Bionic full-thickness gingival flap for implanting and repairing soft and hard tissue defects and preparation method of bionic full-thickness gingival flap

Abstract

The invention discloses a bionic full-thickness gingival flap for implanting and repairing soft and hard tissue defects and a preparation method of the bionic full-thickness gingival flap, and relates to the technical field of biological materials. The functions of the natural gingival valve are simulated through layered design, the antibacterial peptide-collagen hydrogel serves as the bionic gingival valve epithelium to block bacteria, the porous collagen hydrogel serves as the bionic gingival valve intrinsic layer to provide mechanical support, and the compact collagen hydrogel serves as the bionic gingival valve periosteum layer to maintain the osteogenesis space. The material utilizes a honeycomb structure to promote epithelial regeneration, a loose network guides fibroblast migration, a zinc-rich microenvironment enhances osteogenic differentiation, and synergistic repair of soft and hard tissues is realized. By adopting the semi-permeable activation fibroblast paracrine effect of the collagen hydrogel, the limitation of traditional layer-by-layer repair is broken through, the problems of bone grafting area infection, bone meal displacement, space maintenance and the like are effectively solved, secondary trauma of autotransplantation is avoided, and an innovative solution is provided for alveolar bone regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects and a preparation method thereof. Background Art

[0002] Implant restoration has become one of the mainstream options for the treatment of edentulous / missing teeth, and its good prognosis depends on sufficient bone volume in the implant area. However, due to the negative impact of diseases and aging changes on the alveolar bone, more than 60% of patients need bone augmentation to meet the bone volume requirements for dental implants. As the most widely used bone augmentation method, guided bone regeneration completes the treatment by implanting bone replacement materials in the target area, covering and fixing the barrier membrane, and finally suturing the gums to seal. However, if there is a lack of immediate gingival closure after bone grafting, the homeostasis of the bone replacement graft will be difficult to maintain, and alveolar bone regeneration will be at risk of bacterial infection in the surgical area, loss of bone implants, and invasion of fibrous tissue.

[0003] To solve the problem of insufficient gums, traditional methods often use autologous free gingival flap transplantation, but this method has obvious limitations: 1) The secondary trauma is large, and the sampling process causes additional damage to the donor area; 2) The operation is complicated, and the gingival flap shape needs to be trimmed and matched with the defect area, and a barrier membrane still needs to be used in combination; 3) The success rate of transplantation is affected by the transplantation area and infection, and the survival rate is low when the transplantation is carried out on a large area.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to provide a biomimetic full-thickness gingival flap for implant restoration of soft and hard tissue defects and a method for its preparation. The biomimetic full-thickness gingival flap is designed to address clinical issues such as bacterial infection, bone powder loss, and loss of bone formation space caused by insufficient gingival seal in the bone grafting area. Through a layered biomimetic design, it effectively replaces the autologous gingival flap, rapidly seals and protects the bone grafting area, and broadens the indications for alveolar bone grafting. Furthermore, the biomimetic full-thickness gingival flap mimics the basic functions of the epithelial, endosteal, and periosteum layers of the autologous gingival flap. It also serves as a biological template for soft tissue restoration in the bone grafting area, guiding the rapid regeneration of defective tissue and adapting to the soft and hard tissue repair process in the area to form a multi-tissue collaborative reconstruction repair concept.

[0006] The present invention is achieved in that: The present invention provides a biomimetic full-thickness gingival flap for implant restoration of soft and hard tissue defects. The flap comprises three layers: a biomimetic gingival flap epithelium, a biomimetic gingival flap lamina propria, and a biomimetic gingival flap periosteum. The biomimetic gingival flap epithelium comprises collagen hydrogel and antimicrobial peptides, the biomimetic gingival flap lamina propria comprises porous collagen hydrogel, and the biomimetic gingival flap periosteum comprises dense collagen hydrogel and zinc-nuclear zeolite imidazolate framework-8 (ZIF-8).

