BPDAH / VMA hydrogel sustained-release microsphere for promoting periodontal bone regeneration as well as preparation method and application of BPDAH / VMA hydrogel sustained-release microsphere

Abstract

The invention discloses BPDAH / VMA hydrogel sustained-release microspheres for promoting periodontal bone regeneration as well as a preparation method and application of the BPDAH / VMA hydrogel sustained-release microspheres, and provides a new material selection for clinical treatment of periodontal bone regeneration, so that the BPDAH / VMA hydrogel sustained-release microspheres can continuously and efficiently promote periodontal bone tissue regeneration, regulate and control an osteogenesis microenvironment and reduce postoperative complications such as infection. The method is expected to become a preferable scheme widely applied to clinical treatment materials, has good clinical application potential, and is expected to provide a new direction for design of other periodontal tissue regeneration and bone regeneration repair materials. VMA is innovatively grafted on the surfaces of the GelMA hydrogel microspheres, and B (at) PH NPs are adsorbed and loaded, so that the GelMA hydrogel microspheres have the curative effects of regulating an osteogenesis microenvironment and inducing periodontal osteoblast differentiation. According to the invention, the hydrogel microsphere is directly injected into the periodontal pocket near the periodontal bone tissue defect, and the osteogenesis microenvironment can be regulated and controlled and osteoblast differentiation can be induced through the slow release effect of the hydrogel microsphere on loaded osteogenesis and anti-inflammatory components, so that periodontal bone regeneration is promoted.

Description

Technical Field

[0001] This invention relates to a BPDAH / VMA hydrogel sustained-release microsphere for promoting periodontal bone regeneration, its preparation method, and its application. It belongs to the technical field of periodontal tissue regeneration materials. Background Technology

[0002] Periodontitis is a chronic, progressive, infectious periodontal disease characterized by periodontal attachment loss, periodontal pocket formation, and alveolar bone resorption. It is the leading cause of tooth loss in adults. The global prevalence of severe periodontitis is reported to be 8.5%, affecting a cumulative total of 743 million people, and its prevalence and severity increase with age. Colonization by periodontal pathogens is the initiating factor of periodontitis. Early plaque colonization at the gingival junction can trigger an immune inflammatory response in the host. If left untreated, gingival inflammation can progress to deeper periodontal tissues, leading to progressive destruction of periodontal supporting tissues such as alveolar bone, cementum, and periodontal ligament. As the lesion progresses towards the root, symptoms such as gingival recession, difficulty chewing, and tooth loosening and displacement may occur, ultimately leading to tooth loss. Attachment loss and true periodontal pocket formation are characteristic pathological manifestations of periodontitis. Combined with alveolar bone resorption visible on imaging, as well as clinical manifestations such as calculus, gingival bleeding, and periodontal purulent discharge, these can serve as diagnostic criteria for periodontitis. Controlling the progression of periodontal disease and repairing periodontal tissue defects are the ultimate goals of clinical treatment for periodontitis. However, traditional periodontitis treatment focuses on plaque and inflammation control, halting or delaying disease progression, but rarely achieves satisfactory periodontal tissue regeneration. Therefore, seeking effective and stable periodontal tissue regeneration techniques is of significant clinical importance.

[0003] The challenges of periodontal bone regeneration treatment are as follows: First, long junctional epithelial healing is the primary healing mechanism in periodontium, making regenerative healing difficult to achieve. The healing mechanism of periodontal tissues depends on the growth rate of cells from different periodontal sources and the particle size and absorption rate of the bone graft material. Periodontal ligament precursor cells and alveolar bone marrow stem cells grow more slowly than cells derived from gingival epithelium and connective tissue. New periodontal attachment within the periodontal pocket is often blocked by long junctional epithelium on the root surface, hindering regenerative healing of periodontal tissues.

[0004] II. The osteogenic microenvironment is susceptible to the influence of inflammatory cells and related factors: The destruction and regeneration of periodontal bone tissue are mainly regulated by osteoblasts and osteoclasts. As an important part of the host defense system, periodontal barrier epithelial cells and immune cells such as neutrophils, monocytes / macrophages in the gingival crevicular fluid release pro-inflammatory factors such as interleukins, prostaglandins and proteases in the periodontal inflammatory environment, while activating osteoclasts, destroying the osteogenic microenvironment, and affecting the regeneration effect and speed of periodontal tissue.

[0005] Third, periodontal surgery is highly complex, invasive, and prone to complications: Bone grafting and traditional artificial bone filling procedures are lengthy, significantly invasive, and susceptible to postoperative complications such as bleeding, infection, and rejection. Furthermore, periodontal regeneration surgery is technically demanding and complex, hindering its widespread clinical application. Hydrogel microspheres, also known as microgels, are a novel and advantageous drug-loaded material that can adapt to the complex oral environment and is widely used in the treatment of various oral diseases. However, their application in periodontal bone regeneration is limited, lacking relevant experimental data and clinical translational applications.

[0006] Current research includes patent CN117819530B, which discloses a calcium ion-doped carbon dot and self-triggered sustained-release system, its preparation method, and applications. In this method, aspirin, metformin, and an aqueous solution of calcium chloride are mixed and heated under vacuum to form carbon dots. Based on this, the positive charge of calcium ions in the carbon dots attracts and cross-links with the negative charge of sodium alginate carboxyl groups, achieving a self-triggered gelation process to form a carbon dot-based hydrogel microsphere sustained-release system. This invention's hydrogel microsphere sustained-release system, by adjusting the raw material ratio, can obtain fluorescent carbon dots with high calcium ion doping content and their microsphere sustained-release system. Furthermore, it can be applied in biomedical fields such as inflammation and bone defects, solving the problems of poor water solubility and rapid release of existing non-steroidal anti-inflammatory drugs, which cannot remain at the site of inflammation or defect for extended periods.

[0007] For example, patent application CN117860790A discloses oxygen-releasing hydrogel microspheres loaded with probiotics and their application in periodontitis, specifically hydrogel microspheres loaded with calcium peroxide and Lactobacillus rhamnosus. The hydrogel microspheres described in this invention can maintain the activity of probiotics, stably release oxygen, selectively inhibit the growth of *F. nucleatum* and its biofilm formation, exhibit good cell compatibility and blood compatibility, and simultaneously promote osteoblast differentiation. In rat periodontitis experiments, the hydrogel microspheres can alleviate periodontal tissue inflammation and promote periodontal bone formation, showing promise for synergistic treatment of periodontitis.

