Three-layer composite periodontal repair material, preparation method and application

Abstract

This invention discloses a three-layer composite periodontal restorative material, its preparation method, and its applications, relating to the field of oral medical materials technology. The material employs an asymmetric sandwich three-layer composite structure, consisting of, from top to bottom: an upper hyaluronic acid gel layer loaded with fibroblast growth factor and cellulose nanocrystals; a middle barrier layer of hydrophilic modified polylactic acid; and a lower gel layer composed of dopamine-grafted hyaluronic acid and modified cellulose nanocrystals. Through functional division and deep synergy, the three-layer structure integrates three core functions—soft tissue repair, hard tissue repair, and cell migration barrier—within a single material. This invention utilizes cellulose nanocrystals as a multifunctional through-carrier, achieving mechanical enhancement, controlled release of growth factors, and osteogenic promotion. The material's degradation rate is highly matched to the periodontal tissue regeneration cycle, exhibiting excellent mechanical properties, biocompatibility, antibacterial activity, and self-absorption characteristics, achieving integrated, efficient, and stable repair of periodontal soft and hard tissues.

Description

Technical Field

[0001] This invention relates to the field of oral medical materials technology, and more specifically to a three-layer composite periodontal restorative material, its preparation method, and its application technology. Background Technology

[0002] Periodontitis is one of the most common diseases in the field of oral health. The combined defects resulting from gingival soft tissue recession and alveolar bone hard tissue resorption are a challenging clinical problem. Guided tissue regeneration and guided bone regeneration techniques are currently the mainstream methods for repairing such defects. Their core principle is to use materials such as barrier membranes to prevent rapidly growing soft tissues such as gingival fibroblasts from invading the bone defect area, thus buying time and space for alveolar bone regeneration.

[0003] Currently, periodontal restorative materials used in clinical practice and research can be mainly divided into the following categories, but each has its own insurmountable drawbacks: The first type is single-layer biodegradable barrier membranes, such as polylactic acid membranes and collagen membranes. These materials isolate cells only physically and lack the biological function of actively inducing tissue regeneration. Furthermore, their mechanical strength is relatively low, making them prone to collapse and displacement in vivo, leading to barrier failure. In addition, their degradation behavior is difficult to control precisely; if degradation is too rapid, they cannot maintain their spatial maintenance function for a sufficient time, while if degradation is too slow, they may trigger a foreign body reaction, requiring a second surgery for removal, increasing patient suffering and the risk of infection.

[0004] The second category is single-component hydrogel repair materials, such as hyaluronic acid-based gels. While these materials can be loaded with active ingredients like growth factors or hydroxyapatite to promote single-tissue repair of the gums or alveolar bone, they lack a layered structure and cannot effectively block cell migration. When applied to combined soft and hard tissue defects, soft tissue easily grows into the bone defect area, crowding out bone regeneration space and leading to bone repair failure. Furthermore, single-component hydrogels typically have poor mechanical properties and struggle to maintain stable morphology under complex stress conditions in vivo.

[0005] The third category is simple composite repair materials, such as physically bonding a barrier membrane to a hydrogel. These materials lack integrated functional design and stable interfacial bonding, making them prone to peeling apart. Furthermore, they do not undergo microstructural or functional enhancement, resulting in limited improvement in mechanical properties and making it difficult to achieve precise temporal and spatial matching and synergy between soft and hard tissue repair.

[0006] In summary, existing technologies, due to their simple or singular structures, generally fail to simultaneously meet the triple clinical requirements of active soft tissue repair, efficient hard tissue regeneration, and reliable barrier against cell migration. Furthermore, the mechanical strength, degradation rate, and bioactivity of the materials are difficult to balance and coordinate, severely limiting the clinical application effectiveness of periodontal regeneration technology. Therefore, developing a multifunctional periodontal restorative material that can achieve simultaneous, efficient, and stable repair of both soft and hard tissues, while also exhibiting mechanical compatibility, controllable degradation, and functional integration, is a pressing technical challenge that needs to be addressed by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to address the technical problems of existing periodontal restorative materials, such as their simple structure, insufficient function, and imbalance in soft and hard tissue regeneration. This invention provides a three-layer composite periodontal restorative material, its preparation method, and its application.

[0008] To achieve the above objectives, the present invention specifically adopts the following technical solution: The first aspect of the present invention provides a three-layer composite periodontal restorative material, comprising an upper hydrogel layer, a middle barrier membrane layer and a lower hydrogel layer disposed sequentially. The matrix of the upper hydrogel layer is hyaluronic acid gel, loaded with fibroblast growth factor-2 (FGF-2) and cellulose nanocrystals to induce gingival soft tissue regeneration and provide initial adhesion and sealing. The hyaluronic acid gel is formed by adipic acid dihydrazide-modified hyaluronic acid (ADH-HA) and aldehyde-modified hyaluronic acid (α-HA) through acylhydrazone bonds and / or Schiff base-type dynamic covalent cross-linking. The cellulose nanocrystals are dispersed in the hyaluronic acid gel network through hydrogen bonding, physical entanglement, and surface adsorption, and form multi-point weak interactions with fibroblast growth factor-2, thereby improving the mechanical stability of the upper hydrogel layer and the sustained-release performance of fibroblast growth factor-2. The middle barrier membrane is a hydrophilic modified polylactic acid (PLA) separator, which is used to prevent gingival fibroblasts from migrating downwards and to provide a stable space for bone regeneration. The surface of the hydrophilic modified PLA separator has a polydopamine coating containing catechol and amino groups, which can form hydrogen bonds, covalent / non-covalent interfacial interactions, and wet adhesion with the upper hyaluronic acid gel layer and the lower dopamine-grafted hyaluronic acid gel layer. Thus, the middle barrier membrane layer simultaneously serves as a physical barrier layer, a mechanical support layer, and an interfacial coupling layer. The matrix of the lower hydrogel layer is dopamine-grafted hyaluronic acid (HA-DA) and contains modified cellulose nanocrystals for antibacterial, biocompatibility and alveolar bone repair. The dopamine-grafted hyaluronic acid forms a cross-linked network through dopamine phenol hydroxyl oxidation coupling, and the modified cellulose nanocrystals are dispersed in the cross-linked network as nucleation templates for hydroxyapatite or calcium phosphate minerals, thereby forming a synergistic effect of antibacterial, mineralization induction and osteogenic promotion. An integrated composite structure is formed between the upper hydrogel layer, the middle barrier film layer, and the lower hydrogel layer through interfacial adhesion mediated by polydopamine coating, cellulose nanocrystal reinforcement, and interpenetration / contact curing of hyaluronic acid-based gel network.

[0009] In one embodiment, the amount of cellulose nanocrystals added in the upper hydrogel layer is 2-10 wt% of the matrix, and the loading of fibroblast growth factor (FGF-2) is 10-100 ng / mg gel. In the lower hydrogel layer, the amount of modified cellulose nanocrystals added is 3~12wt% of the matrix.

[0010] In one embodiment, the thickness ratio of the upper hydrogel layer, the middle barrier film layer, and the lower hydrogel layer is (2~5):(0.1~0.5):(3~6).

[0011] Specifically, the above ratio represents the synergistic range of three-layer mechanical matching, degradation matching, and functional complementarity; deviations from this range will result in interlayer delamination, degradation imbalance, barrier failure, or asynchronous repair, and adjustments to a single component cannot compensate for the overall performance decline.

[0012] In one embodiment, the degradation cycle of the upper hydrogel layer is 4 to 6 weeks, the degradation cycle of the middle barrier film layer is 8 to 10 weeks, and the degradation cycle of the lower hydrogel layer is 10 to 12 weeks.