[0007] In some preferred embodiments, the collagen hydrogel of the biomimetic gingival flap epithelium has a honeycomb microstructure on its surface, and / or the collagen hydrogel of the biomimetic gingival flap epithelium is a methacrylated polypeptide hydrogel.

[0008] In some preferred embodiments, the bionic gingival flap lamina propria has a loose porous structure.

[0009] The present invention also provides a method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects, comprising the following steps: Gelatin is reacted with glycidyl methacrylate to form low-substitution light-cured gelatin (Gel-L), which is then mixed with light-cured polylysine (PLMA) and a photoinitiator is added to form the biomimetic gingival epithelial layer (Gel-EPL) material. Gelatin is reacted with glycidyl methacrylate to form low-substitution light-cured gelatin (Gel-L), which is then mixed with polyethylene oxide (PEO) solution and a photoinitiator to form the biomimetic gingival flap lamina propria (ProGel) raw material. The reaction time of gelatin and glycidyl methacrylate was extended to generate high-substitution light-cured gelatin (Gel-H), and zinc-nuclear zeolite imidazolate framework material-8 was dispersed in the high-substitution light-cured gelatin to form the raw material of biomimetic periosteum layer (Gel-Z). An elastic negative mold with a honeycomb-shaped micro-convex structure was prepared by an indirect printing method. The raw material of the bionic gingival flap epithelium was poured into the model and then frozen into shape. The bionic gingival flap propria material was then covered on the surface of the bionic gingival flap epithelium and then frozen into shape. The bionic periosteum material was covered on the surface of the bionic gingival flap epithelium and the bionic gingival flap propria. The whole was demolded by photocuring and soaked to obtain a complete bionic full-thickness gingival flap.

[0010] In some preferred embodiments, the method for preparing the low-substitution light-cured gelatin includes the following steps: dissolving the gelatin under stirring, slowly adding glycidyl methacrylate (GMA) and sodium hydroxide solution until the reaction system is alkaline, stirring the reaction under dark conditions and then dialyzing, and after the dialysis is completed, centrifuging and freeze-drying to prepare the low-substitution light-cured gelatin.

[0011] In some preferred embodiments, the mixing mass ratio of the low-substitution photocurable gelatin and the photocurable polylysine is (2-5):1.

[0012] In some preferred embodiments, the photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0013] In some preferred embodiments, the zinc nucleate zeolite imidazolate framework material-8 is synthesized by a hydrothermal method.

[0014] In some preferred embodiments, the indirect printing method for preparing an elastic negative mold having a honeycomb-shaped micro-protrusion structure includes the following steps: designing a template having a honeycomb-shaped microstructure in a modeling software, exporting the template to an ultra-high-precision light-curing printer to obtain a resin positive film, and then using polydimethylsiloxane (PDMS) to replicate an elastic negative mold having a honeycomb-shaped micro-protrusion structure.

[0015] In some preferred embodiments, the bionic full-thickness gingival flap needs to be soaked in phosphate buffered saline (PBS) for at least 24 hours after being formed.

[0016] The present invention has the following beneficial effects: The bionic full-thickness gingival flap proposed in this invention achieves a multi-dimensional replacement of autologous gingival flap functions through a layered biomimetic strategy, effectively resolving the clinical difficulties encountered in the bone grafting area. It not only mimics the basic functions of the epithelial, lamina propria, and periosteum layers of the autologous gingival flap, but also simulates the regenerative logic of tissue through its microscopic composition and structure. The ultramicroscopic honeycomb structure of the bionic epithelial layer not only accelerates epithelial cell association but also serves as a template to guide cells to form a three-dimensional epithelial spike structure. The loose bionic lamina propria provides pores that match the size of connective tissue, accommodating the rapid migration of fibroblasts. The bionic periosteum layer activates the osteogenic differentiation of stem cells by creating a zinc-rich environment, promoting subperiosteal osteogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 A bionic strategy diagram and preparation flow chart of a bionic full-thickness gingival flap in an embodiment of the present application; Figure 2 Schematic diagram of the honeycomb microstructure of the bionic full-thickness gingival flap epithelium in the embodiment of the present application; Figure 3 This is a diagram of the antibacterial experiment on the bionic full-thickness gingival flap epithelium in the embodiment of this application; Figure 4 This is a diagram of an in vitro epithelialization experiment of a bionic full-thickness gingival flap in an embodiment of this application; Figure 5 This is a diagram showing the in vitro fibroblast and osteogenic ability experiments of the bionic full-thickness gingival flap in the examples of this application. DETAILED DESCRIPTION

