsed ultrasound (LIPUS) was used to stimulate the surgical area in vitro. The LIPUS was generated by an ultrasound therapy device (Sonicator 740, Mettler Electronics Corp., USA), with stimulation parameters set as follows: ultrasound frequency: 1.5 MHz, sound intensity: 30 mW / cm². 2 (SATA), duty cycle: 20%, pulse repetition frequency: 1kHz. Before each stimulation, the animal's lesion area was lightly shaved and coated with medical ultrasound coupling agent to enhance sound energy transmission. The ultrasound transducer was gently applied to the surface of the lesion for stimulation, 20 minutes per stimulation, once a day, for 8 consecutive weeks until the animal was harvested.

[0066] Table 6. Quantitative analysis of bone microstructural parameters in the growth plate injury area.

[0067] The BV / TV (%), Tb.N (1 / mm), Tb.Th (mm), Tb.Sp (mm), and BMD (g / cm) of the sham surgery group were among the values. 3 The expression levels of ) were 5.8±0.9, 0.46±0.05, 0.061±0.006, 1.10±0.09, and 0.09±0.01, respectively; the BV / TV (%), Tb.N (1 / mm), Tb.Th (mm), Tb.Sp (mm), and BMD (g / cm) of the growth plate damage model group were also observed. 3 The expression levels of ) were 42.7±3.6, 2.86±0.22, 0.183±0.013, 0.29±0.03 and 0.46±0.04, respectively.

[0068] Table 6 shows the results: Quantitative analysis of Micro-CT three-dimensional reconstruction at 8 weeks post-operation revealed that the Model group exhibited significantly elevated BV / TV, Tb.N, Tb.Th, and BMD, while Tb.Sp was significantly decreased, indicating that the damaged area was filled with more bony tissue, and the continuity and structural integrity of the normal growth plate were significantly disrupted. In contrast, the Sham group maintained relatively low levels of bone-related parameters, with a relatively high Tb.Sp, consistent with the tissue characteristics of a normal growth plate area dominated by cartilage. After material intervention, all treatment groups reduced abnormal ossification in the damaged area to varying degrees. The example group was generally superior to the control group, demonstrating that the injectable microsphere-assembled dual-network hydrogel constructed in this invention can effectively inhibit bone bridge formation and improve the bone microstructure state of the damaged area. Further analysis revealed that with the optimization of the HAMA-PBA network composition and the enhanced synergistic effects of functional components such as rGO-Au-PHA, BMP-7, and TA, the overall BV / TV, Tb.N, Tb.Th, and BMD in Examples 1 to 5 showed a decreasing trend, while Tb.Sp gradually increased, indicating a continuous reduction in abnormal bone tissue within the damaged area and more effectively inhibiting local bone bridge formation. Examples 4 and 5 showed the best performance, suggesting a better balance between appropriate network framework concentration, piezoelectric heterostructure content, and active factor loading, which can more effectively maintain the low ossification state in the damaged area and provide a favorable microenvironment for cartilage regeneration. Although Example 6 was still significantly better than the Model group, the degree of improvement was slightly lower than that of Examples 4 and 5, suggesting that excessively high rGO-Au-PHA content may not further enhance the repair effect. Comparative Example 1, lacking rGO-Au-PHA, showed a weaker inhibitory effect on abnormal ossification in the damaged area; Comparative Example 2, without BMP-7, exhibited some improvement, but was still inferior to the complete system; Comparative Example 3, without TA, showed poor bone bridge inhibition, further demonstrating the important role of TA in system stability, inflammatory microenvironment regulation, and comprehensive repair. Overall, the results indicate that the injectable microsphere-assembled dual-network hydrogel constructed in this invention can significantly reduce bone bridge formation in the growth plate injury area and improve local bone microstructure parameters 8 weeks post-surgery, thus facilitating regeneration and repair after growth plate injury.