[0013] In one embodiment, the cellulose nanocrystals are one or more of filter paper, sea squirts, or bacterial cellulose; the modified cellulose nanocrystals are surface-modified cellulose nanocrystals that have the function of promoting hydroxyapatite deposition.

[0014] In one embodiment, the upper hydrogel layer and / or the lower hydrogel layer further contain at least one of an antibacterial agent, a color developer, or a developer.

[0015] A second aspect of the present invention provides a method for preparing a three-layer composite periodontal restorative material, comprising the following steps: S1. Place the polylactic acid membrane in a dopamine solution and allow the dopamine to undergo a self-polymerization reaction under weakly alkaline conditions to form a polydopamine coating containing catechol groups and amino groups on the surface of the polylactic acid membrane, thereby obtaining a hydrophilic modified polylactic acid separator membrane. Among them, the polydopamine coating is used to improve the hydrophilicity and surface energy of the polylactic acid membrane, and serves as the interface coupling layer between the subsequent upper and lower hydrogel layers. S2. Adipic acid dihydrazide-modified hyaluronic acid (ADH-HA), aldehyde-modified hyaluronic acid (α-HA), cellulose nanocrystals and fibroblast growth factor (FGF-2) are mixed to form an upper hydrogel precursor solution, and the upper hydrogel precursor solution is placed on the upper surface of the hydrophilic modified polylactic acid separator obtained in step S1. In this process, the hydrazide group in adipic acid dihydrazide-modified hyaluronic acid (ADH-HA) undergoes hydrazone bonding and / or Schiff base-type dynamic covalent cross-linking with the aldehyde group in aldehyde-modified hyaluronic acid (α-HA) to form a hyaluronic acid gel network. Cellulose nanocrystals are dispersed in the gel network through hydrogen bonding, physical entanglement, and surface adsorption, and form multi-point weak interactions with fibroblast growth factor-2 (FGF-2), thereby obtaining an upper hydrogel layer with sustained-release capacity and mechanical enhancement of fibroblast growth factor-2 (FGF-2). S3. Dopamine-grafted hyaluronic acid (HA-DA), modified cellulose nanocrystals, horseradish peroxidase and hydrogen peroxide are mixed to form a lower hydrogel precursor solution, and the lower hydrogel precursor solution is placed on the lower surface of the hydrophilic modified polylactic acid separator obtained in step S1. Among them, the dopamine phenolic hydroxyl groups in the dopamine-grafted hyaluronic acid (HA-DA) undergo oxidative coupling cross-linking reaction under the action of horseradish peroxidase / hydrogen peroxide system to form a lower hydrogel network; modified cellulose nanocrystals are dispersed in the lower hydrogel network and serve as nucleation templates for calcium phosphate minerals or hydroxyapatite to construct osteogenic mineralization microenvironment. S4. The upper hydrogel layer, the middle hydrophilic modified polylactic acid separator, and the lower hydrogel layer continue to cure under wet conditions, so that hydrogen bonding, catechol-mediated interfacial adhesion, and non-covalent / covalent synergistic effects are formed between the polydopamine coating and the upper and lower hydrogel layers, thereby obtaining a three-layer composite periodontal restorative material with the functions of promoting soft tissue repair in the upper layer, cell barrier in the middle layer, and promoting bone regeneration in the lower layer.

[0016] In one embodiment, in step S1, the dopamine solution is a Tris-HCl buffer solution containing dopamine, with a pH of 8.0-8.8, preferably 8.5, and the dopamine self-polymerization reaction time is 12-24 hours. In step S2, the crosslinking temperature of adipic dihydrazide-modified hyaluronic acid (ADH-HA) and aldehyde-modified hyaluronic acid (α-HA) is 25-37℃, and the crosslinking time is 5-30 minutes. In step S3, dopamine-grafted hyaluronic acid (HA-DA) is crosslinked into a gel under the action of horseradish peroxidase / hydrogen peroxide system, with a crosslinking temperature of 25-37℃ and a crosslinking time of 5-30 minutes. The hydrogel layers formed in steps S2 and S3 are solidified in situ on the surface of the polydopamine-modified polylactic acid membrane, so that a stable wet interface bond is formed between the upper and lower hydrogel layers and the middle barrier membrane layer.

[0017] In one embodiment, the polydopamine coating forms a wet interface bonding structure with the upper and lower hydrogel layers, the wet interface bonding structure including one or more of hydrogen bonding, catechol group-mediated adhesion, amino / hydroxyl interaction and polymer segment entanglement.

[0018] The third aspect of this invention is the application of a three-layer composite periodontal restorative material in the preparation of a medical device for treating combined periodontal soft and hard tissue defects; The three-layer composite periodontal restorative material promotes the regeneration of gingival soft tissue through the upper hydrogel layer, physically isolates the migration of gingival fibroblasts through the middle barrier membrane layer to maintain space for bone regeneration through the middle hydrogel layer, and promotes the regeneration of alveolar bone hard tissue through the lower hydrogel layer. The three layers work together to achieve integrated repair of periodontal tissues.

[0019] The beneficial effects of this invention are as follows: Compared with existing technologies, the three-layer hydrogel-film-hydrogel periodontal restorative material and its preparation method provided by this invention achieve significant beneficial effects through innovative asymmetric three-layer structural design and deep synergy of multifunctional material systems: 1. Achieves simultaneous, integrated, and highly efficient restoration of periodontal soft and hard tissues: This invention achieves three core functions simultaneously on a single implant: soft tissue repair, hard tissue repair, and cell migration barrier, through a clear functional division and spatial partitioning of the upper layer promoting gingival regeneration, the middle layer physically isolating cells, and the lower layer promoting bone formation. This overcomes the shortcomings of existing materials with their single function. The three-layer structure supports, protects, and works synergistically to shorten the restoration cycle, significantly improve the quality and compatibility of tissue regeneration, and is expected to greatly increase the clinical success rate.

[0020] 2. Achieving a unified three-layer structure through PDA-PLA interfacial coupling avoids interlayer delamination caused by simple physical bonding: Existing membrane-gel composite materials are often prepared through physical bonding, lacking stable interfacial interactions between the upper and lower layers, making them prone to delamination, slippage, or peeling after implantation in a humid environment. This invention modifies the PLA membrane surface through dopamine self-polymerization, forming a polydopamine coating containing catechol and amino groups on the PLA membrane surface. This PDA coating not only improves the hydrophilicity and surface energy of the PLA membrane but also forms hydrogen bonds, catechol-mediated wet adhesion, and interfacial entanglement between polymer segments with the upper hyaluronic acid hydrogel and the lower HA-DA hydrogel. Therefore, the middle PDA-PLA membrane in this invention is not an inert interlayer but simultaneously serves as a cell barrier layer, a mechanical support layer, and an interfacial coupling layer. Through this interfacial coupling, the upper hydrogel, middle membrane, and lower hydrogel form a stable whole, significantly reducing the risk of interlayer delamination under wet stress conditions in vivo. In the examples, the surface energy of the PLA membrane after PDA modification increased from 22.8±0.8 mN / m to 33.1±0.7 mN / m, the polar component increased from 1.6±0.2 mN / m to 7.9±0.5 mN / m, and the water contact angle decreased from 101.9±0.9° to 77.1±4.0°, indicating that PDA modification significantly improved the interfacial compatibility between the membrane and the hydrogel.