[0008] Current technologies still have some shortcomings: First, the bone regeneration effect is unsatisfactory: Existing bone repair materials lack sufficient bioactivity, making it difficult to achieve a sustained and efficient bone regeneration effect. The degradation rate of conventional bone repair materials such as hydroxyapatite and calcium phosphate ceramics does not match the osteogenic rate, making them prone to ectopic osteoogenesis. Furthermore, they lack the ability to actively regulate the bone regeneration microenvironment, resulting in unstable bone regeneration effects.

[0009] 2. Short bone-inducing regeneration time: The activity of growth factors is unstable, and their local delivery is difficult to induce osteoblast differentiation for a long time; growth factors such as osteogenic growth factor (BMP-2) have short half-lives in vivo, are easily biodegraded, and have short duration of biological activity; although existing carriers can partially improve the drug release rate, they are prone to burst release, resulting in short duration of effective drug concentration, insufficient cumulative drug release, and poor long-term osteogenic effect.

[0010] 3. High risk of infection: Periodontal surgery and bone grafting have a high risk of infection, and existing bone repair materials have poor anti-infection properties, making them prone to secondary infections. Inflammatory cell infiltration activates osteoclast activity, leading to an imbalance in the osteogenic microenvironment, reducing osteogenic efficiency, and even causing bone resorption. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a BPDAH / VMA hydrogel sustained-release microsphere for promoting periodontal bone regeneration, its preparation method and application, so as to solve the technical problems of poor bone regeneration effect, short bone induction regeneration time and easy infection in periodontal bone regeneration treatment.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing BPDAH / VMA hydrogel sustained-release microspheres that promote periodontal bone regeneration, the specific steps of which are as follows: (1) Polydopamine-heparin nanoparticles (PDAHNPs) were prepared using dopamine hydrochloride (DH) and heparin sodium (Hep) as raw materials. (2) Then load the PDAH NPs with osteogenic growth factor BMP-2 to obtain B@PH NPs; (3) Then, methacrylamide gelatin (GelMA) was dissolved in a photoinitiator (LAP) solution to obtain a prepolymer solution; then, B@PH NPs and vanillin methacrylate (VMA) were fully dispersed in the prepolymer solution to obtain a BPDAH / VMA microsphere precursor solution. (4) Finally, using the BPDAH / VMA microsphere precursor solution as the dispersed phase and oil as the continuous phase, the BPDAH / VMA hydrogel sustained-release microspheres were obtained by using a capillary glass microfluidic chip under UV irradiation.

[0013] Preferably, steps (1) to (3) are all performed under light-protected conditions.

[0014] Preferably, the specific method of step (1) is as follows: first, ammonia water, anhydrous ethanol and the first part of deionized water are mixed evenly to obtain a premixed solution; then, dopamine hydrochloride (DH) and heparin sodium (Hep) are dissolved in the second part of deionized water, and then the premixed solution is added, mixed thoroughly, and stirred under light-protected conditions for cross-linking reaction. After post-treatment, polydopamine-heparin nanoparticles (PDAH NPs) are obtained; wherein, the mass concentration of ammonia water is 28-30%.

[0015] A further preferred embodiment uses ammonia, anhydrous ethanol, deionized water (part 1), dopamine hydrochloride (DH), heparin sodium (Hep), and deionized water (part 2) in a ratio of 0.8 mL: 40 mL: 90 mL: 0.5 g: 0.1 g: 10 mL. The purpose of preparing this mixed solution of deionized water, ethanol, and ammonia is to adjust the pH with ammonia, use dissolved oxygen as an oxidant, and reduce the solution polarity with ethanol, thereby promoting the uniform nucleation of nanoparticles and providing chemical reaction conditions for the oxidative self-polymerization of dopamine.

[0016] Further preferred crosslinking reaction conditions are: 500 rpm and 30 h. Stirring under light-protected conditions aims to prevent dopamine from oxidizing under light and to prevent photo-oxidation side reactions from interfering with the polymerization process.

[0017] Further preferred post-processing includes: centrifugation at 14000 rpm for 10 min, collection of precipitate, washing with anhydrous ethanol, repeating the centrifugation and washing steps again, and drying to obtain the final product.

[0018] Preferably, the specific method of step (2) is as follows: PDAH NPs and osteogenic growth factor BMP-2 are dissolved in PBS, and BMP-2 is loaded by stirring at room temperature under light-protected conditions. The purpose of stirring under light-protected conditions is to avoid the dopamine component in the nanoparticles being oxidized and its structure destroyed by light.

[0019] More preferably, the ratio of PDAH NPs, osteogenic growth factor BMP-2, and PBS is 1 mg: 1 μg: 1 mL; The concentration of PBS was 0.01 mol / L, and the pH was 7.4.

[0020] Further preferred stirring conditions at room temperature are: 500 rpm for 24 hours.

[0021] Further preferred methods include post-processing after stirring at room temperature, including: centrifugation at 12000 rpm for 10 min, collection of the precipitate, freezing at -80℃ for 8–12 h, rapid transfer to a freeze dryer for vacuum drying for 8–12 h, and storage at 4℃ in a sealed, light-protected environment. Rapid transfer of the pre-frozen sample is crucial to prevent melting and subsequent collapse of the hydrogel's three-dimensional network structure.

[0022] Preferably, in step (3), the ratio of methacrylamide gelatin (GelMA), photoinitiator LAP solution, B@PH NPs, and vanillin methacrylate (VMA) is 100g:1mL:1mg:1mg, wherein the mass concentration of the photoinitiator LAP solution is 0.05%, and the photoinitiator LAP solution is obtained by dissolving LAP in PBS.

[0023] Preferably, in step (3), the method for preparing the prepolymer solution is: heating at 60°C in the dark for 30 minutes. During raw material mixing, aluminum foil can be used to cover the mixture to prevent light exposure and deactivation of the photoinitiator LAP due to external light and temperature. Heating in the dark aims to prevent LAP from being deactivated by light and to prevent partial crosslinking of GelMA under light.

[0024] Preferably, in step (4), the continuous phase is 2% Drop-Surf microdroplet-generated oil.

[0025] Preferably, in step (4), the flow rate of the dispersed phase is 96 μL / min, and the flow rate of the continuous phase is 120 μL / min, to ensure the formation of microspheres, improve the microsphere yield, and prevent chip blockage caused by the adhesion of the two phases. The microfluidic outlet hose is irradiated with 405 nm UV light for 30 s (to ensure that the photoinitiator LAP and the 405 nm sensitive wavelength UV light excite the methacrylate groups in GelMA to undergo a free radical chain polymerization reaction, so that the hydrogel microspheres are fully cross-linked and cured), to achieve polymerization and curing.

