Biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, preparation method and application thereof

Abstract

This invention provides a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, its preparation method, and its application, relating to the technical field of tissue engineering scaffolds. The biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold comprises, from the inside out, a cementum repair layer, a periodontal ligament repair layer, and an alveolar bone repair layer; the cementum repair layer and the alveolar bone repair layer include a mineralized hydrogel layer; wherein the raw materials for preparing the mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and hydroxyapatite; the alveolar bone repair layer includes a non-mineralized hydrogel layer; wherein the raw materials for preparing the non-mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and fibrin I. This invention precisely matches the mechanical and biological requirements of each layer of the periodontal complex through a three-layer structure, a reversibly hydrazone-regulated viscoelastic gradient, and a differentiated biochemical microenvironment of nHAp / FBN1.

Description

Technical Field

[0001] This invention relates to the technical field of tissue engineering scaffolds, and in particular to a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, its preparation method, and its application. Background Technology

[0002] Periodontal tissue defects have a high incidence rate among patients with oral diseases. The periodontal complex consists of three layers of soft and hard tissues: alveolar bone, periodontal ligament, and cementum, each with significantly different mechanical microenvironments. Existing tissue engineering materials mostly focus on single hard or soft tissue repair, lacking the ability to comprehensively reconstruct the multi-layered structure of the periodontal complex. Furthermore, existing materials typically only focus on elastic modulus, neglecting the dynamic viscoelastic characteristics of physiological tissues. This results in an inability to provide stem cells with mechanical cues close to the physiological environment, making it difficult to achieve orderly and regionalized differentiation of dendritic ligament cells (PDLSCs) in osteogenic, fibrogenic, and cementogenic directions. Therefore, how to simultaneously meet the different viscoelastic requirements of multiple layers of soft and hard tissues within a single scaffold system and achieve precise three-dimensional regulation of PDLSCs remains a key technical challenge that has long remained unsolved in clinical and engineering fields.

[0003] Periodontal tissue defects are a common complication of orthodontic treatment, including alveolar bone fenestration, bone fissures, and gingival recession, among other pathological changes. Periodontal tissue defects not only restrict tooth movement in three-dimensional space during orthodontic treatment but also affect the long-term stability of tooth position and the long-term health of periodontal tissues. In severe cases, they can lead to tooth loosening, periodontal tooth loss, and consequently, significantly impact the patient's stomatognathic system function and oral health-related quality of life. Current clinical procedures can achieve partial periodontal tissue regeneration to some extent, but they suffer from limited indications, poor regeneration effects, and high costs. Periodontal tissue engineering scaffolds have become an important research direction in the field of periodontal regeneration.

[0004] The periodontal complex is composed of layers of hard and soft tissues that support the dentition (e.g., Figure 1 As shown, the periodontal complex comprises alveolar bone, periodontal ligament, and cementum. Given the complex and ordered structure of the mineralized and unmineralized layers of the periodontal complex, a single periodontal tissue engineering scaffold cannot achieve the synchronous regeneration of the multi-layered, ordered soft and hard tissues of the periodontal complex. Therefore, biomimetic multi-layered tissue engineering scaffolds have become an important research direction for periodontal regeneration.

[0005] Park et al. designed a polycaprolactone (PCL)-polyglycolic acid (PGA) scaffold that simulates the periodontal ligament-alveolar bone bilayer structure, achieving the regeneration of periodontal tissue-like structures. Sowmya et al. used a three-layer porous nanocomposite hydrogel scaffold with chitin-polylactic acid-glycolic acid (PLGA) as the main component, which enabled the formation of new cementum, fibrous periodontal ligament, and trabecular bone. However, simply stacking and combining the hydrogel scaffold layers makes it difficult to reproduce the spatial structure of the physiological periodontal complex.

[0006] Previous studies have mostly focused on regulating the cell regeneration microenvironment by combining different materials or loading different bioactive factors. They have paid little attention to the differential mechanical properties of soft and hard tissues in the periodontal complex, such as viscoelasticity. These studies can only form periodontal-like structures to a certain extent. At the same time, the simple mechanical superposition between the various scaffold layers makes it difficult to reproduce the complex spatial structure of the periodontal complex, and functional regeneration of periodontal tissues remains challenging.

[0007] Meanwhile, the shortcomings of existing repair materials include at least the following aspects: First, existing restorative materials and tissue engineering scaffolds for periodontal tissue defects mainly focus on the regeneration of a single type of tissue (such as bone or soft tissue), and generally lack structural designs for the overall restoration of the periodontal complex. Because alveolar bone, periodontal ligament, and cementum differ significantly in mechanical properties and physiological functions, existing scaffolds cannot simultaneously meet the different microenvironmental needs of these three tissue types, resulting in restorative effects limited to local areas and failing to achieve the reconstruction of the overall periodontal complex structure.

[0008] Secondly, traditional scaffold materials generally focus on static elastic parameters while neglecting the dynamic viscoelastic characteristics of physiological tissues. Existing materials cannot transmit signals to stem cells that match the mechanics of real tissues, resulting in a lack of control over the direction of stem cell differentiation, which is not conducive to the precise induction of periodontal ligament stem cells in osteogenic, fibrogenic, and cementogenic directions.

[0009] Thirdly, regarding the control of viscoelasticity, previous materials struggled to maintain consistent initial elastic modulus, porosity, and degradation rate when viscoelasticity was altered, leading to an increase in variables. This material employs a dual-network crosslinking system and dynamic covalent bond regulation of viscoelasticity. Therefore, it can maintain fundamental properties such as initial elastic modulus, porosity, and degradation rate essentially unchanged when viscoelasticity is controlled using a gradient.

[0010] Fourth, traditional manufacturing methods often employ segmented or post-assembly molding, which can easily lead to problems such as interlayer interface fracture, discontinuous mechanical properties, and uneven cell distribution, making it difficult to achieve spatially continuous signal transmission and orderly tissue regeneration.

[0011] In view of this, the present invention is hereby proposed. Summary of the Invention

[0012] The purpose of this invention is to provide a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, its preparation method, and its application. This invention aims to address the technical problems in the regeneration of multilayer soft and hard tissues in periodontal complexes, such as the lack of a unified scaffold system that combines spatial layering structure, tissue-specific viscoelastic matching, and biochemical microenvironment adaptation.

[0013] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, comprising a cementum repair layer, a periodontal ligament repair layer and an alveolar bone repair layer stacked sequentially from the inside out; The cementum repair layer and the alveolar bone repair layer include a mineralized hydrogel layer; wherein, the raw materials for preparing the mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and hydroxyapatite; The alveolar bone repair layer includes a non-mineralized hydrogel layer; wherein, the raw materials for preparing the non-mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and fibrin 1.

[0014] The purpose of this invention is to overcome the shortcomings of the prior art and provide a biomimetic three-layer hydrogel scaffold for the periodontal complex with a viscoelastic gradient. This scaffold constructs three layers—mineralized, unmineralized, and mineralized—within a single structure, achieving spatial correspondence between alveolar bone, periodontal ligament, and cementum, mimicking a physiological structure to promote periodontal complex regeneration. The adjustable viscoelasticity within each scaffold layer enables mechanical adaptation of different tissue layers, creating a biomimetic mechanical microenvironment for each layer of the periodontal complex. This promotes the triaxial orderly differentiation of periodontal ligament stem cells in osteogenic, fibrogenic, and cementogenic directions, achieving overall regeneration of the multi-layered soft and hard tissue structure of the periodontal complex. Furthermore, this invention utilizes projection-based light-curing 3D printing technology to complete the three-layer structure in a single molding process, achieving continuous transitions between layers and precise spatial positioning, avoiding structural separation and performance discontinuities caused by traditional assembly manufacturing. Through the above technical solution, this invention can provide a more effective means of repairing periodontal tissue defects after orthodontic treatment and offer a new solution for the clinical application of periodontal regeneration materials.

[0015] Furthermore, the modified hyaluronic acid includes hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid.

[0016] Furthermore, the mineralized hydrogel layer comprises: a first crosslinking network formed by methacrylamide type I collagen, a second crosslinking network formed by crosslinking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and hydroxyapatite doped within the first and second crosslinking networks.

[0017] Furthermore, the non-mineralized hydrogel layer comprises: a first cross-linked network formed by methacrylamide type I collagen, a second cross-linked network formed by cross-linking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and protofibrin 1 loaded within the first and second cross-linked networks.

[0018] Furthermore, the structural formula of the hydrazine-modified hyaluronic acid is shown in Formula I below: ; Formula I; Where n is an integer between 249 and 998.

[0019] Furthermore, the aldehyde-modified hyaluronic acid includes hyaluronic acid modified with fatty aldehydes and / or hyaluronic acid modified with benzaldehyde.

[0020] Furthermore, the structural formula of the fatty aldehyde-modified hyaluronic acid is shown in Formula II-1 below:

[0021] Formula II-1; Where a is an integer between 249 and 998.

[0022] Furthermore, the structural formula of the benzaldehyde-modified hyaluronic acid is shown in Formula II-2 below:

[0023] Formula II-2; Where b is an integer between 249 and 998.

[0024] Furthermore, in the mineralized hydrogel layer, the mass ratio of the modified hyaluronic acid, methacrylamide type I collagen and hydroxyapatite is (14~16):(1~3):(24~26).

[0025] Furthermore, in the non-mineralized hydrogel layer, the mass ratio of the modified hyaluronic acid, methacrylamide type I collagen, and fibrin 1 is (28~32):(3~5):(0.2~2).