[0021] 3. Significantly improves mechanical properties and wet structural stability, meeting the complex stress environment of periodontal defect areas: Single hyaluronic acid hydrogels typically suffer from low mechanical strength, easy deformation in wet conditions, and difficulty in maintaining space within the body; while simple PLA membranes have some support capabilities, they lack active repair functions and have weak interfacial bonding with hydrogels. This invention provides a supporting framework through a middle PDA-PLA membrane and forms a nano-reinforcing network through CNC machining in the upper and lower hydrogels, significantly improving the three-layer composite material's performance in compression, tension, and wet peel resistance. In the examples, the compressive strength of the three-layer composite material is 0.75 MPa, approximately 837.5% higher than that of pure HA gel (0.08 MPa); the tensile strength is 1.20 MPa, approximately 300% higher than that of pure HA gel (0.30 MPa); and the interlaminar peel strength is approximately 0.58 N / cm, significantly higher than that of physically bonded three-layer materials. These results demonstrate that the material of this invention not only has better compressive and tensile strength but also stronger wet interfacial stability, reducing the risk of post-implantation collapse, displacement, delamination, and barrier failure.

[0022] 4. Programmed restoration matching the periodontal tissue regeneration rhythm through stratified degradation: Periodontal tissue regeneration is not a single-point event, but a continuous process including early gingival soft tissue closure, intermediate barrier maintenance, and late alveolar bone reconstruction. Existing single-layer materials usually have only a single degradation curve, making it difficult to simultaneously match the different cycles of soft tissue healing and bone tissue regeneration. This invention achieves stratified degradation behavior by controlling the material composition, crosslinking density, and thickness ratio of the upper HA / CNC / FGF-2 hydrogel, the middle PDA-PLA membrane, and the lower HA-DA / modified CNC hydrogel: the upper hydrogel layer gradually degrades mainly within 4-6 weeks, matching the early gingival soft tissue closure and repair cycle; the middle PDA-PLA membrane maintains its barrier function within 8-10 weeks, matching the bone regeneration space maintenance window; and the lower hydrogel layer persists within 10-12 weeks, matching the alveolar bone regeneration and mineralization stages. Therefore, this invention does not simply pursue the "degradability" of materials, but achieves temporal matching between different functional layers and different tissue regeneration stages, thereby improving the stability and coordination of soft and hard tissue combined repair.

[0023] 5. Controllable growth factor loading and release behavior, and long-lasting biological activity: This invention utilizes cellulose nanocrystals as a highly efficient carrier for fibroblast growth factors, uniformly immobilizing them in the upper hydrogel layer. This effectively solves the problems of low growth factor embedding rate and easy burst release loss in traditional gels. Combined with the physical protection of the middle barrier membrane and the support of the lower stable substrate, the slow, stable, and long-term release of fibroblast growth factors is achieved, significantly prolonging the effective time of their biological action and improving the utilization efficiency of growth factors, thereby inducing higher-quality gingival soft tissue regeneration.

[0024] 6. A stable, clean, and undisturbed bone regeneration microenvironment is constructed: The hydrophilic modified polylactic acid separator in the middle layer of this invention provides a durable and tight physical barrier, effectively blocking the migration of gingival fibroblasts and providing a fundamental guarantee for the dedicated space for bone regeneration below. Simultaneously, the upper gel seals the wound, and the lower HA-DA gel provides an antibacterial osteogenic interface. These three layers synergistically construct a sterile, stable, and soft tissue-free ideal bone regeneration microenvironment, greatly improving osteoblast activity and the success rate and quality of bone defect repair.

[0025] 7. Excellent antibacterial and anti-inflammatory properties and biocompatibility: The dopamine-grafted hyaluronic acid and modified cellulose nanocrystals in the lower hydrogel of this invention synergistically endow the material with good antibacterial and anti-inflammatory properties, effectively reducing the risk of postoperative infection. All components are biodegradable or bio-derived medical-grade materials with good cell compatibility and low tissue irritation, meeting the safety standards for oral implant materials. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a schematic cross-sectional view of the three-layer composite periodontal restorative material provided in Embodiment 1 of the present invention.

[0028] Figure 2 This is a verification diagram of the gelation effect of the upper hydrogel in Embodiment 1 of the present invention.

[0029] Figure 3 This is the Fourier transform infrared spectrum of the upper hydrogel component in Example 1 of the present invention.

[0030] Figure 4 The image shows the X-ray photoelectron spectroscopy spectrum of the polylactic acid film modified with dopamine in Example 1 of this invention.

[0031] Figure 5 This is a diagram showing the water contact angle of the polylactic acid film before and after dopamine modification in Example 1 of the present invention.

[0032] Figure 6 This is the 1H NMR spectrum of hyaluronic acid grafted with dopamine in the lower hydrogel matrix of Example 1 of the present invention.

[0033] Figure 7 This is a photograph of the lower hydrogel layer in Example 1 of the present invention.

[0034] Figure 8 This is the X-ray photoelectron spectrum of cellulose nanocrystals derived from sea squirts loaded with hydroxyapatite in Example 1 of the present invention.

[0035] Figure 9 The figure shows the mechanical property test results of the three-layer composite periodontal restorative material in Example 1 of the present invention.

[0036] Figure 10 This is a diagram showing the interlayer peel strength test results of the three-layer composite periodontal restorative material in Embodiment 1 of the present invention.

[0037] Figure 11 This is a graph showing the loading rate of fibroblast growth factor and its in vitro release curve in the upper hydrogel of Example 1 of the present invention.

[0038] Figure 12 This is a schematic diagram of the timeline of the layered degradation of the three-layer composite periodontal restorative material in Embodiment 1 of the present invention.

[0039] Figure 13 This is a diagram showing the CCK-8 assay results of the upper hydrogel promoting gingival fibroblast proliferation in Example 1 of this invention.

[0040] Figure 14 This is a Transwell experiment result diagram showing the effect of the three-layer composite periodontal restorative material in Example 1 of the present invention on blocking the migration of gingival fibroblasts.

[0041] Figure 15 This is a qPCR detection result of the expression of osteogenic differentiation-related genes RUNX2, ALP and OCN in the lower hydrogel of Example 1 of the present invention.

[0042] Figure 16 This is a graph showing the colony count (CFU) quantitative antibacterial rate test results of the lower HA-DA hydrogel in Example 1 of the present invention.

[0043] Figure 17 Micro-CT images and quantitative analysis of bone regeneration in rat periodontitis models using different treatment groups.

[0044] Figure 18 HE staining and Masson staining images of rat periodontitis models using different treatment groups. Detailed Implementation

[0045] To make the technical problems, technical solutions, and technical effects of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0046] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0047] To facilitate understanding of the various embodiments, the abbreviations used are explained in the following table: Example 1

[0048] This embodiment provides a three-layer composite periodontal restorative material, including an upper hydrogel layer, a middle barrier membrane layer, and a lower hydrogel layer.

[0049] The matrix of the upper hydrogel layer is hyaluronic acid, in which 5wt% cellulose nanocrystals are uniformly dispersed and loaded with fibroblast growth factor at a loading of 50ng / mg gel.

[0050] The middle barrier layer is a hydrophilic modified polylactic acid separator with a thickness of 0.3 mm.

[0051] The matrix of the lower hydrogel layer is hyaluronic acid grafted with dopamine, in which 10wt% of modified cellulose nanocrystals are uniformly dispersed.

[0052] The thickness ratio of the upper hydrogel layer, the middle barrier film layer, and the lower hydrogel layer is 3:0.3:4.

[0053] The specific steps for preparing this three-layer composite periodontal restorative material are as follows: S1. Preparation of cellulose nanocrystals: An acid hydrolysis method was used. Filter paper was cut into small pieces and added to a 64 wt% sulfuric acid solution. The mixture was vigorously stirred at 60°C for 2 hours. After the reaction was complete, the reaction was terminated with a large amount of deionized water. The acid solution was removed by repeated centrifugation and washing. The milky white suspension was collected, dialyzed until neutral, and finally freeze-dried to obtain cellulose nanocrystal powder.