[0019] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0020] The following is a detailed description of a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects and its preparation method.

[0021] Firstly, the present application proposes a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects, which comprises the following three layers: a bionic gingival flap epithelium, a bionic gingival flap lamina propria, and a bionic gingival flap periosteum. The bionic strategy diagram is shown in FIG. Figure 1 -As shown in A.

[0022] In some embodiments, the biomimetic gingival flap epithelial layer comprises collagen hydrogel and antibacterial polypeptide; the gel surface of the biomimetic gingival flap epithelial layer has a honeycomb microstructure.

[0023] Among them, the biomimetic gingival flap epithelium can isolate bacteria and, as a biological substitute for the gingival epithelium, resist external stimuli. Collagen hydrogels, such as methacryloylated polypeptide hydrogel (GelMA), have excellent plasticity and compatibility. They can flow and adapt to the defect shape and, through light curing, protect the wound surface and block stimuli, making them ideal biomimetic epithelial substrates. Antimicrobial peptides are safe and have low drug resistance. As antimicrobial ingredients in the biomimetic gingival flap epithelium, they are more effective than common antibiotics. Therefore, cross-linking and grafting antimicrobial peptides into the methacryloylated polypeptide hydrogel network can impart antimicrobial capabilities to the biomimetic gingival flap epithelium.

[0024] Furthermore, the biomimetic gingival flap epithelium can serve as a biological template for tissue regeneration, guiding the rapid migration of gingival epithelial cells and recreating the epithelial spike structure. Cell-scale directional microstructures (such as microgrooves) can accelerate epithelial cell migration and growth. Therefore, the honeycomb-like microstructure on the gel surface of the biomimetic gingival flap epithelium can accelerate the directional growth of epithelial cells and the reconstruction of spikes.

[0025] In some embodiments, the antimicrobial polypeptide can be selected as ε-polylysine (EPL).

[0026] In some embodiments, the biomimetic gingival flap lamina propria comprises a porous collagen hydrogel; the biomimetic gingival flap lamina propria has a loose porous structure.

[0027] The biomimetic gingival flap's lamina propria needs to have a certain thickness and elasticity to immediately serve as a biological replacement for the connective tissue layer after transplantation, connecting the epithelium and periosteum and buffering external forces. Therefore, this application uses light-cured collagen hydrogel as the substrate and constructs a porous collagen hydrogel (PorGel) using polyethylene oxide (PEO) pore-forming technology to provide space for the migration of fibroblasts and vasculature, allowing fibroblasts to quickly migrate and form tissue connections.

[0028] In some embodiments, the biomimetic gingival flap periosteal layer includes a dense collagen hydrogel and a zinc-nuclear zeolite imidazolate framework material-8.

[0029] Among them, the biomimetic gingival flap periosteum layer (Gel-Z) is designed to provide a stable support space for bone powder in the bone graft area, effectively block the invasion of fibroblasts, and act as a biological template to promote alveolar bone regeneration. To achieve this goal, a dense collagen hydrogel with a dense structure and excellent mechanical properties was prepared by precisely controlling the GMA reaction substitution ratio of the collagen side chains. This hydrogel not only has good biocompatibility but also effectively blocks the invasion of fibroblasts. To further enhance its biological activity, this application introduces the zinc-core MOF zeolite imidazole framework-8 (ZIF-8) with osteogenesis-promoting effects. It can create an osteogenic microenvironment by slowly releasing zinc ions. Therefore, by loading it into the hydrogel network, a biomimetic gingival flap periosteum layer with both barrier function and osteogenic activity can be constructed, providing an optimized biological microenvironment for alveolar bone regeneration.