[0026] Preferably, in step (4), after polymerization and solidification, fresh microspheres are collected and purified. The specific method is as follows: fresh microspheres are mixed with demulsifier at a volume ratio of 1:1, allowed to stand, centrifuged, soaked in deionized water, centrifuged, soaked in 75% ethanol solution, centrifuged, soaked in PBS, centrifuged, frozen at -80℃ for 8-12 hours, quickly transferred to a freeze dryer for vacuum drying for 8-12 hours, and stored at 4℃ in the dark. The demulsifier is Drop-Surf demulsifier, purchased from Chengdu Paier Chip Technology Co., Ltd.

[0027] Further preferably, the fresh microspheres are mixed with the demulsifier and allowed to stand for 5 minutes to remove the continuous phase oil solution.

[0028] In a further preferred embodiment, the centrifugation conditions were all 2700 rpm for 3 min.

[0029] In a further preferred embodiment, the soaking and corresponding centrifugation steps are both repeated once.

[0030] A BPDAH / VMA hydrogel sustained-release microsphere that promotes periodontal bone regeneration was obtained by the aforementioned preparation method.

[0031] The aforementioned application of BPDAH / VMA hydrogel sustained-release microspheres in the preparation of restorative materials that promote periodontal bone regeneration.

[0032] Technical principle of the invention: 1. Application and Polymerization Principle of PDAH NPs: Heparin has a strong affinity for BMP-2 and can stably bind to BMP-2 through electrostatic interactions, preventing its rapid degradation or inactivation and prolonging its biological activity cycle. Simultaneously, the catechol structure of polydopamine (PDA) can chemically enhance the binding of nanoparticles to BMP-2. Combined with the regulation by heparin, this achieves the continuous and slow release of BMP-2, ensuring its effective concentration is maintained in the local microenvironment.

[0033] Dopamine molecules consist of an amino group linked to catechol (or phenol) via an ethylamino group. The catechol structure of the dopamine monomer can be oxidized to benzoquinone in an alkaline environment (with ammonia providing pH adjustment) by an oxidizing agent (dissolved oxygen or trace amounts of oxidizing agents in ethanol). This benzoquinone then forms PDA nanoparticles through non-covalent interactions such as π-π stacking and hydrogen bonding, as well as covalent crosslinking reactions such as Michael addition. Heparin is a highly sulfated glycosaminoglycan with strongly negatively charged sulfate (-SO3⁻) and carboxyl (-COO⁻) groups, which can bind to protonated amino groups (-NH3⁺) on the PDA surface to form ionic bonds. Simultaneously, the hydroxyl (-OH) group of heparin forms hydrogen bonds with the quinone or amino groups of PDA, further enhancing the stability of the polydopamine three-dimensional network structure. Furthermore, the introduction of heparin can produce a steric hindrance effect, inhibiting the spatial extension of polydopamine and thus regulating the nanoparticle size, causing the nanoparticle diameter to decrease with increasing heparin concentration.

[0034] 2. Microfluidic Technology Principle: Microfluidics utilizes microchannel structures such as chips and coaxial needles to regulate fluid flow and mixing. In the preparation of microspheres, two or more immiscible liquids are introduced into the microchannels. At the confluence of the channels, fluid shearing causes one phase to disperse into another, forming uniformly sized emulsion droplets (dispersed phase), which are carried forward by a continuously flowing liquid (mobile phase). Subsequently, the droplets solidify into spheres through physicochemical processes such as polymerization, solvent evaporation, and phase separation. To meet different needs, materials with different functions can be added to the microspheres as carriers to synthesize polymer microspheres or polymer particles. This invention adds BMP-2@PDAH NPs and VMA to the continuous phase to achieve loading of BMP-2@PDAH NPs and grafting of VMA, thereby endowing the microspheres with anti-inflammatory and osteogenic properties.

[0035] The capillary glass microfluidic chip (purchased from Luxe Microcontroller (Shenzhen) Technology Co., Ltd.) consists of two glass substrates. The main structural units include inlet microchannels for the dispersed and mobile phases, an outlet microchannel for the collecting phase, and an intermediate microstructure responsible for shearing the droplets. The core principle is to inject a hydrogel precursor liquid as the dispersed phase and an oil phase as the continuous phase into two independent microfluidic chip channels. When the two phases come into contact at the channel junction, the continuous phase encapsulates the dispersed phase droplets and detaches from the fluid through shear force and surface tension, forming uniform monodisperse droplets. Subsequently, the droplets are cured into hydrogel microspheres through photocrosslinking and other curing methods.

[0036] 3. Photocuring Principle of GelMA Hydrogel Microspheres and VMA Grafting: Gelatin (Gel) is a common hydrogel matrix material, rich in active groups such as amino and hydroxyl groups, and has good biocompatibility. Vanillin is an aromatic aldehyde extracted from lignin, which can scavenge excess ROS, reduce oxidative stress, and decrease inflammatory responses. Its molecular chain has a large number of active groups such as hydroxyl groups, providing active sites for chemical modification. Both can be functionalized with methacrylates through esterification, covalently linking methacrylate groups to corresponding sites to form polymerizable double bond structures, thus producing GelMA and VMA. Under the action of a photoinitiator, GelMA can undergo free radical chain polymerization, triggering chain initiation, chain growth, and chain termination processes, thereby forming a three-dimensional network structure between molecules and achieving microsphere curing. Simultaneously, VMA is grafted onto GelMA via a Schiff base reaction.

[0037] The functionalization of methacrylates not only retains the original bioactivity of gelatin and vanillin, but also introduces photocuring properties. Its crosslinking density can be controlled by parameters such as light intensity and initiator concentration, providing a basis for the customization of material properties.

[0038] The beneficial effects of this invention are as follows: This invention provides a BPDAH / VMA hydrogel sustained-release microsphere for promoting periodontal bone regeneration, its preparation method, and its application. It offers a new material option for the clinical treatment of periodontal bone regeneration, enabling it to continuously and efficiently promote periodontal bone tissue regeneration, regulate the osteogenic microenvironment, and reduce postoperative complications such as infection. It is expected to become a preferred solution for widely used clinical treatment materials, with great clinical application potential, and is also expected to provide new directions for the design of other periodontal tissue regeneration and bone regeneration repair materials.