[0054] S2. Hydrophilic Modification and Surface Energy Characterization of Polylactic Acid (PLA) Membrane: Commercially available PLA membranes were immersed in Tris-HCl buffer (pH=8.5) containing dopamine and reacted with shaking at room temperature in the dark for 24 hours. After the reaction, the membrane was removed and ultrasonically cleaned several times with deionized water to remove unbound dopamine, resulting in a hydrophilic modified PLA separator membrane coated with polydopamine. The surface energy of the obtained PDA-PLA membrane was tested. The results are shown in Table 1. The total surface energy of the unmodified PLA membrane was 22.8±0.8 mN / m, and the polar component was 1.6±0.2 mN / m. After dopamine modification, the total surface energy of the PDA-PLA membrane increased to 33.1±0.7 mN / m, and the polar component increased to 7.9±0.5 mN / m. The above results indicate that dopamine self-polymerization treatment significantly improves the hydrophilicity and interfacial activity of the PLA membrane surface, which is beneficial to the spreading, wetting and curing of the upper HA / CNC / FGF-2 hydrogel and the lower HA-DA / modified CNC hydrogel on the membrane surface, thereby enhancing the interlayer bonding stability of the three-layer composite material.

[0055] S3. Preparation of the upper hydrogel: Adipic acid dihydrazide-modified hyaluronic acid and aldehyde-modified hyaluronic acid were dissolved in PBS buffer, and the cellulose nanocrystal powder prepared above was added and ultrasonically dispersed evenly. Then, fibroblast growth factor solution was added, mixed evenly, and quickly poured onto the upper surface of a horizontally placed hydrophilic modified polylactic acid separator membrane, and cross-linked at 37°C.

[0056] S4. Preparation of the lower layer hydrogel: Mix 6wt% dopamine-grafted hyaluronic acid hydrogel precursor solution with modified cellulose nanocrystals, add horseradish peroxidase and hydrogen peroxide as crosslinking initiation system, mix evenly and immediately coat it on the lower surface of the hydrophilic modified polylactic acid separator membrane, and crosslink at room temperature for 5 minutes to form.

[0057] S5. Composite and Curing: The three-layer composite structure, after the preparation of the upper and lower gels, is cured at room temperature for 30 minutes to ensure a strong bond between the interlayer interfaces, resulting in the final three-layer composite periodontal restorative material.

[0058] Performance test results: In vitro cell migration experiments showed that the material had a blocking rate of over 85% against gingival fibroblasts.

[0059] Degradation experiments showed that the material underwent an overall degradation cycle of approximately 10 weeks in simulated body fluids, with the degradation rates of each layer meeting expectations.

[0060] Mechanical tests show that the tensile strength of this material is more than 60% higher than that of the control pure hydrogel group without cellulose nanocrystals.

[0061] Cytotoxicity and antibacterial experiments confirmed that the material has good biocompatibility and antibacterial properties.

[0062] The main properties of the material obtained in this embodiment are as follows: the compressive strength is 0.75 MPa, the tensile strength is 1.20 MPa, and the elastic modulus is 25 kPa; the interlaminar peel strength is approximately 0.58 N / cm; the release of FGF-2 from the upper gel lasts up to 40 days, with a cumulative release rate of approximately 93% over 40 days; the degradation cycle of the upper hydrogel layer is 6 weeks, the degradation cycle of the middle PDA-PLA membrane is 10 weeks, and the degradation cycle of the lower HA-DA / CNC-nHAP gel layer is 12 weeks; the proliferation rate of gingival fibroblasts on day 7 is approximately 160%; the transwell assay shows a migration barrier rate of approximately 96% for gingival fibroblasts; the relative expression levels of osteogenic-related genes OCN, RUNX2, and ALP are approximately 1.75, 1.70, and 1.78, respectively; and the antibacterial rate against Porphyromonas gingivalis is approximately 40%.

[0063] Example 2

[0064] The material composition and preparation method of this embodiment are basically the same as those of Example 1, the difference being that some key parameters have been adjusted: In the upper hydrogel, the amount of cellulose nanocrystals added is 2wt%, and the loading of fibroblast growth factor is 10ng / mg gel.

[0065] The amount of modified cellulose nanocrystals added in the lower hydrogel is 3 wt%.

[0066] The thickness of the middle polylactic acid film is 0.1 mm.

[0067] The thickness ratio of the upper hydrogel layer, the middle barrier film layer, and the lower hydrogel layer is 2:0.1:3.

[0068] The material prepared in this embodiment is relatively soft and degrades slightly faster than that in Example 1. Tests showed that its compressive strength was approximately 0.42 MPa, tensile strength was approximately 0.75 MPa, elastic modulus was approximately 18 kPa, and interlaminar peel strength was approximately 0.38 N / cm. The degradation cycle of the upper hydrogel layer was approximately 4 weeks, the middle PDA-PLA membrane was approximately 8 weeks, and the lower HA-DA / TCNC-nHAP gel layer was approximately 10 weeks. On day 7, the gingival fibroblast proliferation rate was approximately 145%, the gingival fibroblast migration barrier rate was approximately 88%, and the relative expression levels of OCN, RUNX2, and ALP were approximately 1.50, 1.42, and 1.55, respectively. The antibacterial rate against *Porphyromonas gingivalis* was approximately 35%.

[0069] The material produced by this method is relatively softer and degrades slightly faster, making it suitable for clinical scenarios with smaller defects, abundant blood supply to soft tissues, and shorter expected healing periods.

[0070] Example 3

[0071] The material composition and preparation method of this embodiment are basically the same as those of Example 1, the difference being that some key parameters have been adjusted: In the upper hydrogel, the amount of cellulose nanocrystals added is 10wt%, and the loading of fibroblast growth factor is 100ng / mg gel.

[0072] The amount of modified cellulose nanocrystals added in the lower hydrogel is 12 wt%.

[0073] The thickness of the middle polylactic acid film is 0.5 mm.

[0074] The thickness ratio of the upper hydrogel layer, the middle barrier film layer, and the lower hydrogel layer is 5:0.5:6.

[0075] The material prepared in this embodiment exhibits high mechanical strength and a long growth factor release period. Tests showed that its compressive strength is approximately 0.98 MPa, tensile strength is approximately 1.45 MPa, elastic modulus is approximately 32 kPa, and interlaminar peel strength is approximately 0.72 N / cm. The degradation period of the upper hydrogel layer is approximately 6 weeks, the middle PDA-PLA membrane is approximately 10 weeks, and the lower HA-DA / TCNC-nHAP gel layer is approximately 12 weeks. On day 7, the gingival fibroblast proliferation rate was approximately 165%, the gingival fibroblast migration barrier rate was approximately 97%, and the relative expression levels of OCN, RUNX2, and ALP were approximately 1.85, 1.78, and 1.88, respectively. The antibacterial rate against *Porphyromonas gingivalis* was approximately 42%.

[0076] The material produced by this method has higher mechanical strength and a longer growth factor release period, and its degradation rate is relatively slower. It is suitable for posterior teeth that are subjected to greater occlusal forces, large-scale bone defects, or complex cases with a long expected healing period.

[0077] In addition, the source of cellulose nanocrystals in Examples 1 to 3 was replaced Replacing the filter paper with sea squirts or bacterial cellulose does not change the process; the mechanical and osteogenic effects are similar.