[0026] Furthermore, the periodontal ligament repair layer surrounds the cementum repair layer and forms a first cubic structure; the alveolar bone repair layer surrounds the first cubic structure and forms a second cubic structure.

[0027] Furthermore, at least two of the first cube structures are disposed within the second cube structure.

[0028] Furthermore, the width of the cementum repair layer is 200~400 μm, and the thickness of the cementum repair layer is 400~600 μm.

[0029] Furthermore, the thickness of the periodontal ligament repair layer is 100~300 μm.

[0030] Furthermore, the spacing between each of the first cubic structures disposed within the second cubic structure is independently 300~500 μm.

[0031] Furthermore, the total width of the second cubic structure is 3000~3200 μm, and the total height of the second cubic structure is 1400~1600 μm.

[0032] In a second aspect, the present invention provides a method for preparing a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold as described in the first aspect, the method comprising: Modified hyaluronic acid, methacrylamide type I collagen, hydroxyapatite, photoinitiator and solvent were mixed to obtain a mineralized hydrogel precursor solution; The modified hyaluronic acid, methacrylamide type I collagen and protoporphyrin 1, photoinitiator and solvent were mixed to obtain a non-mineralized hydrogel precursor solution; The alveolar bone repair layer, periodontal ligament repair layer, and cementum repair layer are sequentially printed using photopolymerization 3D printing to obtain the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold.

[0033] Furthermore, the preparation method of the modified hyaluronic acid includes the following steps: Hyaluronic acid and propyne amine undergo a condensation reaction to yield alkynylated hyaluronic acid; Alkyne-modified hyaluronic acid and an azide are subjected to a click chemical reaction to obtain the modified hyaluronic acid; wherein the azide includes hydrazine azidohydrazine and / or azidoaldehyde.

[0034] Furthermore, the molecular weight of the hyaluronic acid is 100~400kDa.

[0035] Furthermore, the condensation reaction is carried out in the presence of a composite condensing agent; wherein the composite condensing agent comprises N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0036] Furthermore, the molar ratio of hyaluronic acid, propyneamine, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:(0.8~1):(0.8~1):(0.8~1).

[0037] Furthermore, the condensation reaction is carried out in a solvent; wherein the solvent includes MES buffer.

[0038] Furthermore, the pH of the condensation reaction is 5.5~6.5, the temperature of the condensation reaction is 20~30℃, and the time of the condensation reaction is 3~5 h.

[0039] Furthermore, the azidohydrazine is N-(3-azidopropyl)-2-hydrazinoacetamide.

[0040] Further, the azide aldehyde includes azide-polyethylene glycol-acetaldehyde and / or N-(2-azidoethyl)-4-carboxybenzamide.

[0041] Furthermore, the click chemical reaction is carried out in the presence of a catalyst and a reducing agent; wherein the catalyst comprises copper sulfate; and the reducing agent comprises sodium ascorbate.

[0042] Furthermore, the molar ratio of the alkynylated hyaluronic acid, the azide catalyst, and the reducing agent is 1:(0.8~1.2):(0.001~0.01):(0.01~0.1).

[0043] Furthermore, the click chemistry reaction is carried out in a solvent; wherein the solvent includes PBS buffer.

[0044] Furthermore, the temperature of the click chemical reaction is 20~30℃, and the time of the click chemical reaction is 18~30 h.

[0045] Furthermore, the mineralized hydrogel precursor solution comprises, by mass percentage: 1-5% modified hyaluronic acid, 2-5% methacrylamide type I collagen, 1-10% hydroxyapatite, 0.1-0.5% photoinitiator, with the remainder being solvent.

[0046] Furthermore, the non-mineralized hydrogel precursor solution comprises, by mass percentage: 1-5% modified hyaluronic acid, 2-5% methacrylamide type I collagen, 0.1-5% fibrin, 0.1-0.5% photoinitiator, with the remainder being solvent.

[0047] Furthermore, the photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl)phosphate.

[0048] Furthermore, the solvent includes PBS buffer.

[0049] Furthermore, the photopolymerization 3D printing step includes: Add 0.2~0.3 wt% of light blocker to the mineralized hydrogel precursor solution to obtain material 1; add 0.005~0.02 wt% of light blocker to the non-mineralized hydrogel precursor solution to obtain material 2; the printing process adopts an alternating mode of material 1-material 2-material 1 to finally achieve the printing of a three-dimensional structure of three-layer hydrogel, thus obtaining the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold.

[0050] Furthermore, the light-blocking agent includes food dyes and / or biocompatible dyes.

[0051] Furthermore, the light-blocking agent is tartrazine and / or curcumin.

[0052] Furthermore, the parameters of the material 1 obtained by photopolymerization 3D printing include: layer height 50~200 μm; light intensity 200~220 PWM; exposure time 7~9 s; number of base layers 1~5; base layer exposure time 7~9 s; peel distance 1~5 mm; peel speed 20~30 mm / min; peel recovery speed 80~120 mm / min; lifting speed 160~200 mm / min.

[0053] Furthermore, the parameters of the material 2 obtained by photopolymerization 3D printing include: layer height 50~200 μm; light intensity 260~300 PWM; exposure time 10~14 s; number of base layers 1~5; base layer exposure time 10~14 s; peel distance 1~5 mm; peel speed 20~30 mm / min; peel recovery speed 80~120 mm / min; lifting speed 160~200 mm / min.

[0054] Thirdly, the present invention provides the application of the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold as described in the first aspect in the preparation of materials for repairing alveolar bone, periodontal ligament and cementum.

[0055] Fourthly, the present invention provides a viscoelastically adjustable multilayer photocurable hydrogel scaffold for defect repair, wherein the viscoelastically adjustable multilayer photocurable hydrogel scaffold for defect repair includes the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold as described in the first aspect, or the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold prepared by the preparation method described in the second aspect. Furthermore, the viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair also includes cultured cells.

[0056] Furthermore, the cells include any one of periodontal ligament stem cells, osteoblast stem cells, fibroblast stem cells, and cementoblast stem cells, preferably periodontal ligament stem cells.

[0057] Fifthly, the present invention provides a method for preparing the viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair, the method comprising: Cells were cultured to the logarithmic growth phase, then collected, washed, and digested with trypsin. After the reaction was terminated, the cells were collected to obtain a cell suspension. Modified hyaluronic acid, methacrylamide type I collagen, hydroxyapatite, photoinitiator and solvent were mixed to obtain a mineralized hydrogel precursor solution; The modified hyaluronic acid, methacrylamide type I collagen and protoporphyrin 1, photoinitiator and solvent were mixed to obtain a non-mineralized hydrogel precursor solution; The cell suspension was added to the mineralized hydrogel precursor solution and the non-mineralized hydrogel precursor solution, respectively, to obtain a cell-containing mineralized hydrogel precursor solution and a cell-containing non-mineralized hydrogel precursor solution. The alveolar bone repair layer, periodontal ligament repair layer and cementum repair layer were printed sequentially by photopolymerization 3D printing to obtain a framework. The scaffold was subjected to in vitro static culture, allowing the cells to transport nutrients and maintain survival within the gel, resulting in the viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair.

[0058] Furthermore, the cells include any one of periodontal ligament stem cells, osteoblast stem cells, fibroblast stem cells, and cementoblast stem cells, preferably periodontal ligament stem cells.

[0059] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention proposes a viscoelastically adapted biomimetic three-layer hydrogel scaffold. Based on the physiological viscoelasticity differences of different layers of tissue in the periodontal complex, the tunable viscoelastic gradient induces the osteogenic, fibrogenic and cementogenic differentiation of stem cells, thereby achieving the orderly regeneration of the multi-layer soft and hard tissue structure of the periodontal complex.

[0060] (2) This invention utilizes a reversible hydrazone bond regulation strategy to construct mineralized and non-mineralized hydrogels, achieving precise control of viscoelasticity. Furthermore, it employs projection-based photopolymerization 3D printing technology to construct a three-layer scaffold with spatially matched structure. Further optimization of viscoelastic parameters enables controllable adjustment of the differentiation direction of PDLSCs. This invention overcomes the technical bottleneck in existing technologies, namely the lack of viscoelastically adapted multi-layered regeneration carriers and the inability to achieve triaxial differentiation and spatially ordered reconstruction of PDLSCs. It can promote the development of periodontal tissue regeneration from single-layer repair to complete complex reconstruction, providing an effective solution for periodontal injury repair after orthodontic treatment. Attached Figure Description

[0061] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0062] Figure 1 This is a schematic diagram of the periodontal complex provided by the present invention.

[0063] Figure 2 This is a schematic diagram showing the structural composition and functional zoning of the biomimetic periodontal composite multilayer hydrogel tissue engineering scaffold provided by the present invention.

[0064] Figure 3 This is a schematic diagram showing the structure and dimensions of the biomimetic periodontal composite multilayer hydrogel tissue engineering scaffold provided by the present invention.

[0065] Among them, 100 is the cementum repair layer, 200 is the periodontal ligament repair layer, and 300 is the alveolar bone repair layer.

[0066] Figure 4 is a comparison of the infrared spectra of unmodified hyaluronic acid, hydrazine-modified hyaluronic acid provided in Preparation Example 1, fatty aldehyde-modified hyaluronic acid provided in Preparation Example 2, and benzaldehyde-modified hyaluronic acid provided in Preparation Example 3.

[0067] Figure 5 This is a 3D printing model of the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold provided in Example 1.

[0068] Figure 6A This is a schematic diagram of the disassembly of the layered construction of the 3D printed hydrogel provided in Example 1.

[0069] Figure 6B This is a schematic diagram of the overall structure of the 3D-printed hydrogel provided in Example 1.