[0039] This invention innovatively grafts VMA onto the surface of GelMA hydrogel microspheres and adsorbs and loads B@PH NPs, which has the therapeutic effect of regulating the osteogenic microenvironment and inducing periodontal osteoblast differentiation. In this invention, the hydrogel microspheres described herein are directly injected into the periodontal pocket near the periodontal bone tissue defect. Through the sustained release effect of the hydrogel microspheres on the loaded osteogenic and anti-inflammatory components, the osteogenic microenvironment can be regulated and osteoblast differentiation induced, thereby promoting periodontal bone regeneration.

[0040] Material properties characterization of the hydrogel sustained-release microspheres obtained in this invention: ① Nanoparticle morphology characterization: PDAH NPs exhibit a monodisperse spherical structure with an average diameter of 288.2 nm; ② Energy-dispersive X-ray spectroscopy (EDS) characterization of nanoparticles: The sulfur element in heparin is uniformly distributed on the surface of PDHANPs; ③ Morphological characterization of hydrogel microspheres: SEM observation revealed that the average particle size of BPDAH / VMA was 126 μm; ④ Fourier transform infrared (FTIR) characterization: PDAH NPs in the 1500-1510 cm⁻¹ range -1 1380-1385cm -1 and 1610-1611cm -1 A characteristic peak appears at 1393.67 cm⁻¹. GM-V reaches this peak at 1393.67 cm⁻¹. -1 1343.68 cm -1 Nearby and 1643.56cm -1 An absorption peak appears at this location; ⑤ Hydrophilic properties: The hydrogel microspheres reached swelling equilibrium after 24 h of swelling, with an equilibrium swelling rate of 775.75%, indicating good hydrophilic properties; ⑥ Biodegradability: The hydrogel microspheres showed a degradation rate of 66.8% after 72 hours, indicating excellent biodegradability in the oral microenvironment. ⑦ Drug sustained-release performance: The hydrogel microspheres rapidly release BMP-2 in the early stage to achieve the effective drug concentration and achieve sustained release of BMP-2 within 48 hours. The cumulative drug release rate reaches 86.5% after 72 hours of release.

[0041] Biological performance characterization of hydrogel sustained-release microspheres: ① Cell compatibility: Except for the 150 mg / mL group, the cell survival rate of all experimental groups was higher than 70%. BPDAH / VMA extract had little effect on cell proliferation, and BPDAH / VMA showed good cell compatibility; ②Cytotoxicity: Cell survival rates in all experimental groups were above 85%. BPDAH / VMA extract showed no significant cytotoxicity, and BPDAH / VMA exhibited good biocompatibility. ③ Anti-inflammatory properties: Intracellular ROS levels in each experimental group gradually decreased with increasing extract concentration; in the 200 mg / mL experimental group, intracellular ROS levels were close to those in the control group. High-concentration BPDAH / VMA extract can effectively scavenge ROS, and BPDAH / VMA can exert anti-inflammatory effects by regulating the periodontal inflammatory microenvironment by reducing ROS levels. ④ Osteogenic performance: Compared with the BMP-2 group, the ALP activity of the B@PH NPs group was significantly increased, indicating that PDAH NPs can maintain BMP-2 activity and achieve long-term osteogenic effects. The ALP activity of the BPDAH / VMA group was significantly higher than that of the BMP-2 group and the B@PHNPs group, indicating that there is a synergistic effect between VMA and BMP-2, and the introduction of VMA can significantly enhance the osteogenic induction ability of the system.

[0042] This invention has the following advantages: 1. The hydrogel sustained-release microspheres for promoting periodontal bone regeneration described in this invention possess the characteristic three-dimensional network structure of hydrogels, which can regulate the release of biomolecules and simulate the cell growth environment. They are not only an ideal local drug delivery carrier but also beneficial for cell adhesion and growth. The hydrogel microspheres described in this invention have excellent hydrophilicity, biodegradability, and drug sustained-release properties, good biocompatibility, and significant anti-inflammatory and osteogenic properties, showing promising application prospects.

[0043] 2. The present invention discloses a method for preparing hydrogel sustained-release microspheres for promoting periodontal bone regeneration. This method uses GelMA as the hydrogel matrix, utilizes the free radical polymerization reaction of methacrylate groups and a photoinitiator to photocure and cross-link the hydrogel network, and grafts VMA onto GelMA via a Schiff base reaction to effectively improve its fluidity and thermal stability, making it easy to inject. Simultaneously, microfluidic technology is applied to generate highly monodisperse microspheres at an adjustable production rate, achieving efficient drug encapsulation and slow release. By adjusting parameters such as the flow rate and channel diameter of the microfluidic system, the size and structure of the microspheres can be precisely controlled. Furthermore, the method requires low-cost processing equipment, is easy to assemble, and reduces production costs.

[0044] 3. The application of the hydrogel sustained-release microspheres for promoting periodontal bone regeneration described in this invention. The hydrogel microspheres prepared by this method have the potential to promote periodontal bone tissue repair. In this invention, the hydrogel microspheres described in this invention are directly injected into the periodontal pocket near the periodontal bone tissue defect using a microsyringe. Through the sustained-release effect of the hydrogel microspheres on osteogenic and anti-inflammatory components, an innovative method is provided to regulate the osteogenic microenvironment and induce osteoblast differentiation, thereby promoting periodontal bone regeneration.

[0045] This invention achieves the following innovations: 1. Innovation in drug selection: Clinically, antibacterial treatment for periodontitis, besides mechanical scaling, mainly involves oral antibiotics, but this is prone to bacterial resistance and adverse reactions. This study utilizes vanillin grafted onto a three-dimensional network framework of porous hydrogel microspheres to fully leverage its advantages as a natural active pharmaceutical ingredient with low cytotoxicity, exhibiting antibacterial, anti-inflammatory, and antioxidant effects against periodontitis in the early stages of drug release. Furthermore, for bone resorption caused by periodontitis, current research often employs osteogenic stem cell delivery therapy as a treatment for periodontal bone regeneration, but this is easily affected by pathological factors such as the inflammatory microenvironment, resulting in poor osteogenic stability and sustainability. This study uses BMP-2 loaded with PDAH nanoparticles to maintain drug activity, continuously recruit autologous stem cells, and achieve a sustained and stable osteogenic effect. This invention innovatively combines the above-mentioned drugs, exhibiting a significant synergistic osteogenic effect and greatly improving the efficacy of periodontal bone regeneration.