[0078] Replacing ordinary hyaluronic acid with adipic acid dihydrazide-modified hyaluronic acid or aldehyde-modified hyaluronic acid maintains the same cross-linking method but results in stronger adhesion.

[0079] Antibacterial agents and developing agents can be omitted, and the core three-layer structure and function remain unaffected.

[0080] With adjustments to the crosslinking conditions, the crosslinking temperature can be adjusted from 25-37℃ and the time from 5-30 minutes, and molding can be achieved in both cases.

[0081] This solution works based on a deep collaboration of a three-layer architecture: Mechanical Synergy and Structural Stability: The middle layer of hydrophilic modified polylactic acid separator acts as the "skeleton" of the entire material, providing crucial rigid support and preventing the collapse of the upper and lower hydrogel layers in vivo. Cellulose nanocrystals in the upper and lower layers, acting as nanofillers, significantly enhance the mechanical strength of the hydrogel itself. Simultaneously, the active groups on their surface enhance the interfacial bonding with the middle polydopamine coating, forming a cohesive composite structure with progressively transitioning mechanical properties and strong interfacial bonding, ensuring morphological stability and reliable barrier function after implantation.

[0082] Spatial Isolation and Zonal Repair: The middle polylactic acid membrane acts as a physical barrier, precisely separating the upper soft tissue repair zone from the lower hard tissue repair zone. The upper hydrogel loaded with fibroblast growth factor actively induces gingival fibroblasts and epithelial cells to proliferate and migrate above the membrane, achieving orderly regeneration of gingival soft tissue. Simultaneously, this gel layer covers the wound, providing initial closure and hemostasis. The lower dopamine-grafted hyaluronic acid hydrogel contains dopamine groups that provide excellent cell adhesion sites and exhibit antibacterial activity. Modified cellulose nanocrystals effectively induce the deposition of calcium phosphate minerals, providing an ideal microenvironment for osteoblast differentiation and bone matrix formation, thereby promoting the regeneration of alveolar bone hard tissue. The presence of the middle membrane ensures that the underlying bone regeneration space is not disturbed by the rapidly growing soft tissue cells above.

[0083] Functional Synergy and Microenvironment Regulation: The three layers support and amplify each other's functions. The initial occlusion and continuous release of fibroblast growth factor from the upper gel ensure gingival healing; the middle membrane creates a stable and undisturbed osteogenic environment for the lower layer; the dopamine-grafted hyaluronic acid gel in the lower layer, with its antibacterial and osteogenic induction properties, in turn protects the upper and middle layers from infection risks and ensures efficient bone regeneration. Simultaneously, cellulose nanocrystals permeate the upper and lower layers, serving a triple function as mechanical reinforcement, fibroblast growth factor carrier, and nucleation template. Through precise control of the components and proportions of each layer, the degradation rate of the three layers can be matched with their respective tissue regeneration cycles, achieving dynamic integration and eventual complete replacement of the material-tissue organism.

[0084] In summary, this solution, through its ingenious structural design and synergistic use of a multifunctional material system, achieves a unity of mechanical properties, biological functions, and degradation behavior that is difficult to attain with a single material, representing an innovative and efficient solution in the field of periodontal tissue engineering.

[0085] To verify the structural design, interfacial stability, mechanical properties, growth factor release performance, stratified degradation behavior, cellular function, and in vivo repair effects of the three-layer composite periodontal restorative material of this invention, the following experiments were conducted. The experimental results show that the material of this invention is not a simple physical superposition of an upper hydrogel, a middle membrane, and a lower hydrogel, but rather achieves multi-dimensional synergy through PDA interfacial coupling, CNC through-and-through reinforcement, FGF-2 sustained release, stratified degradation, and upper and lower functional partitioning.

[0086] I. Material structure and chemical characterization experiments of the present invention: Experimental Example 1: Morphological Observation of a Three-Layer Composite Structure A three-layer composite periodontal restorative material was prepared according to Example 1, and its cross-sectional structure was observed.

[0087] The results are as follows Figure 1As shown, the material of this invention comprises an upper hydrogel layer, a middle barrier membrane layer, and a lower hydrogel layer. The upper hydrogel layer promotes gingival soft tissue repair; the middle PDA-PLA barrier membrane prevents gingival fibroblasts from migrating downwards and maintains space for bone regeneration; the lower HA-DA / modified CNC hydrogel layer provides an antibacterial, mineralization-inducing, and osteogenic microenvironment. This figure demonstrates that the material of this invention has a clearly defined asymmetric three-layer structure, rather than being a single homogeneous material.

[0088] Experimental Example 2: Verification of the gel-forming properties of the upper hydrogel; ADH-HA, α-HA, CNC and FGF-2 were mixed and crosslinked into a gel at 37°C.

[0089] The results are as follows Figure 2 As shown, the upper hydrogel can rapidly form under set conditions and maintain its gel morphology in an inverted state, indicating that a stable cross-linked network can be formed between ADH-HA and α-HA. This result demonstrates the feasibility of in-situ or pre-forming the upper hydrogel before implantation, making it suitable for supporting FGF-2 and CNC, and providing a basis for initial wound closure.

[0090] Experimental Example 3: Fourier transform infrared spectroscopy analysis of the upper hydrogel component: Fourier transform infrared spectroscopy was used to characterize HA, ADH-HA, α-HA and the upper composite hydrogel.

[0091] The results are as follows Figure 3 As shown, characteristic absorption peaks related to the hyaluronic acid backbone, hydrazide groups, aldehyde groups, and cross-linking structure appeared in the upper hydrogel, indicating that ADH-HA and α-HA successfully participated in the cross-linking reaction to form a hyaluronic acid-based hydrogel network. This result proves that the upper HA / CNC / FGF-2 hydrogel is not a simple mixture, but has a stable chemical cross-linking basis.

[0092] Experimental Example 4; XPS analysis of PDA-modified PLA membrane: Surface elemental analysis of unmodified PLA films and dopamine-modified PLA films was performed using X-ray photoelectron spectroscopy.

[0093] The results are as follows Figure 4 As shown, the unmodified PLA membrane surface mainly exhibits C and O element signals; after dopamine modification, a significant N element signal appears on the PLA membrane surface, indicating that polydopamine was successfully deposited on the PLA membrane surface. This result proves that the middle barrier membrane is not a typical PLA membrane, but rather an interfacial coupling membrane with a PDA active coating. The PDA coating can improve the hydrophilicity of the membrane surface and enhance its wet interfacial bonding with the upper and lower hydrogel layers through catechol, amino, and other groups.

[0094] Experimental Example 5; Surface Energy Test of PDA-Modified PLA Film: The surface energy changes of PLA films before and after modification were compared using surface energy testing methods.

[0095] The results are as follows Figure 5 As shown, the surface energy of the PLA membrane is significantly improved after dopamine modification. This increased surface energy indicates enhanced wettability of the membrane surface and improved interfacial compatibility with the hydrogel precursor solution, which is beneficial for the uniform spreading, curing, and bonding of the upper and lower hydrogel layers on the PDA-PLA membrane surface. This result further demonstrates that the PDA-PLA membrane serves not only as a physical barrier in this invention but also as an interfacial coupling platform connecting the upper and lower hydrogels.

[0096] Experimental Example 6: Water Contact Angle Test of DA-Modified PLA Membrane: The hydrophilicity of PLA membranes before and after modification was tested using a water contact angle tester.

[0097] The results are as follows Figure 5 As shown, the unmodified PLA membrane has a large water contact angle, exhibiting hydrophobicity; the PDA-modified membrane has a significantly reduced water contact angle, indicating a significant enhancement in the hydrophilicity of the membrane surface. This result demonstrates that PDA modification can improve the wet bonding conditions between the PLA membrane and the hydrogel layer, thereby reducing the risk of interfacial debonding and interlayer peeling of the three-layer structure in a humid in vivo environment.