[0070] Figure 7 This is a photograph of the actual product during the hydrogel printing process.

[0071] Figure 8A The frequency scan curves of the mineralized hydrogel show the variation trends of G′ and G″ in different frequency ranges.

[0072] Figure 8B The frequency scan curves of the non-mineralized hydrogels show the variation trends of G′ and G″ in different frequency ranges.

[0073] Figure 8CThe statistical results are shown in the graph for the hydrogel G′, G″ and loss tangent (tan = G″ / G′) of the mineralized group; where the data are expressed as mean ± standard deviation (n=3).

[0074] Figure 8D The figure shows the statistical results of G′, G″ and loss tangent (tan = G″ / G′) of the non-mineralized hydrogel; the data are expressed as mean ± standard deviation (n=3).

[0075] Figure 9 The in vivo application and effect diagram of the biomimetic hydrogel provided in Application Example 1. Detailed Implementation

[0076] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0077] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0078] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0079] In a first aspect, the present invention provides a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, comprising a cementum repair layer, a periodontal ligament repair layer and an alveolar bone repair layer stacked sequentially from the inside out; The cementum repair layer and the alveolar bone repair layer include a mineralized hydrogel layer; wherein, the raw materials for preparing the mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and hydroxyapatite; The alveolar bone repair layer includes a non-mineralized hydrogel layer; wherein, the raw materials for preparing the non-mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and fibrin 1.

[0080] It should be noted that the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold described in this invention is based on the viscoelastic properties and biochemical microenvironment of the mineralized layer (alveolar bone, cementum) and the non-mineralized layer (periodontal ligament) in the biomimetic periodontal complex. This invention uses a methacryloyl-1 type I collagen (Col I-MA) and hyaluronic acid (HA) composite system to construct the hydrogel scaffold. Simultaneously, in the biomimetic dynamic mechanical microenvironment, a dual-network cross-linking method is employed: (a) a covalent cross-linking network is constructed through the photocuring reaction of Col I-MA; (b) a secondary cross-linking network is constructed through the hydrazone bond reaction of hyaluronic acid-hydrazide (HA-hydrazide) and hyaluronic acid-aldehyde (HA-aldehyde) under neutral conditions. By controlling the proportion of different aldehyde groups (aliphatic aldehyde ALD, benzaldehyde BLD), the proportion of different hydrazone bonds is adjusted, thereby regulating the viscoelasticity of the hydrogel. (c) In a simulated mineralized / unmineralized microenvironment, nano-hydroxyapatite (nHAp) is incorporated into the mineralized layer to construct a mineralization-inducing microenvironment, while the unmineralized layer is loaded with fibrin 1 (FBN1) to construct a periodontal ligament tissue-inducing microenvironment. Finally, the three-layer hydrogel is effectively mechanically integrated using 3D bioprinting technology to achieve a differentiated biomimetic design of the viscoelasticity and biochemical microenvironment of each scaffold layer.

[0081] It should be noted that the overall biomimetic periodontal complex structure of the scaffold is a strip-shaped embedded three-dimensional structure, consisting of cementum, periodontal ligament, and alveolar bone layers from the inside out. The three layers are physically integrated and chemically interconnected through continuous light-curing printing, avoiding interface fractures and discontinuities in biological properties. The hierarchical tissue correspondence, material properties, and functional positioning are as follows: inner layer – mineralized hydrogel – induces cementum formation; middle layer – non-mineralized hydrogel – provides an environment for periodontal ligament fiber generation; outer layer – mineralized hydrogel – promotes alveolar bone regeneration and provides mechanical support. Furthermore, the incorporation of nano-hydroxyapatite (nHAp) into the mineralized hydrogel aims to construct a bone-like mineralized microenvironment, while the loading of fibrillary protein FBN1 into the non-mineralized hydrogel aims to simulate the periodontal ligament ECM signaling.

[0082] As an optional implementation, the modified hyaluronic acid includes hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid.

[0083] As an optional implementation method, such as Figure 2 As shown, the mineralized hydrogel layer comprises: a first cross-linked network formed by methacrylamide type I collagen, a second cross-linked network formed by cross-linking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and hydroxyapatite doped within the first and second cross-linked networks.

[0084] As an optional implementation method, such as Figure 2 As shown, the non-mineralized hydrogel layer comprises: a first cross-linked network formed by methacrylamide type I collagen, a second cross-linked network formed by cross-linking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and protofibrin 1 loaded within the first and second cross-linked networks.

[0085] It should be noted that the first crosslinking network in this invention is a covalent crosslinking network formed by methacrylamide type I collagen, and the second crosslinking network is a dynamic crosslinking network formed by crosslinking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid. The first crosslinking network, Col I-MA photocrosslinking, forms a structural framework, improving mechanical stability. The second dynamic network, through the hydrazone reaction of HA-HYN with HA-ALD / HA-BLD, provides tunable viscoelasticity and relaxation behavior. Furthermore, by adjusting the ratio of HA-ALD to HA-BLD, different hydrazone content differences are achieved, ultimately obtaining three viscoelastic ranges: low loss modulus, medium loss modulus, and high loss modulus, with the loss tangent tanδ adjusted within the range of 0.1 to 0.3. As an optional embodiment, the structural formula of the hydrazine-modified hyaluronic acid is shown in Formula I below: ; Formula I; Where n is an integer between 249 and 998, for example, it can be 249, 256, 270, 284, 298, 312, 326, 340, 354, 368, 382, ​​396, 410, 424, 438, 452, 466, 480, 494, 508, 522, 536, 550, 564, 578, 592, 606, 620, 634, 648, 662, 676, 690, 704, 718, 732, 746, 760, 774, 788, 802, 816, 830, 844, 858, 872, 886, 900, 914, 928, 942, 956, 970, 984, 998, etc.

[0086] As an optional implementation, the aldehyde-modified hyaluronic acid includes fatty aldehyde-modified hyaluronic acid and / or benzaldehyde-modified hyaluronic acid.

[0087] As an optional implementation, the structural formula of the fatty aldehyde-modified hyaluronic acid is shown in Formula II-1 below:

[0088] Formula II-1; Where 'a' is an integer between 249 and 998, for example, it can be 249, 256, 270, 284, 298, 312, 326, 340, 354, 368, 382, ​​396, 410, 424, 438, 452, 466, 480, 494, 508, 522, 536, 550, 564, 578, 592, 606, 620, 634, 648, 662, 676, 690, 704, 718, 732, 746, 760, 774, 788, 802, 816, 830, 844, 858, 872, 886, 900, 914, 928, 942, 956, 970, 984, 998, etc.

[0089] As an optional implementation, the structural formula of the benzaldehyde-modified hyaluronic acid is shown in Formula II-2 below:

[0090] Formula II-2; Where b is an integer between 249 and 998, for example, it can be 249, 256, 270, 284, 298, 312, 326, 340, 354, 368, 382, ​​396, 410, 424, 438, 452, 466, 480, 494, 508, 522, 536, 550, 564, 578, 592, 606, 620, 634, 648, 662, 676, 690, 704, 718, 732, 746, 760, 774, 788, 802, 816, 830, 844, 858, 872, 886, 900, 914, 928, 942, 956, 970, 984, 998, etc.

[0091] As an optional implementation, in the mineralized hydrogel layer, the mass ratio of the modified hyaluronic acid, methacrylamide type I collagen and hydroxyapatite is (14~16):(1~3):(24~26); Among them, "14~16" can be, for example, 14, 14.2, 14.4, 14.6, 14.8, 15, 15.2, 15.4, 15.6, 15.8, 16, etc.; Among them, "1~3" can be, for example, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, etc.; Among them, "24~26" can be, for example, 24, 24.2, 24.4, 24.6, 24.8, 25, 25.2, 25.4, 25.6, 25.8, 26, etc.

[0092] As an optional implementation, in the non-mineralized hydrogel layer, the mass ratio of the modified hyaluronic acid, methacrylamide type I collagen, and fibrin 1 is (28~32):(3~5):(0.2~2); Among them, "28~32" can be, for example, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, etc.; and "3~5" can be, for example, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, etc. Among them, "0.2~2" can be, for example, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, etc.

[0093] As an optional implementation method, such as Figure 3 As shown, the periodontal ligament repair layer 200 surrounds the cementum repair layer 100 and forms a first cubic structure; the alveolar bone repair layer 300 surrounds the first cubic structure and forms a second cubic structure.

[0094] As an optional implementation method, such as Figure 3 As shown, at least two of the first cube structures are disposed within the second cube structure.

[0095] As an optional implementation method, such as Figure 3 As shown, the width W1 of the cementum repair layer 100 is 200~400 μm, for example, it can be 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, etc.

[0096] As an optional implementation method, such as Figure 3 As shown, the thickness H1 of the cementum repair layer 100 is 400~600 μm, for example, it can be 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, 500 μm, 520 μm, 540 μm, 560 μm, 580 μm, 600 μm, etc.

[0097] As an optional implementation method, such as Figure 3 As shown, the thickness H2 of the periodontal ligament repair layer 200 is 100~300 μm, for example, it can be 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, 20 μm, 22 μm, 24 μm, 26 μm, 28 μm, 30 μm, etc.

[0098] As an optional implementation method, such as Figure 3As shown, the spacing L between each of the first cube structures set within the second cube structure is independently 300~500 μm, for example, it can be 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, 500 μm, etc.

[0099] As an optional implementation method, such as Figure 3 As shown, the total width W of the second cube structure all The value is 3000~3200μm, for example, it can be 3000 μm, 3020 μm, 3040 μm, 3060 μm, 3080 μm, 3100 μm, 3120 μm, 3140 μm, 3160 μm, 3180 μm, 3200 μm, etc.