[0030] Furthermore, some embodiments of the present invention also provide a method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects, comprising the following steps: S1. React gelatin with glycidyl methacrylate to form low-substitution light-cured gelatin, which is then mixed with light-cured polylysine, and a photoinitiator is added to form a bionic gingival flap epithelial layer material.

[0031] Specifically, the preparation method of low-substitution light-cured gelatin includes the following steps: dissolving gelatin under stirring, slowly adding glycidyl methacrylate and sodium hydroxide solution until the reaction system is alkaline, stirring for a period of time under dark conditions and then dialyzing, centrifuging and freeze-drying after the dialysis is completed, thereby preparing low-substitution light-cured gelatin.

[0032] For reference, the mixing mass ratio of low-substituted photocurable gelatin and photocurable polylysine is (2-5):1. For example, the mass ratio of the two can be selected as 2:1, 3:1, 4:1 or 5:1.

[0033] S2. React gelatin with glycidyl methacrylate to form low-substitution light-cured gelatin, and mix it with polyethylene oxide solution and photoinitiator to form bionic gingival flap lamina propria raw material.

[0034] S3. Extend the reaction time of gelatin and glycidyl methacrylate to generate high-substitution light-cured gelatin, and disperse zinc-nuclear zeolite imidazolate skeleton material-8 in the high-substitution light-cured gelatin to form a bionic periosteum layer raw material.

[0035] In some embodiments, the zinc-nucleated zeolite imidazolate framework material-8 is synthesized by a hydrothermal method.

[0036] It should be noted that there is no clear order restriction for steps S1, S2, and S3. In addition, in some embodiments, the photoinitiator includes but is not limited to lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0037] S4. Prepare an elastic negative mold with a honeycomb-shaped micro-convex structure through indirect printing method, pour the bionic gingival flap epithelial layer material into the model and freeze-mold it, then cover the bionic gingival flap epithelial layer surface with the bionic gingival flap propria material and freeze-mold it, cover the bionic gingival flap epithelial layer and bionic gingival flap propria surface with the bionic periosteum material, demold the whole by photocuring, and obtain a complete bionic full-thickness gingival flap after soaking.

[0038] Specifically, in some embodiments, the indirect printing method for preparing an elastic negative mold having a honeycomb-shaped micro-protrusion structure includes the following steps: designing a template having a honeycomb-shaped microstructure in a modeling software, exporting the template to an ultra-high precision light-curing printer to obtain a resin positive film, and then using polydimethylsiloxane to replicate an elastic negative mold having a honeycomb-shaped micro-protrusion structure.

[0039] Furthermore, in some embodiments, the bionic full-thickness gingival flap needs to be soaked in phosphate buffer for at least 24 hours after being formed.

[0040] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0041] Example 1 This embodiment provides a method for preparing a bionic full-thickness gingival flap, and the preparation flow chart thereof is as follows: Figure 1 -B, the steps include: (1) Preparation method of biomimetic gingival flap epithelial layer material: Gelatin was heated and dissolved under stirring conditions, and GMA (0.5% wt) and sodium hydroxide solution (0.1 mol / L) were slowly added until the reaction system was alkaline. The reaction was stirred in the dark for 24 hours and then dialyzed for 4 days. The obtained low-substitution light-cured gelatin (Gel-L) solution was centrifuged to remove impurities and then freeze-dried. Polylysine (EPL) was dissolved in deionized water under stirring conditions, and GMA (0.5% wt) and sodium hydroxide solution (0.1 mol / L) were slowly added to adjust the reaction system to alkaline. The reaction was stirred in the dark for 24 hours and then dialyzed for 4 days. The obtained light-cured polylysine (PLMA) solution was centrifuged to remove impurities and then freeze-dried. Gel-L and PLMA were mixed in a mass ratio of 5:1, fully dissolved in PBS solution, and 0.25% wt of the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) was added to obtain the biomimetic gingival flap epithelial layer Gel-EPL raw material.