[0098] Experimental Example 7: 1H NMR spectroscopy analysis of the lower hydrogel matrix HA-DA: HA and HA-DA were characterized by proton nuclear magnetic resonance spectroscopy.

[0099] The results are as follows Figure 6 As shown, compared with unmodified HA, HA-DA exhibited dopamine-related characteristic peaks, indicating that dopamine was successfully grafted onto the hyaluronic acid molecular chain. This result proves the successful preparation of the lower hydrogel matrix HA-DA, providing a chemical basis for subsequent HRP / H2O2 crosslinking, wet adhesion, antibacterial properties, and the construction of an osteogenic microenvironment.

[0100] Experimental Example 8: Verification of the gel-forming properties of the lower layer hydrogel: A 6 wt% HA-DA precursor solution was mixed with modified CNC, and HRP and H2O2 were added for crosslinking.

[0101] The results are as follows Figure 7 As shown, the lower hydrogel can rapidly gel at room temperature and maintain a stable morphology, indicating that the HA-DA system has good gelling ability. This result proves that the lower HA-DA / modified CNC hydrogel can serve as a functional layer on the side of bone defect, undertaking antibacterial, mineralization, and osteopromoting functions.

[0102] Experimental Example 9: XPS analysis of modified cellulose nanocrystals supported on hydroxyapatite: The elemental composition of cellulose nanocrystals derived from sea squirts before and after loading with hydroxyapatite was determined by X-ray photoelectron spectroscopy.

[0103] The results are as follows Figure 8 As shown, Ca and P elemental signals appeared on the surface of the modified cellulose nanocrystals, indicating that hydroxyapatite or calcium phosphate minerals were successfully loaded onto the surface of the cellulose nanocrystals. This result proves that the modified CNC not only exists as a nano-reinforcing filler, but also as a mineralization nucleation template, providing a materials science basis for promoting bone regeneration in the underlying hydrogel.

[0104] Material mechanical properties and interface stability test Test Example 10: Mechanical Strength Test The compressive strength, tensile strength, and elastic modulus of pure HA gel, physically bonded three-layer material, and the three-layer composite material of Example 1 were tested using a universal testing machine.

[0105] The results are as follows Figure 9 As shown, the compressive strength of pure HA gel is 0.08 MPa, and the tensile strength is 0.30 MPa. The compressive strength of the three-layer composite material in Example 1 is 0.75 MPa, an increase of approximately 837.5% compared to pure HA gel; the tensile strength is 1.20 MPa, an increase of approximately 300% compared to pure HA gel; and the elastic modulus is 25 kPa. These results indicate that the middle PDA-PLA membrane provides structural support, CNC enhances the upper and lower hydrogel networks, and the three-layer composite material possesses both high mechanical strength and suitable flexibility, meeting the basic requirements for implantation, veneer, and in vivo stress environment in periodontal defect areas.

[0106] This result corresponds to the beneficial effect of the present invention: "significantly enhanced mechanical properties and structural stability".

[0107] Test Example 11, Interlayer Peel Strength Test: The interlayer bonding strength of different composite materials was evaluated using a 180° peel test. A physically bonded three-layer material and the three-layer composite material from Example 1 were used as controls. Before testing, the materials were pre-wetted in PBS to simulate a humid in vivo environment.

[0108] The results are as follows Figure 10 As shown, the interlayer peel strength of the physically bonded three-layer material is approximately 0.22 N / cm, while the interlayer peel strength of the three-layer composite material in Example 1 is approximately 0.58 N / cm. Compared with the physically bonded three-layer material, the peel strength of the material in Example 1 is significantly improved, and the peeling process mainly involves the destruction of the hydrogel bulk rather than interfacial debonding.

[0109] These results demonstrate that the PDA-PLA membrane forms a stable wet interface with the upper and lower hydrogel layers through surface active groups such as catechols and amino groups; simultaneously, the CNC-enhanced hydrogel network further improves the overall peel resistance. Therefore, the material of this invention is not a simple stacking of three layers, but a composite repair material with interfacial coupling and structural integrity.

[0110] FGF-2 Load and Release Performance Tests: Experiment 12, FGF-2 loading rate and in vitro release experiment: The loading and release performance of FGF-2 on the upper hydrogel was detected by ELISA. HA / FGF-2 hydrogel group and HA / CNC / FGF-2 hydrogel group were set up as controls. The hydrogels of each group were placed in PBS, and the release solution was collected on days 4, 8, 12, 16, 20, 24, 28, 32, 36, and 40, and the FGF-2 content was measured.

[0111] The results are as follows Figure 11 As shown, the HA / FGF-2 hydrogel group exhibited rapid release in the early stages, with approximately 76% released within the first 12 days and almost complete release by day 28, demonstrating a clear burst release phenomenon. In contrast, the HA / CNC / FGF-2 hydrogel group showed a slower release rate, with a cumulative release rate of approximately 13% on day 4, approximately 68% on day 20, and approximately 93% on day 40, exhibiting a longer-term sustained release characteristic.

[0112] These results indicate that CNC not only plays a mechanical reinforcing role in the upper hydrogel, but also adsorbs and retains FGF-2 through its high specific surface area and surface hydroxyl network, thereby reducing early burst release and prolonging the FGF-2 interaction time. This corresponds to the beneficial effect of this invention: "controllable growth factor loading and release behavior, and sustained biological activity."

[0113] Layered degradation performance test: Experimental Example 13: Stratified Degradation Timeline Analysis: The three-layer composite material of Example 1 was placed in simulated body fluid, and the structural integrity of the material and the mass loss of each layer were observed at different time points. The degradation process of the upper hydrogel layer, the middle PDA-PLA barrier film and the lower HA-DA / modified CNC hydrogel layer were analyzed.

[0114] The results are as follows Figure 12As shown, the material in Example 1 exhibits a distinct stratified degradation characteristic: the upper HA / CNC / FGF-2 hydrogel layer gradually degrades over 4-6 weeks, corresponding to the early sealing and repair stage of the gingival soft tissue; the middle PDA-PLA barrier membrane maintains an intact barrier structure over 8-10 weeks, corresponding to the bone regeneration space maintenance stage; and the lower HA-DA / modified CNC hydrogel layer persists over 10-12 weeks, corresponding to the alveolar bone regeneration and mineralization stage.

[0115] The results demonstrate that the three-layer material of the present invention, through the design of different components, crosslinking densities and thickness ratios, matches the degradation sequence of each layer with the processes of gingival healing, barrier maintenance and bone tissue regeneration, reflecting the characteristics of time-programmed synergistic repair, rather than the passive stacking of the three materials.

[0116] Cellular tests: Experiment 14: Gingival fibroblast proliferation experiment: The effect of the upper hydrogel on the proliferation of gingival fibroblasts was detected using the CCK-8 assay. The experiment included a blank control group, a pure HA hydrogel group, an HA / CNC hydrogel group, and the HA / CNC / FGF-2 upper hydrogel group from Example 1. Gingival fibroblasts were co-cultured with extracts of different materials, and CCK-8 reagent was added on days 1, 3, 5, and 7. The absorbance was measured at 450 nm, and the cell growth rate was calculated.

[0117] The results are as follows Figure 13 As shown, the cell growth rate of the blank control group was set at 100%. The cell growth rate on day 7 was approximately 132% in the pure HA hydrogel group, approximately 136% in the HA / CNC hydrogel group, and approximately 160% in the HA / CNC / FGF-2 hydrogel group of Example 1. Compared with the pure HA hydrogel group and the HA / CNC hydrogel group, the upper hydrogel of Example 1 significantly increased the proliferation level of gingival fibroblasts.