[0100] As an optional implementation method, such as Figure 3 As shown, the total height H of the second cube structure all The value is 1400~1600μm, for example, it can be 1400 μm, 1420 μm, 1440 μm, 1460 μm, 1480 μm, 1500 μm, 1520 μm, 1540 μm, 1560 μm, 1580 μm, 1600 μm, etc.

[0101] In a second aspect, the present invention provides a method for preparing a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold as described in the first aspect, the method comprising: Modified hyaluronic acid, methacrylamide type I collagen, hydroxyapatite, photoinitiator and solvent were mixed to obtain a mineralized hydrogel precursor solution; The modified hyaluronic acid, methacrylamide type I collagen and protoporphyrin 1, photoinitiator and solvent were mixed to obtain a non-mineralized hydrogel precursor solution; It should be noted that this invention uses photopolymerization 3D printing to sequentially print alveolar bone repair layer, periodontal ligament repair layer, and cementum repair layer to obtain the biomimetic periodontal composite multilayer hydrogel assembly. The three-layer integrated molding is achieved using projection photopolymerization (DLP) 3D printing, including: loading mineralized hydrogel precursors and non-mineralized hydrogel precursors into resin tanks with dual material partitions; the printing system projects different pixel light fields into different spatial regions through a digital micromirror array module; sequentially printing material 1 (mineralized) - material 2 (non-mineralized) - material 1 (mineralized); completing the continuous stacking of the three layers and the integrated molding of the viscoelastic gradient. (This is related to) woven engineering scaffolds.

[0102] As an optional implementation, the method for preparing the modified hyaluronic acid includes the following steps: Hyaluronic acid and propyne amine undergo a condensation reaction to yield alkynylated hyaluronic acid; Alkyne-modified hyaluronic acid and an azide are subjected to a click chemical reaction to obtain the modified hyaluronic acid; wherein the azide includes hydrazine azidohydrazine and / or azidoaldehyde.

[0103] As an optional implementation, the molecular weight of the hyaluronic acid is 100~400 kDa, for example, it can be 100 kDa, 120 kDa, 140 kDa, 160 kDa, 180 kDa, 200 kDa, 220 kDa, 240 kDa, 260 kDa, 280 kDa, 300 kDa, 320 kDa, 340 kDa, 360 kDa, 380 kDa, 400 kDa, etc.

[0104] As an optional implementation, the condensation reaction is carried out in the presence of a composite condensing agent.

[0105] As an optional implementation, the composite condensing agent includes N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0106] As an optional embodiment, the molar ratio of hyaluronic acid, propyneamine, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:(0.8~1):(0.8~1):(0.8~1).

[0107] As an optional implementation, the condensation reaction is carried out in a solvent.

[0108] As an optional implementation, the solvent for the condensation reaction includes MES buffer.

[0109] As an optional implementation, the pH of the condensation reaction is 5.5~6.5, for example, 5.5, 5.6, 5.8, 6, 6.2, 6.4, 6.5, etc.; the temperature of the condensation reaction is 20~30℃, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc.; and the time of the condensation reaction is 3~5 h, for example, 3 h, 3.2 h, 3.4 h, 3.6 h, 3.8 h, 4 h, 4.2 h, 4.4 h, 4.6 h, 4.8 h, 5 h, etc.

[0110] As an optional implementation, the condensation reaction may further include post-processing steps: dialysis and freeze-drying.

[0111] As an optional embodiment, the azidohydrazine is N-(3-azidopropyl)-2-hydrazidoacetamide.

[0112] As an optional implementation, the hydrazine azide is .

[0113] As an optional implementation, the azide aldehyde includes azide-polyethylene glycol-acetaldehyde and / or N-(2-azidoethyl)-4-formylbenzamide.

[0114] As an optional implementation, the azide aldehyde includes and / or .

[0115] As an optional implementation, the click chemistry reaction is carried out in the presence of a catalyst and a reducing agent.

[0116] As an optional implementation, the catalyst includes copper sulfate.

[0117] As an optional implementation, the reducing agent includes sodium ascorbate.

[0118] As an optional implementation, the molar ratio of the alkynylated hyaluronic acid, the azide catalyst, and the reducing agent is 1:(0.8~1.2):(0.001~0.01):(0.01~0.1).

[0119] As an optional implementation, the click chemistry reaction is carried out in a solvent.

[0120] As an optional implementation, the solvent for the click chemistry reaction includes PBS buffer.

[0121] As an optional implementation, the temperature of the click chemical reaction is 20~30℃, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc., and the time of the click chemical reaction is 18~30 h, for example, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, etc.

[0122] As an optional implementation, the click chemical reaction may further include post-processing steps: dialysis and freeze-drying.

[0123] As an optional implementation, the mineralized hydrogel precursor solution comprises, by mass percentage: 1-5% modified hyaluronic acid (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.), 2-5% methacrylamide type I collagen (e.g., 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.), 1-10% hydroxyapatite (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc.), 0.1-0.5% photoinitiator (e.g., 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, etc.), with the balance being solvent.

[0124] As an optional implementation, the non-mineralized hydrogel precursor solution comprises, by mass percentage: 1-5% modified hyaluronic acid (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.), 2-5% methacrylamide type I collagen (e.g., 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.), 0.1-5% fibrin (e.g., 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.), 0.1-0.5% photoinitiator (e.g., 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, etc.), with the remainder being solvent.

[0125] As an optional implementation, the photoinitiator in the mineralized hydrogel precursor solution and the non-mineralized hydrogel precursor solution includes lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP).

[0126] As an optional implementation, the solvent in the mineralized hydrogel precursor solution and the non-mineralized hydrogel precursor solution includes PBS buffer.

[0127] As an optional implementation, the photopolymerization 3D printing step in the mineralized hydrogel precursor solution and the non-mineralized hydrogel precursor solution includes: Add 0.2~0.3 wt% (e.g., 0.2 wt%, 0.22 wt%, 0.24 wt%, 0.25 wt%, 0.26 wt%, 0.28 wt%, 0.3 wt%, etc.) of a light blocker to the mineralized hydrogel precursor solution to obtain material 1; add 0.005~0.02 wt% (e.g., 0.005 wt%, 0.006 wt%, 0.008 wt%, 0.01 wt%, 0.012 wt%, 0.014 wt%, 0.015 wt%, 0.016 wt%, 0.018 wt%, 0.02 wt%, etc.) of a light blocker to the non-mineralized hydrogel precursor solution to obtain material 2; the printing process adopts an alternating mode of material 1-material 2-material 1, and finally realizes the printing of a three-dimensional structure of three-layer hydrogel to obtain the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold.

[0128] As an optional implementation, the light-blocking agent includes food dyes and / or biocompatible dyes.

[0129] As an optional implementation, the light-blocking agent is tartrazine and / or curcumin.

[0130] As an optional implementation, the parameters of the material 1 obtained by photopolymerization 3D printing include: layer height of 50~200 μm (e.g., 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, etc.); light intensity of 200~220 PWM (e.g., 200 PWM, 202 PWM, 204 PWM, 206 PWM, 208 PWM, 210 PWM, 212 PWM, 214 PWM, 216 PWM, 218 PWM, 220 PWM, etc.); and exposure time of 7~9 s (e.g., 7 s, 7.2 s, 7.4 s, 7.6 s, 7.8 s, 8 s). Exposure times are 7-9 s (e.g., 7 s, 7.2 s, 7.4 s, 7.6 s, 7.8 s, 8 s, 8 s, 8.2 s, 8.4 s, 8.6 s, 8.8 s, 9 s, etc.); number of substrate layers is 1-5 (e.g., 1, 2, 3, 4, 5); substrate exposure time is 7-9 s (e.g., 7 s, 7.2 s, 7.4 s, 7.6 s, 7.8 s, 8 s, 8.2 s, 8.4 s, 8.6 s, 8.8 s, 9 s, etc.); peeling distance is 1-5 mm (e.g., 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, etc.); peeling speed is 20-30 mm / min (e.g., 20 mm / min, 21 mm / min, 22 mm / min, 23 mm / min, 24 mm / min, 25 mm / min, 26 mm / min, 27 mm / min, 28 mm / min, 29 mm / min, etc.). The speed can be 80-120 mm / min (e.g., 80 mm / min, 85 mm / min, 90 mm / min, 95 mm / min, 100 mm / min, 105 mm / min, 110 mm / min, 115 mm / min, 120 mm / min, etc.); the lifting speed can be 160-200 mm / min (e.g., 160 mm / min, 165 mm / min, 170 mm / min, 175 mm / min, 180 mm / min, 185 mm / min, 190 mm / min, 195 mm / min, 200 mm / min).