[0042] (2) Preparation of biomimetic gingival flap lamina propria raw material: Gelatin was heated and dissolved under stirring conditions, and GMA (0.5% wt) and sodium hydroxide solution (0.1 mol / L) were slowly added until the reaction system was alkaline. The mixture was stirred in the dark for 24 hours and then dialyzed for 4 days. The obtained low-substitution photocurable gelatin (Gel-L) solution was centrifuged to remove impurities and then freeze-dried. Gel-L was dissolved in PBS solution at 10% wt, and an equal volume of 1.5% wt polyethylene oxide (PEO) solution and 0.25% wt photoinitiator LAP were added to the solution to form the biomimetic lamina propria raw material PorGel.

[0043] (3) Preparation method of biomimetic gingival flap periosteum layer raw material: Extend the GMA grafting time to 48 hours and then centrifuge. The obtained high-substitution light-cured gelatin (Gel-H) solution is centrifuged and freeze-dried. Then, 0.04 mol / L dimethylimidazole solution is slowly added dropwise to an equal volume of 0.1 mol / L zinc nitrate hexahydrate solution. After incubation at 45°C for 6-8 hours, high-speed centrifugation is performed. The precipitate is washed with anhydrous ethanol and dried to prepare ZIF-8. 0.2% wt ZIF-8 is dispersed in the Gel-H hydrogel precursor to form the biomimetic periosteum layer Gel-Z raw material.

[0044] (4) Preparation method of bionic full-thickness gingival flap: First, the layered structure of the full-thickness gingival flap was designed using Materialise Magics 24.0. Indirect printing method was used: a micro honeycomb module was constructed in the modeling software. The schematic diagram of the honeycomb module is shown in the figure. Figure 2As shown, the hexagonal pores have a diameter of 200 μm, a wall thickness of 20 μm, and a height of 100 μm. The model was exported to an ultra-high-precision light-curing resin printer to obtain a resin positive mold, which was then replicated using polydimethylsiloxane (PDMS) to create an elastic negative mold. Gel-EPL was vacuum-infused into the PDMS negative mold and freeze-molded. PorGel was then applied to the epithelial layer and freeze-molded over the biomimetic full-thickness gingival flap prototype. Similarly, Gel-Z was applied to the epithelial and fibroblast layers of the biomimetic full-thickness gingival flap prototype. The entire structure was then cured and demolded using 405 nm light. After soaking in PBS for 24 hours, a biomimetic full-thickness gingival flap with a complete, well-organized, and fully functional structure was obtained.

[0045] Test example The bionic full-thickness gingival flap prepared in Example 1 was subjected to in vitro antibacterial experiments, in vitro epithelialization ability experiments, in vitro fibroblast cell experiments, and in vitro osteogenic ability experiments.

[0046] (1) In vitro antibacterial test: 20 μL of Staphylococcus aureus ( S. aureus gingivalis ( P.gingivalis ) The bacterial solution was added to the surface of Gel-L and biomimetic full-thickness gingival flap and placed in a 37°C incubator for 2 hours. 500 μL PBS solution was added to resuspend and diluted 10 6 After the dilution is doubled, 100 μL of the diluted bacterial solution is plated. After incubation at 37°C for 12 hours, the number of colony-forming units (CFU) and the absorbance (OD) value of the bacterial solution at a wavelength of 600 nm are recorded. The antibacterial rate is calculated using formula (1) or formula (2):

[0047]

[0048] “C1” and “C0” are the bacterial colony counts in the cells that received and did not receive biomimetic full-thickness gingival flap treatment. 600 1 ” and “OD 600 0 ” is the OD value of the bacterial solution at a wavelength of 600 nm with and without biomimetic full-thickness gingival flap treatment.