[0046] 2. Drug carrier innovation: Existing local drug delivery systems still suffer from limitations such as insufficient dosage and relatively low bioavailability. This invention innovatively combines three drug carriers—nanoparticles, hydrogels, and microspheres—to prepare hydrogel microspheres loaded with nanoparticles. This combination combines the advantages of nanoparticles (high targeting and delivery efficiency), microspheres (strong encapsulation stability and high drug loading capacity), and hydrogels (high biocompatibility and good sustained-release properties). This combination not only increases the cumulative drug release but also ensures the sequential release of drug components, namely, the initial release of externally grafted VMA followed by the subsequent sustained release of internally encapsulated BPDAH nanoparticles. Furthermore, the drug loading is highly flexible, adaptable to the forefront of clinical drug development and capable of upgrades and iterations, possessing extremely high clinical application potential. This invention combines different drugs and carriers, focusing on the entire process of periodontitis development, pioneering a new approach to local drug delivery, aiming to solve the challenges of clinical periodontitis treatment.

[0047] 3. Innovative Treatment Plans: Current treatments for periodontitis focus on single antibacterial or bone repair therapies, neglecting the inhibitory effect of the inflammatory microenvironment on bone regeneration. This invention focuses on the overall pathogenesis of periodontitis—"plaque adhesion - inflammatory microenvironment dysregulation - periodontal bone resorption"—and proposes a phased treatment model of "anti-inflammatory first, then osteogenic." By rapidly releasing VMA to clear excess ROS and inhibit the secretion of pro-inflammatory factors, the inflammatory microenvironment is reversed. Then, BMP-2 is released slowly to activate the osteogenic differentiation of autologous bone marrow stem cells, synergizing with the anti-inflammatory effect of VMA to break the vicious cycle of "inflammation-bone resorption." Therefore, this approach integrates the anti-inflammatory properties of VMA and the osteogenic induction function of BMP-2 into a multifunctional drug delivery carrier. Through matching the material degradation kinetics with the drug release curve, spatiotemporally and spatially controllable periodic synergistic treatment is achieved. Attached Figure Description

[0048] Figure 1 shows the appearance of B@PH NPs powder in an embodiment of the present invention; Figure 2 is an appearance diagram of BPDAH / VMA microspheres in an embodiment of the present invention; Figure 3 is a scanning electron microscope image of B@PH NPs in an embodiment of the present invention (A: 300nm scale bar, B: 100nm scale bar, C: particle size analysis of B@PH NPs). Figure 4 shows a scanning electron microscope image of BPDAH / VMA microspheres in an embodiment of the present invention (A: 10 μm scale bar, B: 20 ​​μm scale bar, C: BPDAH / VMA microsphere particle size analysis). Figure 5 shows the energy dispersive spectroscopy (EDS) analysis of B@PH NPs in an embodiment of the present invention; Figure 6 shows the infrared spectrum in an embodiment of the present invention (A: infrared spectrum of B@PH NPs, B: infrared spectrum of BPDAH / VMA microspheres). Figure 7 is a swelling curve of BPDAH / VMA microspheres in an embodiment of the present invention; Figure 8 shows the degradation test results of BPDAH / VMA microspheres in the embodiments of the present invention (A: degradation morphology of GM and BPDAH / VMA, B: 72h degradation rate of GM and BPDAH / VMA). The data are expressed as SD ± average, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 9 is an in vitro drug release curve of BPDAH / VMA microspheres in an embodiment of the present invention; Figure 10 is a statistical analysis graph of the effect of different concentrations of BPDAH / VMA extract on the proliferation of MC3T3-E1 cells investigated by CCK-8 assay in this embodiment of the invention. Data are expressed as SD ± mean, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 11 is a statistical analysis graph of the effect of different concentrations of BPDAH / VMA extract on the survival rate of MC3T3-E1 cells investigated by live and dead cell staining in this embodiment of the invention (A: staining results, B: statistical analysis). Data are expressed as SD ± mean, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 12 shows the effect of different concentrations of BPDAH / VMA extract on the in vitro ROS level of MC3T3-E1 by the ROS fluorescent probe in the embodiments of the present invention (A: staining results, B: statistical analysis). The data are expressed as SD ± mean, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 13 shows the data on the effect of different treatments on osteogenic differentiation of MC3T3-E1 by ALP staining in the embodiments of the present invention. The data are expressed as SD ± mean, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 14 is a flowchart of the preparation process of BPDAH / VMA microspheres in an embodiment of the present invention. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0050] Explanation of relevant technical terms.

[0051] Periodontitis: Periodontitis is a chronic, infectious, progressive periodontal disease caused by periodontal pathogens (dental plaque) and associated with various local and systemic contributing factors. It is the leading cause of tooth loss in adults. The pathological changes in periodontitis are mainly characterized by chronic inflammation caused by dental plaque and progressive destruction of periodontal tissues due to immune responses. Clinically, patients often present with symptoms such as gingivitis, periodontal pocket formation, alveolar bone resorption, and tooth loosening. Periodontal bone resorption is a common and serious complication of periodontitis, often leading to tooth loosening and even tooth loss.

[0052] Bone morphogenetic protein-2 (BMP-2): BMP-2 is a clinically used bone repair factor that can mediate osteogenic signaling pathways to promote bone formation. However, its biological activity is unstable and its half-life in vivo is short, resulting in poor efficacy when applied directly.

[0053] Dopamine: The dopamine molecule consists of an amino group linked to catechol (or phenol) via an ethylamino group. The catechol structure of the dopamine monomer can be oxidized to benzoquinone in an alkaline environment (where ammonia provides pH adjustment) by an oxidizing agent (dissolved oxygen or trace amounts of oxidizing substances in ethanol). Through non-covalent interactions such as π-π stacking and hydrogen bonding, as well as covalent crosslinking reactions such as Michael addition, polydopamine (PDA) nanoparticles are synergistically formed.

[0054] Heparin: Heparin is a highly sulfated glycosaminoglycan. Its sulfate group (-SO3⁻) and carboxyl group (-COO⁻) carry strong negative charges, which can combine with the protonated amino group (-NH3⁺) on the PDA surface to form ionic bonds. At the same time, the hydroxyl group (-OH) of heparin forms hydrogen bonds with the quinone group or amino group of PDA, further improving the stability of the polydopamine three-dimensional network structure.

[0055] B@PH NPs: Polydopamine nanoparticles that are heparin-functionalized and loaded with bone morphogenetic protein-2.

[0056] Vanillin: Vanillin, also known as vanillin, is an aromatic aldehyde extracted from lignin. It can scavenge excess ROS, reduce oxidative stress, and decrease inflammatory response. It has a simple structure, is rich in active groups, and easily forms polymers with monomers.