[0131] As an optional implementation, the parameters of the material 2 obtained by photopolymerization 3D printing include: layer height of 50~200 μm (e.g., 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, etc.); and light intensity of 260~300 PWM (e.g., 260 PWM, 262 PWM, 264 PWM, 266 PWM, 268 PWM, 270 PWM, 272 PWM, 274 PWM, 276 PWM, 278 PWM, 280 PWM, 282 PWM, 284 PWM, 286 PWM, 288 PWM, 290 PWM, 292 PWM, 294 PWM). PWM, 296 PWM, 298 PWM, 300 PWM; exposure time 10~14 s (e.g., 10 s, 10.2 s, 10.4 s, 10.6 s, 10.8 s, 11 s, 11.2 s, 11.4 s, 11.6 s, 11.8 s, 12 s, 12.2 s, 12.4 s, 12.6 s, 12.8 s, 13 s, 13.2 s, 13.4 s, 13.6 s, 13.8 s, 14 s, etc.); number of substrate layers 1~5 (e.g., 1 layer, 2 layers, 3 layers, 4 layers, 5 layers); substrate exposure time 10~14 s (e.g., 10 s, 10.2 s, 10.4 s, 10.6 s, 10.8 s, 11 s, 12 s, 12.2 s, 12.4 s, 12.6 s, 12.8 s, 13 s, 13.2 s, 13.4 s, 13.6 s, 13.8 s, 14 s, etc.); number of substrate layers 1~5 (e.g., 1 layer, 2 layers, 3 layers, 4 layers, 5 layers); substrate exposure time 10~14 s (e.g., 10 s, 10.2 s, 10.4 s, 10.6 s, 10.8 s, 12 s, 12.2 s, 12.4 s, 12.6 s, 12.8 s, 13 s, 11.2 s, 11.4 s, 11.6 s, 11.8 s, 12 s, 12.2 s, 12.4 s, 12.6 s, 12.8 s, 13 s, 13.2 s, 13.4 s, 13.6 s, 13.8 s, 14 s, etc.; peeling distance 1~5 mm (e.g., can be 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.Peeling speeds: 20-30 mm / min (e.g., 20 mm / min, 21 mm / min, 22 mm / min, 23 mm / min, 24 mm / min, 25 mm / min, 26 mm / min, 27 mm / min, 28 mm / min, 29 mm / min, 30 mm / min, etc.); peel recovery speeds: 80-120 mm / min (e.g., 80 mm / min, 85 mm / min, 90 mm / min, 95 mm / min, 100 mm / min, 105 mm / min, 110 mm / min, 115 mm / min, 120 mm / min, etc.); lifting speeds: 160-200 mm / min (e.g., 160 mm / min, 165 mm / min, 170 mm / min, 175 mm / min, 180 mm / min, 185 mm / min, 190 mm / min, 195 mm / min, 200 mm / min).

[0132] Thirdly, the present invention provides the application of the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold as described in the first aspect in the preparation of materials for repairing alveolar bone, periodontal ligament and cementum.

[0133] Fourthly, the present invention provides a viscoelastically adjustable multilayer photocurable hydrogel scaffold for defect repair, wherein the viscoelastically adjustable multilayer photocurable hydrogel scaffold for defect repair includes the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold as described in the first aspect, or the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold prepared by the preparation method described in the second aspect. Furthermore, the viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair also includes cultured cells.

[0134] As an optional implementation, the cells include any one of periodontal ligament stem cells, osteoblast stem cells, fibroblast stem cells, and cementoblast stem cells, preferably periodontal ligament stem cells.

[0135] Fifthly, the present invention provides a method for preparing the viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair, the method comprising: Cells were cultured to the logarithmic growth phase, then collected, washed, and digested with trypsin. After the reaction was terminated, the cells were collected to obtain a cell suspension. Modified hyaluronic acid, methacrylamide type I collagen, hydroxyapatite, photoinitiator and solvent were mixed to obtain a mineralized hydrogel precursor solution; The modified hyaluronic acid, methacrylamide type I collagen and protoporphyrin 1, photoinitiator and solvent were mixed to obtain a non-mineralized hydrogel precursor solution; The cell suspension was added to the mineralized hydrogel precursor solution and the non-mineralized hydrogel precursor solution, respectively, to obtain a cell-containing mineralized hydrogel precursor solution and a cell-containing non-mineralized hydrogel precursor solution. The alveolar bone repair layer, periodontal ligament repair layer and cementum repair layer were printed sequentially by photopolymerization 3D printing to obtain a framework. The scaffold was subjected to in vitro static culture, allowing the cells to transport nutrients and maintain survival within the gel, resulting in the viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair.

[0136] As an optional implementation, the cells include any one of periodontal ligament stem cells, osteoblast stem cells, fibroblast stem cells, and cementoblast stem cells, preferably periodontal ligament stem cells.

[0137] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0138] Preparation Example 1 This preparation example provides a hydrazine-modified hyaluronic acid (HA-HYN), the structural formula of which is shown in Formula I below: ; Formula I; Where n is 299.

[0139] The hydrazine-modified hyaluronic acid (HA-HYN) described in this embodiment is prepared by the following steps: (a) Dissolve HA (120 kDa) in MES buffer (0.2 M, pH 4.5) to a concentration of 10 mg / mL. Then, add N-hydroxysuccinimide (0.8 equivalents of HAMA dimer), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.8 equivalents), and propyneamine (0.8 equivalents) sequentially to this solution. Adjust the pH to 6, stir at room temperature for 4 h, dialyze against deionized water for 3 days, and freeze-dry to obtain a white powder, denoted as HA-acetylenite.

[0140] (b) HA-acetylene (300 mg) was dissolved in phosphate-buffered saline (PBS, pH 7.4) at 2 wt%, and hydrazine azide (1 equivalent of HA dimer unit) was added; then, nitrogen was bubbled for 30 min; copper sulfate pentahydrate (0.004 equivalent) and sodium ascorbate (0.06 equivalent) were dissolved in deionized water, bubbled with nitrogen, and added to the above HA-acetylene solution; after stirring at room temperature for 1 day, the solution was dialyzed with deionized water for 3 days and lyophilized to obtain the hydrazine-modified hyaluronic acid (HA-HYN).

[0141] Preparation Example 2 This preparation example provides a fatty aldehyde-modified hyaluronic acid (HA-ALD), the structural formula of which is shown in Formula II-1 below:

[0142] Formula II-1; Where a is 299; The fatty aldehyde-modified hyaluronic acid (HA-ALD) described in this embodiment is prepared by the following steps: (a) Dissolve HA (120 kDa) in MES buffer (0.2 M, pH 4.5) to a concentration of 10 mg / mL. Then, add N-hydroxysuccinimide (0.8 equivalents of HAMA dimer), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.8 equivalents), and propyneamine (0.8 equivalents) sequentially to this solution. Adjust the pH to 6, stir at room temperature for 4 h, dialyze against deionized water for 3 days, and lyophilize to obtain a white powder, denoted as HA-acetylenite.

[0143] (b) HA-acetylene (300 mg) was dissolved at 2 wt% in phosphate-buffered saline (PBS, pH 7.4), and then azidoaldehyde (1 equivalent of HA dimer unit) was added separately. Before adding azidoaldehyde to the HA solution, a small amount of DMSO was used to dissolve azidoaldehyde; then nitrogen was bubbled for 30 min. Copper sulfate pentahydrate (0.004 equivalent) and sodium ascorbate (0.06 equivalent) were dissolved in deionized water, bubbled with nitrogen, and added to the above HA-acetylene solution. After stirring at room temperature for 1 day, the solution was dialyzed with deionized water for 3 days and lyophilized to obtain the aliphatic aldehyde-modified hyaluronic acid (HA-ALD).

[0144] Preparation Example 3 This preparation example provides a benzaldehyde-modified hyaluronic acid (HA-BLD), the structural formula of which is shown in Formula II-1 below:

[0145] Formula II-2; Where b is 299; The benzaldehyde-modified hyaluronic acid (HA-ALD) described in this embodiment is prepared by the following steps: (a) Dissolve HA (120 kDa) in MES buffer (0.2 M, pH 4.5) to a concentration of 10 mg / mL. Then, add N-hydroxysuccinimide (0.8 equivalents of HAMA dimer), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.8 equivalents), and propyneamine (0.8 equivalents) sequentially to this solution. Adjust the pH to 6, stir at room temperature for 4 h, dialyze against deionized water for 3 days, and lyophilize to obtain a white powder, denoted as HA-acetylenite.

[0146] (b) HA-acetylene (300 mg) was dissolved at 2 wt% in phosphate-buffered saline (PBS, pH 7.4), and then azidoaldehyde (1 equivalent of HA dimer unit) was added separately. Before adding azidoaldehyde to the HA solution, a small amount of DMSO was used to dissolve azidoaldehyde. Then, nitrogen was bubbled for 30 min. Copper sulfate pentahydrate (0.004 equivalent) and sodium ascorbate (0.06 equivalent) were dissolved in deionized water, bubbled with nitrogen, and added to the above HA-acetylene solution. After stirring at room temperature for 1 day, the solution was dialyzed with deionized water for 3 days and lyophilized to obtain the benzaldehyde-modified hyaluronic acid (HA-BLD).

[0147] To verify the successful construction of hyaluronic acid (HA) side chain modification, Fourier transform infrared spectroscopy (FTIR) was used to characterize the structure of HA and its modified products. Figure 4 shows a comparison of the infrared spectra of unmodified hyaluronic acid, hydrazine-modified hyaluronic acid provided in Preparation Example 1, aliphatic aldehyde-modified hyaluronic acid provided in Preparation Example 2, and benzaldehyde-modified hyaluronic acid provided in Preparation Example 3.