[0069] In summary, the key technical points of this invention are: addressing the problems of persistent inflammation, cartilage matrix loss, abnormal ossification, and bone bridge formation that easily occur after growth plate injury, by constructing a microsphere-assembled hydrogel system that combines injectable in-situ filling, piezoelectric stimulation, dynamic dual-network assembly, continuous delivery of active factors, and microenvironment regulation. This system is not simply a physical mixture of inorganic particles, active factors, and a hydrogel matrix. Instead, it first prepares HAMA-PBA functional microspheres loaded with piezoelectric heterostructures and bioactive factors using microfluidic technology, and then utilizes the dynamic interaction between TA and the microsphere surface to construct a dual-network hydrogel. This ensures injectability while also considering uniform distribution of functional components, network structure stability, tissue adhesion, and long-term repair effects. Its core innovation lies in organically coupling the electroactivity of the rGO-Au modified piezoelectric hydroxyapatite heterostructure, the drug loading and dynamic response capabilities of HAMA-PBA microspheres, and the secondary assembly and anti-inflammatory and antioxidant capabilities imparted by TA into the same system, thereby achieving multidimensional regulation of the growth plate injury repair process.

[0070] Compared with existing single scaffold materials, conventional injectable hydrogels, and passively released growth factor delivery systems used for bone defect or cartilage repair, the injectable microsphere-assembled dual-network hydrogel system based on piezoelectric hydroxyapatite heterostructure provided by this invention has the following advantages: (1) It has a targeted design for growth plate damage repair, which can simultaneously promote cartilage regeneration and inhibit bone bridge formation. Guided by the growth plate damage microenvironment and repair target, this invention can better promote cartilage-like matrix deposition, maintain chondrocyte phenotype and reduce abnormal bone connections in the damaged area through the synergistic effect of piezoelectric heterostructure stimulation, active factor delivery and microenvironment regulation, thereby improving the targeting and effectiveness of growth plate regeneration and repair.

[0071] (2) An integrated system of "functional microspheres + dynamic dual network" was constructed, which combines injectability, structural stability and uniform component distribution. In this invention, HAMA-PBA functional microspheres loaded with rGO-Au-PHA and BMP-7 were first prepared by microfluidic control, and then dynamically assembled with TA solution to form a dual network hydrogel. This design not only facilitates the uniform dispersion of functional components in the system and reduces the risk of local aggregation and burst release, but also takes into account both injection fluidity and overall stability after gelation, forming a microsphere-assembled hydrogel system that is more suitable for in-situ filling of irregular damaged areas.

[0072] (3) It has good dynamic response capability and continuous delivery capability, which can achieve more gentle and effective local regulation. The present invention uses HAMA-PBA and TA to construct a dynamic dual network structure, which, while maintaining the integrity of the system, endows the material with a certain degree of reconfigurability, self-healing and environmental adaptability; at the same time, active ingredients such as BMP-7 are preferentially encapsulated inside the microspheres, which can achieve a more continuous and stable release process, thereby avoiding the rapid loss of active substances, improving the local utilization efficiency, and being more conducive to the regulation of tissue regeneration in the long-term repair process.

[0073] (4) It possesses multiple functions such as anti-oxidation, anti-inflammation, and tissue adhesion, which is beneficial for improving the adverse microenvironment after growth plate damage. Growth plate damage is often accompanied by problems such as inflammation activation, enhanced oxidative stress, and tissue interface instability. In this invention, TA not only participates in the construction of the dual network, but also endows the system with good antioxidant and anti-inflammatory capabilities, which helps to downregulate the expression of inflammation-related factors such as TNF-α, IL-1β, and IL-6, and reduce the accumulation of reactive oxygen species; at the same time, its polyphenol structure can enhance the adhesion performance of the material at the wet tissue interface, which is conducive to the stable residence and interface adhesion of the material in the damaged area, thereby creating more favorable conditions for tissue repair.