[0057] Vanillin methacrylate (VMA): The methacrylate group (-MA) is a common active group. In its carbon-carbon unsaturated double bond (C=C), the π bond is relatively weak and easily broken under external conditions such as heat, light, and initiators. After being attacked by free radicals, it can initiate a chain polymerization reaction. The electron-withdrawing inductive effect of the ester group (-COOR) enhances the electrophilicity of the double bond, making it more susceptible to free radical attack. At the same time, the carbonyl group (C=O) forms a conjugated system with the carbon-carbon double bond, which delocalizes the electron cloud, reduces molecular energy, increases stability, and can also stabilize polymerization intermediates and promote the continuous progress of the reaction. Therefore, it is widely used in material synthesis. By modifying vanillin with the methacrylate group, vanillin methacrylate can be obtained, which retains its anti-inflammatory and antioxidant properties and can crosslink with other polymers containing methacrylate groups through free radical chain polymerization.

[0058] Methacrylic anhydride gelatin (GelMA): GelMA is a photosensitive bio-hydrogel material prepared from methacrylic anhydride (MA) and gelatin. This material exhibits excellent biocompatibility and can be activated by ultraviolet or visible light to form a three-dimensional structure with sufficient strength suitable for cell growth and differentiation. It is primarily used in tissue engineering and bio-3D printing materials.

[0059] Hydrogel microspheres: Hydrogels are polymeric materials formed by cross-linking of polymers. They have a three-dimensional network structure similar to the natural extracellular matrix (ECM). Due to their excellent plasticity and biocompatibility, they have been widely used in tissue engineering and regenerative medicine. Currently, novel injectable in-situ hydrogels such as thermosensitive hydrogels and photocrosslinked hydrogels can be directly injected to the lesion through minimally invasive means, and can also achieve in-situ solidification in response to external environmental factors such as temperature and light. Hydrogel microspheres, also known as microgels, are micron-sized microspheres formed by the solidification of in-situ hydrogel matrix droplets. They retain the characteristics of injectable in-situ hydrogels, such as minimally invasive and flexible drug delivery and efficient and convenient solidification, while also achieving high cross-linking through high-throughput synthesis, thus improving mechanical strength and stability.

[0060] Example 1: Figure 14 illustrates a method for preparing BPDAH / VMA hydrogel sustained-release microspheres that promote periodontal bone regeneration: (I) Preparation of nanoparticles 1. Preparation of PDAH NPs (1) Oxidative self-polymerization of PDAH NPs: ① Weigh 90 mL of deionized water, 40 mL of ethanol and 0.8 mL of ammonia, transfer them to a beaker and mix thoroughly at room temperature for 10 min; ② Dissolve 0.5 g of dopamine hydrochloride and 0.1 g of heparin sodium in 10 mL of deionized water, and mix thoroughly with the above solution; ③ Use a magnetic stirrer to stir the mixed solution at a speed of 500 rpm and polymerize for 30 h in a dark environment at room temperature.

[0061] (2) Collection and storage of PDAH NPs: ① Dispense the nanoparticle solution into 50mL centrifuge tubes; ② After balancing, place the mixture in a centrifuge and centrifuge at 14000 rpm for 10 min, then discard the supernatant; ③ Add 10 mL of anhydrous ethanol to the precipitate after centrifugation and wash thoroughly; ④ Centrifuge and wash again under the same conditions and discard the supernatant. The resulting precipitate is PDAH NPs. ⑤ After drying at room temperature, store in a dry, sealed container at 4°C in the dark.

[0062] 2. Preparation of B@PH NPs: ① Dissolve 100 mg PDAH NPs and 100 μg BMP-2 in 100 mL PBS; ② Use a magnetic stirrer to stir the mixed solution at a speed of 500 rpm for 24 hours in a dark environment at room temperature; ③ Transfer the mixture to a 50 mL centrifuge tube; ④ After balancing, place the mixture in a centrifuge and centrifuge at 12,000 rpm for 10 min. Discard the supernatant and transfer the precipitate to an EP tube. ⑤ Place the EP tube in an ultra-low temperature freezer at -80℃ for 8 hours; ⑥ Remove the EP tube, open the cap, and quickly transfer it to a freeze dryer for vacuum drying for 10 hours; ⑦ Remove the dried sample, seal it, and store it in a dry, sealed container at 4 ℃ in the dark.

[0063] Experimental results: As shown in Figure 1, dried nanoparticles were successfully prepared, and no obvious impurities were observed with the naked eye. No container sticking occurred during storage.

[0064] The finished product appears as powder or lumps, with a uniform texture and a uniform black color.

[0065] Figure 1 shows the appearance of B@PH NPs powder.

[0066] (II) Preparation of hydrogel microspheres

[0067] 1. Preparation of dispersed and continuous phase solutions: ① Weigh 5 mg LAP and dissolve it in 10 mL PBS phosphate buffer to obtain a 0.05 wt% LAP solution. Store the solution in a foil-coated centrifuge tube for later use. ② Weigh 100 mg of lyophilized GelMA, dissolve it in 1 mL of 0.05 wt% LAP solution, heat at 60°C in the dark for 30 min until GelMA is completely dissolved, and obtain the hydrogel prepolymer solution. ③ Weigh 1 mg of BMP-2@PDAH NPs and 1 mg of VMA and fully disperse them in GelMA to prepare a BPDAH / VMA microsphere precursor solution, which is used as the dispersed phase; ④ The continuous phase solution was generated using Drop-Surf 2% microdroplet oil (purchased from Chengdu Paier Chip Technology Co., Ltd.).

[0068] 2. Install the microfluidic device (the microfluidic chip was purchased from Luxue Microcontroller (Shenzhen) Technology Co., Ltd., model Luxue-PD-wo): ① Connect the CNC equipment to the power supply; ② Connect the CNC equipment connector to the injection pump interface; ③ Use a 5 mL sterile syringe to draw 1 mL of the dispersed phase solution and use a 5 mL syringe to draw 1.5 mL of the continuous phase solution; ④ Attach the two syringes to the syringe pump respectively; ⑤ Connect the syringe needles of the dispersed phase and the continuous phase to the inlet of the chip microchannel using two capillary plastic tubing respectively; ⑥ Connect the collection tubing to the microchannel outlet, and fix the free end to the EP tube port to collect the microspheres; ⑦ Adjust the microfluidic parameters to set the flow rate of the dispersed phase to 96 μL / min and the flow rate of the continuous phase to 120 μL / min.