[0148] As shown in Figure 4, the FTIR results show that HA is at 3389.8 cm⁻¹. -1 2887.7 cm -1 1647.8 cm -1 1392.4cm -1 and 1060.1 cm -1 Characteristic absorption peaks are observed at these locations, corresponding to OH stretching vibration, CH stretching vibration, amide I / carboxylate vibration, and COO2, respectively. - Symmetric stretching vibrations and COC stretching vibrations in the polysaccharide backbone. Compared to HA, benzaldehyde-modified hyaluronic acid (HA-BLD) exhibits higher stretching vibrations in the 1500–1600 cm⁻¹ range. -1 The change in absorption peaks in the region is attributed to aromatic ring skeletal vibrations, indicating the successful introduction of the benzaldehyde group. Polyethylene glycol aldehyde-modified hyaluronic acid (HA-ALD) shows absorption peaks in the 1000–1150 cm⁻¹ range. -1The enhanced absorption in this region corresponds to the characteristic vibrations of the COC ether bond in the PEG chain, indicating that PEG-aldehyde was successfully grafted onto the HA molecular chain. For hydrazine-modified hyaluronic acid (HA-HYN), the absorption at approximately 1551 cm⁻¹... -1 A new absorption peak appears at 1739 cm⁻¹, corresponding to the amide II (NH bending) vibration, and also at 1739 cm⁻¹. -1 A distinct carbonyl absorption peak was observed, indicating that the hydrazide structure was successfully introduced into the HA molecular backbone. In summary, the FTIR results confirm that benzaldehyde, PEG-aldehyde, and hydrazide groups have all been successfully grafted onto the HA backbone.

[0149] Example 1 This embodiment provides a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, comprising, from the inside out, a cementum repair layer, a periodontal ligament repair layer, and an alveolar bone repair layer; the cementum repair layer and the alveolar bone repair layer include a mineralized hydrogel layer; wherein, the raw materials for preparing the mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and hydroxyapatite; the alveolar bone repair layer includes a non-mineralized hydrogel layer; wherein, the raw materials for preparing the non-mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and fibrin I. Figure 5 As shown, a typical example of the three-layer hydrogel dimensions is as follows: the inner cementum repair layer is 300 μm wide and 500 μm thick; the middle periodontal ligament repair layer surrounds the cementum layer and is 200 μm thick; the outer alveolar bone repair layer surrounds the two parts of the hydrogel to form a cubic structure. The final overall dimensions of the three-layer hydrogel are 3100 μm wide and 1500 μm high.

[0150] The mineralized hydrogel layer comprises: a first cross-linked network formed by methacrylamide type I collagen, a second cross-linked network formed by cross-linking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and hydroxyapatite doped within the first and second cross-linked networks; the non-mineralized hydrogel layer comprises: a first cross-linked network formed by methacrylamide type I collagen, a second cross-linked network formed by cross-linking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and fibrin 1 loaded within the first and second cross-linked networks.

[0151] The modified hyaluronic acid is HA-HYN provided in Preparation Example 1, with a molar ratio of hydrazine group to aldehyde group of 1:1, and HA-BLD provided in Preparation Example 3.

[0152] The biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold described in this embodiment is prepared by the following steps: (1) Preparation of mineralized hydrogel precursor solution: The mineralized hydrogel precursor solution was prepared from methacrylamide type I collagen (Col I-MA), modified hyaluronic acid, and nano-hydroxyapatite (nHAp). The amounts of each component are shown in the table below. The hydrogel was photocrosslinked using lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP) as the photoinitiator. Nano-hydroxyapatite was dissolved in PBS, ultrasonically dispersed, and then added to the gel system. The mineralized hydrogel precursor solution comprised the following components by mass percentage:

[0153] (2) Preparation of non-mineralized hydrogel precursor solution: The non-mineralized hydrogel precursor solution was prepared from methacrylamide type I collagen (Col I-MA), modified hyaluronic acid, and fibrin 1 (FBN1). The amounts of each component are shown in the table below. The hydrogel was photocrosslinked using lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP) as the photoinitiator. Fibrin 1 (FBN1) was dissolved in PBS, ultrasonically dispersed, and then added to the gel system. The non-mineralized hydrogel precursor solution comprised the following components by mass percentage:

[0154] (3) 3D model construction: This model is constructed based on the multi-layered soft and hard tissue structure of the periodontal complex, using 3D printing to form a three-layer hydrogel scaffold with spatial partitioning and mechanical gradients. The model has an overall strip-shaped embedded structure, with the alveolar bone layer, periodontal ligament layer, and cementum layer arranged longitudinally to simulate the tissue composition and physiological layout of the real periodontal complex. The scaffold is anatomically mapped to the periodontal complex defect model, and the repair area is designed with differentiated composition and mechanical properties according to different tissue types: the scaffold consists of three layers of hydrogel: mineralized-unmineralized-mineralized, arranged from the inside out: outer layer (alveolar bone repair layer): mineralized hydrogel, viscoelasticity matching bone tissue; middle layer (periodontal ligament repair layer): unmineralized hydrogel, viscoelasticity matching periodontal ligament; inner layer (cementum repair layer): mineralized hydrogel, used for targeted cementum regeneration.

[0155] Projection-based photopolymerization 3D printing: This invention employs projection-based light-curing (DLP) 3D printing technology to construct a three-layer viscoelastic gradient structure of a biomimetic periodontal complex hydrogel scaffold. The printing system consists of an ultraviolet LED light source (405 nm), a digital micromirror array (MMD) imaging module, a resin tank, and a lifting platform. The hydrogel precursor is placed in the resin tank and cured layer by layer using a digital light field. To achieve spatial partitioning and viscoelastic gradient control of different hydrogel layers, different hydrogel fluid formulations with varying amounts of light-blocking agents were selected for printing (Material 1: mineralized hydrogel; Material 2: non-mineralized hydrogel). A regional exposure strategy was used to sequentially print the alveolar bone layer, periodontal ligament layer, and cementum layer, as shown below. Figure 6A and Figure 6B As shown. The light-blocking agent is lemon yellow.

[0156] The specific process parameters for projection-based photopolymerization 3D printing are shown in Table 1. Table 1

[0157] The printing process is as follows: Hydrogel precursor solutions with different amounts of light-blocking agent were pre-prepared and used in the printing steps of the mineralized and non-mineralized layers, respectively. For example... Figure 7 As shown, the hydrogel printing process employs a 1-2-1 material alternation pattern, ultimately achieving the printing of a three-dimensional hydrogel structure. This invention can complete the overall construction of the three-layer scaffold in a single printing process, avoiding interface discontinuities and insufficient functional coupling caused by traditional assembly-based manufacturing, and ensuring the continuous presentation of the viscoelastic gradient in the spatial dimension.

[0158] Example 2 This embodiment provides a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, which differs from Embodiment 1 only in that, in step (1), a mineralized hydrogel precursor solution is prepared: the mineralized hydrogel precursor solution includes the following components by mass percentage:

[0159] The other steps are the same as in Example 1.

[0160] Example 3 This embodiment provides a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, which differs from Embodiment 1 only in that, in step (1), a mineralized hydrogel precursor solution is prepared: the mineralized hydrogel precursor solution includes the following components by mass percentage:

[0161] The other steps are the same as in Example 1.

[0162] Example 4 This embodiment provides a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, which differs from Embodiment 1 only in that, in step (2), a non-mineralized hydrogel precursor solution is prepared: the non-mineralized hydrogel precursor solution includes the following components by mass percentage:

[0163] The other steps are the same as in Example 1.

[0164] Example 5 This embodiment provides a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, which differs from Embodiment 1 only in that, in step (2), a non-mineralized hydrogel precursor solution is prepared: the non-mineralized hydrogel precursor solution includes the following components by mass percentage:

[0165] The other steps are the same as in Example 1.

[0166] Test Example 1 Rheological testing Test samples: mineralized hydrogel precursor solution and non-mineralized hydrogel precursor solution provided in Example 1; mineralized hydrogel precursor solution provided in Examples 2 and 3; non-mineralized hydrogel precursor solution provided in Examples 4 and 5.

[0167] Preparation of hydrogel samples: The above-mentioned hydrogel precursor solution samples were subjected to irradiation crosslinking; crosslinking was then performed by ultraviolet irradiation (25 mW / cm). 2 After 40 nm, the corresponding group of hydrogel samples were obtained.

[0168] Rheological testing methods: Hydrogel precursor solutions were prepared according to the composition table and divided into high / medium / low loss modulus groups (n=3). The precursor solutions were uniformly dropped into cylindrical PDMS molds, and after photocrosslinking, hydrogel samples (8 mm in diameter and 2 mm in thickness) were obtained for later use. Each sample was loaded into a rheometer to analyze its viscoelasticity. First, an amplitude scan was performed within a strain range of 1 rad / s to determine the linear viscoelastic region from 0.1% to 10% strain. Then, a frequency scan was performed from 0.1% to 10 Hz at a constant strain of 1% to obtain the storage modulus (G') and loss modulus (G'') of the samples at different frequencies. Finally, stress relaxation experiments were performed at a strain of 10%.

[0169] The specific test results are as follows: Figures 8A-8D As shown: like Figures 8A-8DAs shown, the frequency scanning results of mineralized hydrogel (MG) and non-mineralized hydrogel (NMG) indicate that G' has a weak frequency dependence in the range of 0.1–10 Hz and remains relatively stable overall, suggesting that the constructed hydrogel network structure has good structural stability under dynamic loading conditions. Specifically, at a frequency of 1 Hz, the storage modulus G' of the mineralized group remained at approximately 2 kPa, with loss moduli of 267.8±60.7 Pa, 405.3±84.7 Pa, and 567.3±39.0 Pa, respectively; while the storage modulus G' of the non-mineralized group remained at approximately 1.2 kPa, with loss moduli of 144.2±35.0 Pa, 198.3±8.0 Pa, and 271.8±30.2 Pa, respectively. In the mineralized group, the loss tangent tanδ (G' / G''), which measures the ratio of viscosity to elasticity, is 0.12, 0.19, and 0.28, respectively; in the unmineralized group, the loss tangent tanδ is 0.10, 0.17, and 0.22, respectively. Both the mineralized and unmineralized groups achieved gradient control of viscoelasticity while maintaining a basically consistent elasticity.