[0074] (5) It has good mechanical adaptability and structural support capabilities, which can improve the stability of local repair in the damaged area.

[0075] This invention utilizes the synergistic effect of a microsphere framework and a dynamic cross-linking network of TA to form a continuous, porous, and viscoelastic three-dimensional structure. This structure not only meets the requirements for injection delivery and in-situ gelation but also provides necessary physical support and spatial maintenance in the damaged area. This helps to reduce tissue collapse, stabilize local structures, and provide a suitable scaffold environment for cell migration, matrix deposition, and tissue reconstruction.

[0076] (6) The treatment method is minimally invasive and easy to operate, and has good potential for clinical translation. The system described in this invention can be directly delivered to the growth plate injury area by injection, and a stable assembled hydrogel is rapidly formed locally to achieve in situ filling and targeted treatment. It has the advantages of small trauma, adaptability to irregular defects, simple operation and good repeatability, and is especially suitable for application scenarios such as growth plate injury where the protection of local structure and tissue is highly required.

[0077] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0078] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A hydrogel based on a piezoelectric hydroxyapatite heterostructure, characterized in that, The hydrogel comprises a dual network structure formed by cross-linking a backbone with natural polyphenols, wherein the backbone is loaded with a nano-heterogeneous structure; the nano-heterogeneous structure is loaded onto the backbone using microfluidic technology. The nanostructure includes reduced graphene oxide, gold nanoparticles, and piezoelectric hydroxyapatite; the piezoelectric hydroxyapatite is prepared by in-situ deposition of metal on the surface of hydroxyapatite. The skeleton comprises 3-aminophenylboronic acid and methacrylamide sodium hyaluronate; The natural polyphenols include tannins.

2. The hydrogel based on a piezoelectric hydroxyapatite heterostructure according to claim 1, characterized in that, According to the concentration ratio, the nano-heterogeneous structure: the skeleton: the natural polyphenol is (1~3) mg / mL: (2~4) wt%: 3 wt%.

3. The hydrogel based on a piezoelectric hydroxyapatite heterostructure according to claim 1, characterized in that, The hydrogel further includes a chondroitinducing factor loaded on the framework; in the hydrogel based on the piezoelectric hydroxyapatite heterostructure, the content of the chondroitinducing factor is 100 μg / mL. The chondrogenic factors include KGN or BMP-7.

4. The method for preparing a hydrogel based on a piezoelectric hydroxyapatite heterostructure as described in any one of claims 1-3, characterized in that, Includes the following steps: Preparation of piezoelectric hydroxyapatite: Dissolve anhydrous magnesium chloride in water, add hydroxyapatite, stir, add alkaline solution, adjust pH, continue stirring, wash, collect precipitate, and dry to obtain piezoelectric hydroxyapatite. Preparation of nano-heterostructures: Graphene oxide was dispersed in water, sonicated, and piezoelectric hydroxyapatite was added and mixed. The mixture was heated to react, cooled, centrifuged, washed, and dried to obtain a reduced graphene oxide-piezoelectric hydroxyapatite composite. The reduced graphene oxide-piezoelectric hydroxyapatite composite was dispersed in water, sonicated, tetrachloroauric acid was added, stirred, sodium citrate was added, reacted, centrifuged, washed, and dried to obtain nano-heterostructures. Preparation of the skeleton: Sodium hyaluronate was dissolved in water, the pH was adjusted, methacrylic anhydride was added, the reaction was carried out in an ice bath, the reaction was terminated, dialyzed, and freeze-dried to obtain methacrylamide hyaluronic acid; methacrylamide hyaluronic acid was dissolved in water, 3-aminophenylboronic acid was added, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added, the pH was adjusted, the mixture was stirred in the dark, the pH was adjusted again, dialyzed, and freeze-dried to obtain the skeleton; Preparation of hydrogels: Dissolve photoinitiator in water, add to the skeleton and dissolve, add nano-heterogeneous structure and mix evenly to obtain a mixed solution. Place the mixed solution in a microfluidic device to prepare microdroplets, photocur, wash, and centrifuge to obtain skeleton microgel microspheres loaded with nano-heterogeneous structure; dissolve natural polyphenols to obtain a natural polyphenol solution, add to the skeleton microgel microspheres loaded with nano-heterogeneous structure, mix evenly to obtain hydrogel based on piezoelectric hydroxyapatite heterostructure.