[0118] These results demonstrate that the hyaluronic acid-based hydrogel exhibits good cell compatibility, the addition of CNC improves the gel microenvironment and structural stability, and the sustained release of FGF-2 further promotes gingival fibroblast proliferation. This experiment proves at the cellular level that the upper hydrogel plays a role in promoting gingival soft tissue repair.

[0119] Experiment 15: Gingival fibroblast migration barrier experiment: The Transwell assay was used to evaluate the ability of different materials to block the downward migration of gingival fibroblasts. The experiment consisted of a control group, an HA / CNC hydrogel group, a PDA-PLA membrane group, and the three-layer composite material group from Example 1. Gingival fibroblasts were seeded in the upper chamber of a Transwell apparatus, and the test material was placed between the upper and lower chambers. Culture medium containing chemokines was added to the lower chamber. After a certain period of culture, cells that migrated to the submembrane surface or the lower chamber were stained, counted, and the cell migration barrier rate was calculated.

[0120] The results are as follows Figure 14 As shown, the blank group could hardly block the migration of gingival fibroblasts, with a blocking rate of approximately 5%; the HA / CNC hydrogel group had a blocking rate of approximately 70%; the PDA-PLA membrane group had a blocking rate of approximately 85%; and the three-layer composite material group of Example 1 had a blocking rate of approximately 96%. These results indicate that the three-layer composite material of the present invention can effectively prevent the migration of gingival fibroblasts to the bone defect area.

[0121] Compared to a standalone PDA-PLA membrane, the three-layer composite material in Example 1 exhibits a further improved barrier efficiency, indicating that the cell barrier effect of this invention does not solely rely on the middle membrane, but is achieved through the combined action of the upper hydrogel sealing, the middle membrane barrier, and the lower hydrogel support. This result corresponds to the beneficial effect of this invention in "constructing a stable bone regeneration space free from soft tissue interference."

[0122] Experiment 16: Osteogenesis-related gene expression detection: The expression levels of osteogenic genes OCN, RUNX2, and ALP after treatment with different materials were detected by qPCR. The experiment was divided into a blank group, a HA-DA hydrogel group, a HA-DA / modified CNC hydrogel group, and a three-layer composite material group as described in Example 1.

[0123] The results are as follows Figure 15 As shown, the relative expression levels of OCN, RUNX2, and ALP in the blank group were close to 1.0; the HA-DA hydrogel group showed limited improvement in the expression of osteogenic-related genes; the HA-DA / modified CNC hydrogel group showed that the expression of OCN, RUNX2, and ALP increased to approximately 1.55, 1.42, and 1.62, respectively; and the three-layer composite material group of Example 1 further increased to approximately 1.75, 1.70, and 1.78.

[0124] These results demonstrate that modified CNCs, especially those loaded with hydroxyapatite, can serve as mineralization nucleation templates to promote osteogenic differentiation. Simultaneously, the three-layer structure further enhances the expression of osteogenic-related genes by blocking interference from gingival fibroblasts, maintaining a stable bone regeneration space, and providing the underlying osteogenic microenvironment. This experiment demonstrates at the molecular level that the underlying hydrogel and the overall three-layer structure possess osteogenic capabilities.

[0125] Test Example 17, Antibacterial Performance Test: The antibacterial activity of the lower HA-DA hydrogel and HA-DA / modified CNC hydrogel against Staphylococcus aureus, Escherichia coli, and Porphyromonas gingivalis was determined using the colony counting CFU method. After co-incubating each material with bacterial suspension, samples were taken, diluted, spread, and cultured. The number of colonies was counted, and the antibacterial rate was calculated.

[0126] The results are as follows Figure 16 As shown, the HA-DA hydrogel exhibits some inhibitory effect on all three bacteria, with an antibacterial rate of approximately 35% against *Porphyromonas gingivalis*. The addition of modified CNC further enhances the antibacterial rate of the HA-DA / modified CNC hydrogel, reaching approximately 40% against *Porphyromonas gingivalis*. These results indicate that the lower HA-DA hydrogel possesses certain antibacterial activity, and the addition of modified CNC helps improve the local microenvironment, thereby enhancing the material's inhibitory ability against periodontal-associated bacteria.

[0127] This result corresponds to the beneficial effect of the present invention: "reducing the risk of postoperative infection and providing a relatively clean microenvironment for bone regeneration".

[0128] Animal experiments: Experimental Example 18: Micro-CT Evaluation of a Rat Model of Periodontal Defects: A rat model of periodontal defect was established, divided into a periodontitis model group, a simple PLLA membrane group, a layered hydrogel group, and a three-layer composite material group (Example 1). Samples were harvested 8 weeks post-surgery, and micro-CT was used to observe new bone formation in the alveolar bone defect area.

[0129] The results are as follows Figure 17 As shown, the periodontitis model group showed obvious bone defects and limited bone recovery; the simple PLLA membrane group had a certain barrier effect, and a small amount of new bone formed in the bone defect area, but the repair was insufficient; the layered hydrogel group showed improved bone repair level compared with the PLLA membrane group; the three-layer composite material group in Example 1 showed the most obvious new bone formation in the bone defect area, better recovery of alveolar bone contour, and enhanced continuity of the bone defect area.

[0130] The results demonstrate that the three-layer composite material of the present invention can maintain bone regeneration space in vivo and provide a mineralization-promoting and osteogenic microenvironment through the lower HA-DA / modified CNC hydrogel, thereby significantly improving the quality of alveolar bone repair.

[0131] Experiment 19: Evaluation of periodontal defect model in rats using HE staining and Masson staining: Eight weeks after surgery, rat tissue from the periodontal defect area was decalcified, embedded, and sectioned, and then stained with HE and Masson staining to evaluate soft tissue repair, inflammatory response, collagen deposition, and new bone formation.

[0132] The results are as follows Figure 18As shown, the periodontitis model group showed extensive inflammatory cell infiltration, disordered periodontal tissue structure, and insufficient repair of bone defect areas; the simple PLLA membrane group, due to its certain barrier function, showed reduced inflammatory infiltration compared to the model group, but soft and hard tissue repair was still not ideal; the layered hydrogel group showed more collagen deposition and some tissue repair, but the maturity of the tissue structure was still limited; the three-layer composite material group in Example 1 showed milder inflammatory response, more complete gingival soft tissue structure, richer and more orderly collagen fiber deposition, and the newly formed tissue in the bone defect area was closer to the normal periodontal tissue structure.

[0133] HE staining results indicate that the material of this invention can reduce local inflammatory response and promote the recovery of periodontal tissue structure; Masson staining results indicate that the material of this invention can promote collagen deposition and tissue maturation, which is beneficial to the synergistic repair of gingival soft tissue and alveolar bone regeneration.

[0134] The results of the animal experiments show that the three-layer composite periodontal repair material of the present invention can simultaneously achieve soft tissue sealing, cell migration barrier, anti-infection microenvironment construction and alveolar bone repair in vivo, and has a comprehensive repair effect that is superior to simple membrane materials and single hydrogel materials.