[0170] It can be seen that both mineralized hydrogel precursor solutions crosslinked by ultraviolet light irradiation to form mineralized hydrogels and non-mineralized hydrogel precursor solutions crosslinked by ultraviolet light irradiation to form non-mineralized hydrogels can have their viscoelasticity controlled and adjustable (the loss tangent tanδ can be adjusted between 0.1 and 0.3).

[0171] Application Example 1 This application example provides a viscoelastically tunable multilayer light-cured hydrogel scaffold for defect repair, comprising, from the inside out, a cementum repair layer, a periodontal ligament repair layer, and an alveolar bone repair layer; the cementum repair layer and the alveolar bone repair layer include mineralized hydrogel layers; the scaffold consists of a three-layer structure composed of mineralized hydrogel, non-mineralized hydrogel, and mineralized hydrogel, achieving synchronous biomimetic construction of viscoelastic gradient and spatial structure during a single molding process. This scaffold is used to induce tri-directional differentiation of periodontal ligament stem cells (PDLSCs) in osteogenic, fibrogenic, and cementogenic directions, achieving the overall reconstruction of alveolar bone, periodontal ligament, and cementum.

[0172] The mineralized hydrogel layer is prepared from modified hyaluronic acid, methacrylamide type I collagen, and hydroxyapatite; the alveolar bone repair layer includes a non-mineralized hydrogel layer; wherein the non-mineralized hydrogel layer is prepared from modified hyaluronic acid, methacrylamide type I collagen, and fibrin I. Figure 5As shown, a typical example of the three-layer hydrogel dimensions is as follows: the inner cementum repair layer is 300 μm wide and 500 μm thick; the middle periodontal ligament repair layer surrounds the cementum layer and is 200 μm thick; the outer alveolar bone repair layer surrounds the two parts of the hydrogel to form a cubic structure. The final overall dimensions of the three-layer hydrogel are 3100 μm wide and 1500 μm high. Furthermore, the viscoelastically tunable multilayer light-cured hydrogel scaffold for defect repair also includes cultured periodontal ligament stem cells.

[0173] The mineralized hydrogel layer comprises: a first cross-linked network formed by methacrylamide type I collagen, a second cross-linked network formed by cross-linking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and hydroxyapatite doped within the first and second cross-linked networks; the non-mineralized hydrogel layer comprises: a first cross-linked network formed by methacrylamide type I collagen, a second cross-linked network formed by cross-linking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and fibrin 1 loaded within the first and second cross-linked networks.

[0174] Among them, the mineralized group uses the low loss modulus formula, and the non-mineralized group uses the high loss modulus formula (preliminary experimental verification results show that this formula has the best repair effect). That is, the mineralized group is 3% modified hyaluronic acid (HA-BLD and HA-HYN molar ratio is 1:1), and the non-mineralized group is 3% modified hyaluronic acid (HA-ALD and HA-HYN molar ratio is 1:1).

[0175] The viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair described in this embodiment is prepared by the following steps: (1) Preparation of periodontal ligament stem cell (PDLSC) suspension: Periodontal ligament stem cells were routinely cultured to the logarithmic growth phase and then collected. The cells were washed with PBS and digested with 0.25% trypsin. After terminating the reaction, the cells were collected by centrifugation to obtain a PDLSC suspension for scaffold construction.

[0176] (2) Preparation of cell-mineralized hydrogel precursor solution: The mineralized hydrogel precursor solution was prepared from methacrylamide type I collagen (Col I-MA), modified hyaluronic acid, and nano-hydroxyapatite (nHAp). The amounts of each component are shown in the table below. The hydrogel was photocrosslinked using lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP) as the photoinitiator. Nano-hydroxyapatite was dissolved in PBS, ultrasonically dispersed, and then added to the gel system. The mineralized hydrogel precursor solution comprised the following components by mass percentage:

[0177] Subsequently, the PDLSCs suspension obtained in step (1) was added to the corresponding precursor solution and mixed thoroughly to ensure that the cells were evenly distributed in the gel fluid, with a cell encapsulation volume of 5 × 10⁻⁶. 6 / mL precursor solution.

[0178] (3) Preparation of cell-containing non-mineralized hydrogel precursor solution: The non-mineralized hydrogel precursor solution was prepared from methacrylamide type I collagen (Col I-MA), modified hyaluronic acid, and fibrin 1 (FBN1). The amounts of each component are shown in the table below. The hydrogel was photocrosslinked using lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP) as the photoinitiator. Fibrin 1 (FBN1) was dissolved in PBS, ultrasonically dispersed, and then added to the gel system. The non-mineralized hydrogel precursor solution comprised the following components by mass percentage:

[0179] Subsequently, the PDLSCs suspension obtained in step (1) was added to the corresponding precursor solution and mixed thoroughly to ensure that the cells were evenly distributed in the gel fluid, with a cell encapsulation volume of 5 × 10⁻⁶. 6 / mL precursor solution.

[0180] (4) 3D model construction: The three precursor solutions are loaded into the printing cavity of the DLP photopolymerization 3D printing system and printed sequentially in a structured manner through a regional exposure strategy: the entire process is solidified layer by layer under 405 nm ultraviolet light and the temperature is controlled at 37 degrees Celsius, ultimately forming a three-layer scaffold with physiologically differentiated structures, spatial mechanical gradients and cell partition distribution, achieving one-time whole printing without interface splicing.

[0181] The specific process parameters for projection-based photopolymerization 3D printing are shown in Table 2. Table 2

[0182] The light-blocking agent is lemon yellow.

[0183] (5) Scaffold in vitro culture: Immediately after printing, the scaffold was transferred into a 6-well plate containing complete culture medium for in vitro static culture. The cells were maintained at 37°C and 5% CO2 to ensure nutrient transport and cell survival within the gel, in preparation for subsequent in vivo scaffold implantation.

[0184] (6) Stent application After establishing periodontal complex defects in experimental animal models or surgically exposing the defect area in clinical subjects, pre-cultured 3D-printed scaffolds are precisely implanted into the defect site under aseptic conditions to achieve in vivo repair of periodontal complex defects.

[0185] After establishing periodontal complex defects in experimental animal models or surgically exposing the defect area in clinical subjects, pre-cultured 3D-printed scaffolds are precisely implanted into the defect site under aseptic conditions to achieve in vivo repair of periodontal complex defects. (Note: For clinical applications, in cases of irregular defects, CBCT scans can be performed beforehand to generate a 3D reconstruction model of the periodontal defect area. Based on the reconstruction model, the size and structure of each scaffold layer can be customized. After 3D printing, the scaffold can better fit the defect area, promoting scaffold repair of the defect.) like Figure 9 As shown, the in vivo application and effect of biomimetic hydrogel: Periodontal complex defects were constructed in a rat model, and then 3D-printed biomimetic scaffolds were implanted in the defect area. Micro-CT was taken 3 months later to observe the defect repair effect. The results showed that the hydrogel repair group could repair the periodontal complex defects in rats well.

[0186] As can be seen, this invention constructs a three-layer structure of mineralized-unmineralized-mineralized within a single scaffold system, corresponding to the physiological zones of alveolar bone, periodontal ligament, and cementum, achieving spatial correspondence among the three tissue types and simultaneous repair of soft and hard tissues. Simultaneously, a reversible hydrazone bond regulation strategy achieves a controllable viscoelasticity adjustment mechanism based on the cross-linked network. While regulating viscoelasticity, the initial elastic modulus, porosity, degradation rate, and other basic physicochemical properties of the material are not altered, allowing the viscoelasticity of each scaffold layer to match the mechanical properties of different tissues, thus achieving controllable induction of the three-way differentiation of periodontal ligament stem cells. Specifically, nano-hydroxyapatite (nHAp) is added to the mineralized layer, and fibrin 1 is added to the unmineralized layer, forming a differentiated biochemical microenvironment to achieve bidirectional biomimetic induction of mineralized and fibrous tissues. Furthermore, projection-based light-curing (DLP) 3D printing technology is used to form the three-layer structure in one step, achieving a continuous spatial transition and avoiding interface fractures and insufficient functional coupling caused by spliced ​​scaffolds.

[0187] In summary, the biomimetic periodontal composite multilayer hydrogel tissue engineering scaffold provided by this invention has at least the following advantages over existing scaffolds: (1) The previous scaffolds were formed under different conditions, including temperature-controlled molding and extrusion molding. The hydrogel scaffold in this invention has photocuring properties, so it can be adapted to projection-type photocuring bio-3D printing for high-resolution molding. Especially for tissues with fine structures and complex layers, such as periodontal complexes, it is easier to make biomimetic manufacturing of physiological structures.