5. The preparation method according to claim 4, characterized in that, The method for preparing the hydroxyapatite includes the following steps: Calcium nitrate tetrahydrate and diammonium hydrogen phosphate were dissolved in water and acid-adjusted to form a colloidal solution as the aqueous phase; cyclohexane was added to the oil phase as an emulsifier; the aqueous phase was added dropwise to the oil phase, stirred to react, washed, subjected to hydrothermal reaction, cooled, centrifuged, washed, and dried to obtain the final product. According to the mass ratio, the calcium content of the calcium nitrate tetrahydrate to the phosphorus content of the diammonium hydrogen phosphate is (1~10):1; The hydrothermal reaction is carried out at a temperature of 100-120°C for 4-10 hours.

6. The preparation method according to claim 4, characterized in that, In the preparation of piezoelectric hydroxyapatite, the mass ratio of anhydrous magnesium chloride to hydroxyapatite is (0.1~1):1; In the preparation of the nano-heterostructure, the mass ratio of graphene oxide to piezoelectric hydroxyapatite is 1:(2~10); the mass ratio of the reduced graphene oxide-piezoelectric hydroxyapatite composite to tetrachloroauric acid is 1:(0.01~0.05).

7. The preparation method according to claim 4, characterized in that, In the preparation of the skeleton, the ratio of hyaluronic acid to methacrylic anhydride is 1 g: (1~5) mL, according to the dosage ratio. The mass ratio of the methacryloyl hyaluronic acid to the 3-aminophenylboronic acid is 0.5:(0.1~0.3).

8. The preparation method according to claim 4, characterized in that, In the preparation of the hydrogel, the contents of the nano-heterostructure, framework, and natural polyphenols in the hydrogel based on the piezoelectric hydroxyapatite heterostructure are (1~3) mg / mL, (2~4) wt%, and 3 wt%, respectively. The volume ratio of the loaded nanostructured framework microgel microspheres to the natural polyphenol solution is 1:

1.

9. The preparation method according to any one of claims 4-8, characterized in that, The preparation of the hydrogel also includes loading chondroitin-inducing factors, and the preparation of the hydrogel includes the following steps: A photoinitiator was dissolved in water, added to the framework for dissolution, and then mixed with a nano-heterostructure and chondrogenic factor to obtain a homogeneous solution. The homogeneous solution was placed in a microfluidic device to prepare microdroplets, which were then photocured, washed, and centrifuged to obtain framework microgel microspheres loaded with nano-heterostructure and chondrogenic factor. Natural polyphenols were dissolved to obtain a natural polyphenol solution, which was then added to the framework microgel microspheres loaded with nano-heterostructure and chondrogenic factor and mixed to obtain a hydrogel based on a piezoelectric hydroxyapatite heterostructure. In the hydrogel based on the piezoelectric hydroxyapatite heterostructure, the contents of the nano-heterostructure, the framework, the natural polyphenols, and the chondroitin inducing factor are (1~3) mg / mL, (2~4) wt%, 3 wt%, and 100 μg / mL, respectively. The volume ratio of the skeletal microgel microspheres loaded with nano-heterogeneous structures and chondrogenic factors to the natural polyphenol solution is 1:

1.

10. The application of the hydrogel based on the piezoelectric hydroxyapatite heterostructure as described in any one of claims 1-3 or the hydrogel based on the piezoelectric hydroxyapatite heterostructure obtained by the preparation method described in any one of claims 4-9 in the preparation of growth plate damage repair drugs.

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