[0069] 3. Preparation of BPDAH / VMA microspheres: ① The injection pump is started by CNC equipment to shear the dispersed phase with a continuous flow at a uniform speed, so as to obtain dispersed phase microsphere droplets of uniform size. ② Irradiate the microfluidic outlet tube with 405 nm UV light for about 30 s to obtain polymerized microspheres; ③After the liquid in the syringe is emptied, the unpurified solid BPDAH / VMA microspheres are collected in the EP tube.

[0070] 4. Collection and preservation of BPDAH / VMA microspheres: ① Add demulsifier 1:1 to EP tube containing BPDAH / VMA microspheres, let stand for 5 min, centrifuge at 2700 rpm for 3 min, and discard the lower layer liquid; ② Soak in deionized water for 3 min, centrifuge at 2700 rpm for 3 min, discard the supernatant, and repeat once; ③ Soak in 75% ethanol for 3 min, centrifuge at 2700 rpm for 3 min, discard the supernatant, and repeat once; ④ Soak in PBS for 3 min, centrifuge at 2700 rpm for 3 min, discard the supernatant, and repeat once; ⑤ Place the EP tube in an ultra-low temperature freezer at -80℃ for 4 to 12 hours; ⑥ Remove the EP tube, open the cap, and quickly transfer it to a freeze dryer for vacuum drying for 8-12 hours; ⑦ Remove the dried sample, seal it, and store it in a dry, sealed container at 4 ℃ in the dark.

[0071] Experimental results: As shown in Figure 2, the hydrogel microsphere droplets can rapidly transform from a liquid state to a gel state after being cured under ultraviolet light for 30 seconds, which is in line with the experimental expectations.

[0072] Figure 2 shows the appearance of BPDAH / VMA microspheres; (III) Material performance testing

[0073] 1. Material properties characterization: (1) PDAH NPs SEM test: As shown in Figure 3, the PDAH NPs prepared in this application have a monodisperse spherical structure with an average diameter of 288.2 nm.

[0074] (2) BPDAH / VMA SEM test: As shown in Figure 4, SEM observation revealed that the average particle size of BPDAH / VMA was 126 μm, making it suitable for injection.

[0075] (3) Energy-dispersive X-ray spectroscopy (EDS) analysis of PDAH NPs: As shown in Figure 5, PDAH NPs contain 82.63% C, 9.67% O, 7.63% N and 0.08% S, and the S element in heparin is evenly distributed on the surface of PDAH NPs, which is beneficial for loading BMP-2 and enhancing its activity.

[0076] (4) Fourier transform infrared spectroscopy (FTIR) analysis: As shown in Figure 6A, the FT-IR spectra further confirm the successful synthesis of PDAH NPs. The PDAH NPs exhibited high spectral density at 1500-1510 cm⁻¹. -1 1380-1385 cm -1 and 1610-1611cm -1 The characteristic peaks appearing at these locations are attributed to the amino ions (-NH3) on DH. + ), the sulfate group (-SO4) on Hep - ) and carboxyl ions (-COO) - ).

[0077] As shown in Figure 6B, compared to GM, GM-V is at 1643.56 cm⁻¹. -1 The sharp peak at this point may be due to the increased stretching vibrations of the unsaturated C=C structure of the aromatic compound caused by the introduction of VMA; 1393.67 cm⁻¹ -1 1343.68 cm -1 The nearby absorption peaks are related to complex methyl rocking vibrations, suggesting that the introduction of VMA has complicated the methyl environment, indicating that the grafting of VMA was successful.

[0078] (5) In vitro swelling experiment: As shown in Figure 7, the microspheres reached swelling equilibrium after 24 hours of swelling, with an equilibrium swelling rate of 775.75%, indicating good hydrophilicity.

[0079] (6) In vitro degradation experiment: As shown in Figure 8, after 72 hours, the volumes of GM and BPDHA / VMA microspheres in PBS increased while maintaining their spherical shape, although slight indentations appeared at the edges. This indicates that PBS promoted microsphere degradation to some extent. In artificial saliva used to simulate the oral environment, the structures of GM and BPDHA / VMA microspheres were destroyed after 72 hours. After freeze-drying and weighing the residue, the degradation rate was found to be 66.8%, indicating that the microspheres exhibited good biodegradability in the oral microenvironment.

[0080] (7) In vitro drug release experiment: As shown in Figure 9, the cumulative drug release rate of BMP-2 / GM reached 70% in the first 4 hours, demonstrating a significant drug burst release. After introducing VMA into the system, the initial drug release rate slowed down, and the release process became more uniform and stable. The early release of BPDAH / VMA was further controlled, and there was no significant difference between the two groups in the middle and late stages, with a cumulative release rate exceeding 80% at 72 hours. These results indicate that VMA modification further enhanced the sustained-release effect of the hydrogel.

[0081] 2. Biological performance characterization: (1) CCK-8 detection: As shown in Figure 10, compared with the control group, except for the 150 mg / mL group, the cell survival rate of all experimental groups was higher than 70%. There was no statistically significant difference between the 100 mg / mL group, the 200 mg / mL group and the 50 mg / mL group (p>0.05), indicating that the BPDAH / VMA extract has little effect on cell proliferation and that BPDAH / VMA has good cell compatibility.

[0082] (2) Staining of live and dead cells: As shown in Figure 11, compared to the control group, cells in each experimental group exhibited dense green fluorescence. With increasing extract concentration, the number of dead cells labeled with red fluorescence increased. Analysis of the fluorescence signals revealed that the cell viability in each experimental group was higher than 85%. There was no statistically significant difference in cell viability between the 50 mg / mL and 100 mg / mL groups and the control group (p>0.05), indicating that the BPDAH / VMA extract had no significant cytotoxicity and that BPDAH / VMA possessed good biocompatibility.

[0083] (3) ROS level detection: As shown in Figure 12, compared with the control group, the model group cells showed a large amount of green fluorescence, indicating that LPS effectively induced the production of ROS in the cells, thus constructing a cellular inflammatory microenvironment model. The green fluorescence in the experimental group gradually weakened with increasing extract concentration.

[0084] ImageJ analysis of fluorescence signals showed that the ROS level in the model group was significantly higher than that in the control group (p<0.0001). The intracellular ROS level in each experimental group gradually decreased with increasing extract concentration. In the 200 mg / mL experimental group, the intracellular ROS level was close to that in the control group, indicating that high-concentration BPDAH / VMA extract can effectively scavenge ROS. BPDAH / VMA can exert an anti-inflammatory effect by regulating the periodontal inflammatory microenvironment by reducing ROS levels.