[0188] (2) Compared with existing bionic periodontal tissue engineering scaffolds, which are difficult to simultaneously take into account the interface between soft and hard tissues, this invention achieves synchronous regeneration of the corresponding physiological zones of alveolar bone, periodontal ligament and cementum by constructing a mineralized-non-mineralized-mineralized three-layer structure in a single scaffold system. This fundamentally overcomes the limitation of traditional materials that can only repair locally and cannot achieve overall reconstruction of the periodontal complex. (3) Existing materials generally focus on elastic regulation, making it difficult to provide stem cells with dynamic mechanical signals that are consistent with physiology. This invention uses a reversible hydrazone bond regulation strategy to achieve independent regulation of viscoelasticity in each layer, enabling the scaffold to continuously transmit tissue-specific mechanical cues to stem cells, while not changing the material's porosity, degradation rate, and other basic physicochemical properties. (4) Traditional multilayer scaffolds have poor interlayer continuity, which can easily lead to mechanical property discontinuity and uneven cell distribution. This invention utilizes projection-based photopolymerization 3D printing technology to form a three-layer structure in one step, achieving spatially continuous viscoelasticity and biochemical microenvironment gradient, which significantly improves structural stability, cell viability and the orderliness of regenerated tissues. Therefore, this invention not only achieves simultaneous repair of multiple soft and hard tissues in the periodontal complex, but also provides a biomechanical solution that enables zoned cell fate regulation, with an overall regeneration effect significantly superior to existing technologies. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold, characterized in that, It includes, from the inside out, the cementum repair layer, the periodontal ligament repair layer, and the alveolar bone repair layer; The cementum repair layer and the alveolar bone repair layer include a mineralized hydrogel layer; wherein, the raw materials for preparing the mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and hydroxyapatite; The alveolar bone repair layer includes a non-mineralized hydrogel layer; wherein, the raw materials for preparing the non-mineralized hydrogel layer include modified hyaluronic acid, methacrylamide type I collagen, and fibrin 1.

2. The biomimetic periodontal composite multilayer hydrogel tissue engineering scaffold according to claim 1, characterized in that, The modified hyaluronic acid includes hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid; Preferably, the mineralized hydrogel layer comprises: a first crosslinking network formed by methacrylamide type I collagen, a second crosslinking network formed by crosslinking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and hydroxyapatite doped within the first and second crosslinking networks; Preferably, the non-mineralized hydrogel layer comprises: a first cross-linked network formed by methacrylamide type I collagen, a second cross-linked network formed by cross-linking hydrazine-modified hyaluronic acid and aldehyde-modified hyaluronic acid, and protofibrin 1 loaded within the first and second cross-linked networks; Preferably, the structural formula of the hydrazine-modified hyaluronic acid is shown in Formula I below: ； Formula I; Where n is an integer between 249 and 998; Preferably, the aldehyde-modified hyaluronic acid includes fatty aldehyde-modified hyaluronic acid and / or benzaldehyde-modified hyaluronic acid; Preferably, the structural formula of the fatty aldehyde-modified hyaluronic acid is shown in Formula II-1 below: Formula II-1; Where a is an integer between 249 and 998; Preferably, the structural formula of the benzaldehyde-modified hyaluronic acid is shown in Formula II-2 below: Formula II-2; Where b is an integer between 249 and 998; Preferably, in the mineralized hydrogel layer, the mass ratio of the modified hyaluronic acid, methacrylamide type I collagen and hydroxyapatite is (14~16):(1~3):(24~26); Preferably, in the non-mineralized hydrogel layer, the mass ratio of the modified hyaluronic acid, methacrylamide type I collagen and fibrin 1 is (28~32):(3~5):(0.2~2).

3. The biomimetic periodontal composite multilayer hydrogel tissue engineering scaffold according to claim 1, characterized in that, The periodontal ligament repair layer surrounds the cementum repair layer and forms a first cubic structure; the alveolar bone repair layer surrounds the first cubic structure and forms a second cubic structure. Preferably, at least two of the first cube structures are disposed within the second cube structure.

4. A method for preparing a biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold according to any one of claims 1 to 3, characterized in that, The preparation method includes: Modified hyaluronic acid, methacrylamide type I collagen, hydroxyapatite, photoinitiator and solvent were mixed to obtain a mineralized hydrogel precursor solution; The modified hyaluronic acid, methacrylamide type I collagen and fibrin 1, photoinitiator and solvent were mixed to obtain a non-mineralized hydrogel precursor solution; The alveolar bone repair layer, periodontal ligament repair layer, and cementum repair layer are sequentially printed using photopolymerization 3D printing to obtain the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold.

5. The method for preparing the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold according to claim 4, characterized in that, The preparation method of the modified hyaluronic acid includes the following steps: Hyaluronic acid and propyne amine undergo a condensation reaction to yield alkynylated hyaluronic acid; Alkyne-modified hyaluronic acid and an azide are subjected to a click chemical reaction to obtain the modified hyaluronic acid; wherein the azide includes hydrazine azidohydrazine and / or azidoaldehyde; Preferably, the molecular weight of the hyaluronic acid is 100~400 kDa; Preferably, the condensation reaction is carried out in the presence of a composite condensing agent; wherein the composite condensing agent comprises N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Preferably, the molar ratio of hyaluronic acid, propyneamine, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:(0.8~1):(0.8~1):(0.8~1); Preferably, the condensation reaction is carried out in a solvent; wherein the solvent includes MES buffer. Preferably, the pH of the condensation reaction is 5.5-6.5, the temperature of the condensation reaction is 20-30°C, and the time of the condensation reaction is 3-5 h; Preferably, the hydrazine azide is N-(3-azidopropyl)-2-hydrazinoacetamide; Preferably, the azide aldehyde comprises azide-polyethylene glycol-acetaldehyde and / or N-(2-azidoethyl)-4-formylbenzamide; Preferably, the click chemical reaction is carried out in the presence of a catalyst and a reducing agent; wherein the catalyst comprises copper sulfate; and the reducing agent comprises sodium ascorbate. Preferably, the molar ratio of the alkynylated hyaluronic acid, the azide catalyst, and the reducing agent is 1:(0.8~1.2):(0.001~0.01):(0.01~0.1); Preferably, the click chemistry reaction is carried out in a solvent; wherein the solvent includes PBS buffer. Preferably, the temperature of the click chemical reaction is 20~30℃, and the time of the click chemical reaction is 18~30 h.

6. The method for preparing the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold according to claim 4, characterized in that, The mineralized hydrogel precursor solution comprises, by mass percentage: 1-5% modified hyaluronic acid, 2-5% methacrylamide type I collagen, 1-10% hydroxyapatite, 0.1-0.5% photoinitiator, with the remainder being solvent; Preferably, the non-mineralized hydrogel precursor solution comprises, by mass percentage: 1-5% modified hyaluronic acid, 2-5% methacrylamide type I collagen, 0.1-5% fibrin, 0.1-0.5% photoinitiator, with the remainder being solvent; Preferably, the photoinitiator comprises lithium phenyl (2,4,6-trimethylbenzoyl)phosphate; Preferably, the solvent includes PBS buffer.

7. The method for preparing the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold according to claim 4, characterized in that, The steps of the photopolymer 3D printing include: Add 0.2~0.3 wt% of light blocker to the mineralized hydrogel precursor solution to obtain material 1; add 0.005~0.02 wt% of light blocker to the non-mineralized hydrogel precursor solution to obtain material 2; the printing process adopts an alternating mode of material 1-material 2-material 1 to finally achieve the printing of a three-dimensional structure of three-layer hydrogel, thus obtaining the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold; Preferably, the light-blocking agent includes food dyes and / or biocompatible dyes, preferably tartrazine and / or curcumin; Preferably, the parameters for photopolymerization 3D printing of material 1 include: layer height 50~200 μm; light intensity 200~220 PWM; exposure time 7~9 s; number of base layers 1~5; base layer exposure time 7~9 s; peel distance 1~5 mm; peel speed 20~30 mm / min; peel recovery speed 80~120 mm / min; lifting speed 160~200 mm / min; Preferably, the parameters of the material 2 for photopolymerization 3D printing include: layer height 50~200 μm; light intensity 260~300 PWM; exposure time 10~14 s; number of base layers 1~5; base layer exposure time 10~14 s; peel distance 1~5 mm; peel speed 20~30 mm / min; peel recovery speed 80~120 mm / min; lifting speed 160~200 mm / min.

8. The use of a biomimetic periodontal composite multilayer hydrogel tissue engineering scaffold according to any one of claims 1 to 3 in the preparation of materials for repairing alveolar bone, periodontal ligament and cementum.

9. A viscoelastically adjustable multilayer photocurable hydrogel scaffold for defect repair, characterized in that, The viscoelastically adjustable multilayer photocurable hydrogel scaffold for defect repair includes the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold as described in any one of claims 1 to 3, or the biomimetic periodontal complex multilayer hydrogel tissue engineering scaffold prepared by the preparation method described in any one of claims 4 to 7. Furthermore, the viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair also includes cultured cells; And / or, the cells include any one of periodontal ligament stem cells, osteoblast stem cells, fibroblast stem cells, and cementoblast stem cells, preferably periodontal ligament stem cells.

10. A method for preparing a viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair according to claim 9, characterized in that, The preparation method includes: Cells were cultured to the logarithmic growth phase, then collected, washed, and digested with trypsin. After the reaction was terminated, the cells were collected to obtain a cell suspension. Modified hyaluronic acid, methacrylamide type I collagen, hydroxyapatite, photoinitiator and solvent were mixed to obtain a mineralized hydrogel precursor solution; The modified hyaluronic acid, methacrylamide type I collagen and fibrin 1, photoinitiator and solvent were mixed to obtain a non-mineralized hydrogel precursor solution; The cell suspension was added to the mineralized hydrogel precursor solution and the non-mineralized hydrogel precursor solution, respectively, to obtain a cell-containing mineralized hydrogel precursor solution and a cell-containing non-mineralized hydrogel precursor solution. The alveolar bone repair layer, periodontal ligament repair layer and cementum repair layer were printed sequentially by photopolymerization 3D printing to obtain a framework. The scaffold was subjected to in vitro static culture, allowing the cells to transport nutrients and maintain survival within the gel, resulting in the viscoelastically tunable multilayer photocurable hydrogel scaffold for defect repair. And / or, the cells include any one of periodontal ligament stem cells, osteoblast stem cells, fibroblast stem cells, and cementoblast stem cells, preferably periodontal ligament stem cells.

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