Claims

1. A three-layer composite periodontal restorative material, characterized in that, include: The upper hydrogel layer has a matrix of hyaluronic acid gel and is loaded with fibroblast growth factor and cellulose nanocrystals to induce gingival soft tissue regeneration and provide initial adhesion and sealing. The hyaluronic acid gel is formed by adipic acid dihydrazide-modified hyaluronic acid and aldehyde-based hyaluronic acid through acylhydrazone bonds and / or Schiff base-type dynamic covalent cross-linking. The cellulose nanocrystals are dispersed in the hyaluronic acid gel network through hydrogen bonding, physical entanglement, and surface adsorption, and form multi-point weak interactions with fibroblast growth factor, thereby improving the mechanical stability of the upper hydrogel layer and the sustained-release performance of fibroblast growth factor. The middle barrier membrane layer is a hydrophilic modified polylactic acid (PLA) separator membrane used to prevent gingival fibroblasts from migrating downwards and to provide a stable space for bone regeneration. The surface of the hydrophilic modified PLA separator membrane has a polydopamine coating containing catechol and amino groups, which can form hydrogen bonds, covalent / non-covalent interfacial interactions, and wet adhesion with the upper hyaluronic acid gel layer and the lower dopamine-grafted hyaluronic acid gel layer. Therefore, the middle barrier membrane layer simultaneously serves as a physical barrier layer, a mechanical support layer, and an interfacial coupling layer. The lower hydrogel layer has a matrix of dopamine-grafted hyaluronic acid and contains modified cellulose nanocrystals for antibacterial, biocompatibility, and promotion of alveolar bone repair. The dopamine-grafted hyaluronic acid forms a cross-linked network through dopamine phenol hydroxyl oxidation coupling, and the modified cellulose nanocrystals are dispersed in the cross-linked network as nucleation templates for hydroxyapatite or calcium phosphate minerals, thereby forming a synergistic effect of antibacterial, mineralization induction, and osteogenic promotion. The upper hydrogel layer, the middle barrier film layer, and the lower hydrogel layer form an integrated composite structure through interfacial adhesion mediated by the polydopamine coating, cellulose nanocrystal reinforcement, and interpenetration / contact curing of the hyaluronic acid-based gel network.

2. The three-layer composite periodontal restorative material according to claim 1, characterized in that, In the upper hydrogel layer, the amount of cellulose nanocrystals added is 2~10wt% of the matrix, and the loading of fibroblast growth factor is 10~100ng / mg gel. In the lower hydrogel layer, the amount of modified cellulose nanocrystals added is 3~12wt% of the matrix.

3. A three-layer composite periodontal restorative material according to claim 1 or 2, characterized in that, The thickness ratio of the upper hydrogel layer, the middle barrier film layer and the lower hydrogel layer is (2~5):(0.1~0.5):(3~6).

4. The three-layer composite periodontal restorative material according to claim 1, characterized in that, The degradation cycle of the upper hydrogel layer is 4-6 weeks, the degradation cycle of the middle barrier film layer is 8-10 weeks, and the degradation cycle of the lower hydrogel layer is 10-12 weeks.

5. The three-layer composite periodontal restorative material according to claim 1, characterized in that, The cellulose nanocrystals are one or more of filter paper, sea squirts, or bacterial cellulose; the modified cellulose nanocrystals are surface-modified cellulose nanocrystals that promote hydroxyapatite deposition.

6. The three-layer composite periodontal restorative material according to claim 1, characterized in that, The upper hydrogel layer and / or the lower hydrogel layer further contain at least one of an antibacterial agent, a color developer, or a developer.

7. A method for preparing a three-layer composite periodontal restorative material, used to prepare the three-layer composite periodontal restorative material according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Place the polylactic acid membrane in a dopamine solution and allow the dopamine to undergo a self-polymerization reaction under weakly alkaline conditions to form a polydopamine coating containing catechol groups and amino groups on the surface of the polylactic acid membrane, thereby obtaining a hydrophilic modified polylactic acid separator membrane. The polydopamine coating is used to improve the hydrophilicity and surface energy of the polylactic acid membrane and serves as an interface coupling layer between the subsequent upper and lower hydrogel layers. S2. Adipic acid dihydrazide-modified hyaluronic acid, aldehyde-modified hyaluronic acid, cellulose nanocrystals and fibroblast growth factor are mixed to form an upper hydrogel precursor solution, and the upper hydrogel precursor solution is placed on the upper surface of the hydrophilic modified polylactic acid separator membrane obtained in step S1. In this process, the hydrazide group in adipic acid dihydrazide-modified hyaluronic acid undergoes hydrazone bonding and / or Schiff base-type dynamic covalent cross-linking with the aldehyde group in aldehyde-modified hyaluronic acid to form a hyaluronic acid gel network; cellulose nanocrystals are dispersed in the gel network through hydrogen bonding, physical entanglement and surface adsorption, and form multi-point weak interactions with fibroblast growth factor, thereby obtaining an upper hydrogel layer with fibroblast growth factor sustained-release capacity and mechanical enhancement effect; S3. Dopamine-grafted hyaluronic acid, modified cellulose nanocrystals, horseradish peroxidase and hydrogen peroxide are mixed to form a lower hydrogel precursor solution, and the lower hydrogel precursor solution is placed on the lower surface of the hydrophilic modified polylactic acid separator obtained in step S1. In this process, the dopamine phenolic hydroxyl groups in the dopamine-grafted hyaluronic acid undergo oxidative coupling crosslinking under the action of horseradish peroxidase / hydrogen peroxide system to form a lower hydrogel network; modified cellulose nanocrystals are dispersed in the lower hydrogel network and serve as nucleation templates for calcium phosphate minerals or hydroxyapatite to construct an osteogenic mineralization microenvironment. S4. The upper hydrogel layer, the middle hydrophilic modified polylactic acid separator, and the lower hydrogel layer continue to cure under wet conditions, so that hydrogen bonding, catechol-mediated interfacial adhesion, and non-covalent / covalent synergistic effects are formed between the polydopamine coating and the upper and lower hydrogel layers, thereby obtaining a three-layer composite periodontal restorative material with the functions of promoting soft tissue repair in the upper layer, cell barrier in the middle layer, and promoting bone regeneration in the lower layer.

8. The method for preparing a three-layer composite periodontal restorative material according to claim 7, characterized in that: In step S1, the dopamine solution is a Tris-HCl buffer solution containing dopamine, with a pH of 8.0-8.8, preferably 8.5, and the dopamine self-polymerization reaction time is 12-24 hours; In step S2, the crosslinking temperature of adipic dihydrazide-modified hyaluronic acid and aldehyde-modified hyaluronic acid is 25-37℃, and the crosslinking time is 5-30 minutes. In step S3, dopamine-grafted hyaluronic acid is crosslinked into a gel under the action of horseradish peroxidase / hydrogen peroxide system, with a crosslinking temperature of 25-37℃ and a crosslinking time of 5-30 minutes. The hydrogel layers formed in steps S2 and S3 are solidified in situ on the surface of the polydopamine-modified polylactic acid membrane, so that a stable wet interface bond is formed between the upper and lower hydrogel layers and the middle barrier membrane layer.

9. The method for preparing a three-layer composite periodontal restorative material according to claim 7, wherein the polydopamine coating forms a wet interface bonding structure with the upper hydrogel layer and the lower hydrogel layer, and the wet interface bonding structure includes one or more of hydrogen bonding, catechol group-mediated adhesion, amino / hydroxyl interaction and polymer chain segment entanglement.

10. The use of the three-layer composite periodontal restorative material according to any one of claims 1 to 6 in the preparation of a medical device for treating combined periodontal soft and hard tissue defects; The three-layer composite periodontal restorative material promotes the regeneration of gingival soft tissue through the upper hydrogel layer, physically isolates the migration of gingival fibroblasts through the middle barrier membrane layer to maintain space for bone regeneration, and promotes the regeneration of alveolar bone hard tissue through the lower hydrogel layer. The three layers work together to achieve integrated repair of periodontal tissues.

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