[0085] (4) Quantitative detection of ALP: As shown in Figure 13, compared with the BMP-2 group, the ALP activity in the B@PH NPs group was significantly increased, indicating that PDAH NPs can maintain BMP-2 activity and achieve long-term osteogenic effects. The ALP activity in the BPDAH / VMA group was significantly higher than that in the BMP-2 group and the B@PH NPs group, indicating that there is a synergistic effect between VMA and BMP-2, and the introduction of VMA can significantly enhance the osteogenic induction capacity of the system.

[0086] Comparative Example 1: Based on the results of in vitro drug release experiments, such as Figure 9 As shown, the BMP-2 / GM hydrogel microspheres exhibited a significant burst release behavior in the initial stage, with the cumulative release amount rapidly increasing within the first 12 hours and then stabilizing after 12 hours, indicating that BMP-2 was mainly released within a short period. The release curve of the BPDAH / VMA hydrogel microspheres of this invention showed a gradual upward trend throughout the 0-48 hours, with a low initial release amount, only reaching a significant plateau at 48 hours. This indicates that the hydrogel microspheres, through the three-dimensional network structure of B@PH nanoparticles and VMA crosslinked GelMA, achieved sustained release for up to 48 hours in an in vitro simulated environment via a nano-micro dual-size network and swelling-controlled release. Compared with existing hydrogel sustained-release microspheres, this invention successfully extended the BMP-2 sustained-release time from 12 hours to 48 hours, demonstrating a significant osteogenic induction advantage and a highly significant effect on promoting periodontal bone regeneration.

[0087] Comparative Example 2: Based on the ROS fluorescent probe results, such as Figure 12 As shown, under LPS-induced conditions, the intracellular ROS level in the model group cells was significantly increased, with a relative ROS level more than three times that of the normal group, indicating that the cells were under significant oxidative stress. After adding different concentrations of the hydrogel microsphere extract of this invention, the intracellular ROS level gradually decreased with increasing extract concentration. The ROS level in the 200 mg / mL extract treatment group was reduced to near or even below the normal control group, indicating that the VMA grafted onto the GelMA surface effectively exerted anti-inflammatory and antioxidant effects in the early stages of LPS induction, clearing intracellular ROS levels to control levels and effectively regulating the osteogenic microenvironment. Combined with the results of in vitro ALP staining experiments, as shown... Figure 13As shown, the ALP activity levels in the osteogenic induction group and the BMP-2-only treatment group were relatively low, while the ALP activity was significantly increased after the introduction of BPDAH / VMA hydrogel microspheres. The hydrogel microspheres achieved a sustained osteogenic induction effect nearly four times that of the control group through inflammation regulation and sustained release of osteogenic factors. Compared with existing periodontal bone regeneration materials, VMA and BMP-2 have a significant synergistic osteogenic effect, successfully increasing the osteogenic induction effect of the medium to twice that of the osteogenic induction medium, demonstrating a significant long-term bone regeneration advantage and a highly significant effect on promoting periodontal bone regeneration.

[0088] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A method for preparing BPDAH / VMA hydrogel sustained-release microspheres that promote periodontal bone regeneration, characterized in that, The specific steps are as follows: (1) Polydopamine-heparin nanoparticles (PDAHNPs) were prepared using dopamine hydrochloride (DH) and heparin sodium (Hep) as raw materials. (2) Then load the PDAH NPs with osteogenic growth factor BMP-2 to obtain B@PH NPs; (3) Then, methacrylamide gelatin (GelMA) was dissolved in a photoinitiator (LAP) solution to obtain a prepolymer solution; then, B@PH NPs and vanillin methacrylate (VMA) were fully dispersed in the prepolymer solution to obtain a BPDAH / VMA microsphere precursor solution. (4) Finally, using the BPDAH / VMA microsphere precursor solution as the dispersed phase and oil as the continuous phase, the BPDAH / VMA hydrogel sustained-release microspheres were obtained by using a capillary glass microfluidic chip under UV irradiation.

2. The preparation method according to claim 1, characterized in that, The specific method of step (1) is as follows: First, ammonia water, anhydrous ethanol and the first part of deionized water are mixed evenly to obtain a premixed solution; then, dopamine hydrochloride (DH) and heparin sodium (Hep) are dissolved in the second part of deionized water, and then the premixed solution is added. After mixing thoroughly, the cross-linking reaction is carried out under light-protected conditions, and then the polydopamine-heparin nanoparticles (PDAH NPs) are obtained.

3. The preparation method according to claim 1, characterized in that, The specific method of step (2) is as follows: Dissolve PDAHNPs and osteogenic growth factor BMP-2 in PBS, and load BMP-2 by stirring at room temperature under light-protected conditions.

4. The preparation method according to claim 1, characterized in that, In step (3), the ratio of methacrylamide gelatin (GelMA), photoinitiator LAP solution, B@PH NPs, and vanillin methacrylate (VMA) is 100g:1mL:1mg:1mg. The mass concentration of the photoinitiator LAP solution is 0.05%, and the photoinitiator LAP solution is obtained by dissolving LAP in PBS.

5. The preparation method according to claim 1, characterized in that, In step (3), the method for preparing the prepolymer solution is: heating at 60°C in the dark for 30 minutes.

6. The preparation method according to claim 1, characterized in that, In step (4), the continuous phase is 2% Drop-Surf microdroplet-generated oil.

7. The preparation method according to claim 1, characterized in that, In step (4), the flow rate of the dispersed phase is 96 μL / min and the flow rate of the continuous phase is 120 μL / min; the microfluidic outlet hose is irradiated with 405 nm UV light for 30 s to achieve polymerization and curing.

8. The preparation method according to claim 1, characterized in that, In step (4), after the polymerization and solidification are completed, fresh microspheres are collected and purified. The specific method is as follows: mix fresh microspheres and demulsifier at a volume ratio of 1:1, let stand, centrifuge, soak in deionized water, centrifuge, soak in 75% ethanol solution, centrifuge, soak in PBS, centrifuge, freeze at -80℃ for 8-12 hours, vacuum dry for 8-12 hours, and store at 4℃ in the dark.

9. A BPDAH / VMA hydrogel sustained-release microsphere for promoting periodontal bone regeneration, characterized in that, It is obtained by the preparation method described in any one of claims 1 to 8.

10. The use of the BPDAH / VMA hydrogel sustained-release microspheres of claim 9 in the preparation of a restorative material that promotes periodontal bone regeneration.

Images