[0049] The antibacterial properties of the bionic full-thickness gingival flap were further analyzed using live-dead bacterial staining, and the morphology of bacteria was observed using scanning electron microscopy (SEM).

[0050] The results are as follows Figure 3 As shown, Figure 3 -A and Figure 3 -B shows that as the concentration of polylysine (EPL) in Gel-EPL increases, the survival rates of Staphylococcus aureus and Porphyromonas gingivalis decrease significantly; Figure 3 -C and Figure 3-D showed that with the increase of EPL concentration in Gel-EPL, the antibacterial rate of Gel-EPL against Staphylococcus aureus increased significantly, among which the antibacterial rate of 5% Gel-EPL was close to 100%; Figure 3 -E and Figure 3 -F showed that with the increase of EPL concentration in Gel-EPL, the antibacterial rate of Gel-EPL against Porphyromonas gingivalis increased significantly, and the antibacterial rate of 5% EPL was close to 100%; Figure 3 -G observed that the untreated bacteria had intact morphology, while the Gel-EPL-treated bacteria had obvious morphological changes, with depressions and damage on the bacterial surface, which further demonstrated the killing effect of EPL on bacteria; Figure 3 -H is a schematic diagram of the antibacterial mechanism of Gel-EPL. Therefore, Gel-EPL has significant antibacterial effects against both Staphylococcus aureus and Porphyromonas gingivalis, and the antibacterial effect becomes more pronounced as the EPL concentration in Gel-EPL increases.

[0051] (2) In vitro epithelialization ability: Human gingival epithelial cells (HGEs) were inoculated on biomimetic full-thickness gingival flaps with or without honeycomb microstructures and cultured under the same culture conditions for 3 days. Cell viability was assessed by live-dead staining. Cell morphology was observed using TRITC-phalloidin and DAPI fluorescence staining combined with confocal scanning electron microscopy (CLSM) and scanning electron microscopy (SEM). Cell migration images were observed using optical microscopy on days 1, 3, and 7 after inoculation, and the proliferation activity of HGEs was detected by CCK8 assay on day 7.

[0052] Furthermore, HGEs were cultured on biomimetic full-thickness gingival flaps with or without honeycomb microstructures for 3 days, and total RNA was extracted and then subjected to single-cell transcriptome sequencing. The expression of key genes related to epithelialization was verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence (IF) staining.

[0053] The results are as follows Figure 4 As shown, Figure 4 -A. Figure 4 -B and Figure 4 -D bacterial live-dead staining results and SEM images showed that the growth pattern of HGEs on the bionic full-thickness gingival flap with honeycomb microstructure was better, the cells were more densely arranged, and the morphology was more complete; Figure 4 -C CCK8 test results showed that the honeycomb microstructure had a positive effect on cell survival, among which the survival rate of HGEs on the bionic full-thickness gingival flap with honeycomb microstructure was significantly higher than that on the bionic full-thickness gingival flap without honeycomb microstructure; Figure 4-E Optical microscopy images show the migration of HGEs on the bionic full-thickness gingival flap at different time points (days 1, 3, and 7). Cell migration is more obvious on the bionic full-thickness gingival flap with honeycomb microstructure. Figure 4 -F and Figure 4 -I heat map and RT-qPCR results showed that the expression of multiple key genes related to epithelialization in HGEs on bionic full-thickness gingival flaps with honeycomb microstructure was significantly upregulated, indicating that the honeycomb microstructure has a promoting effect on the epithelialization ability of cells; Figure 4 -G and Figure 4 The immunofluorescence (IF) staining results of -H showed that the expression of TGF-α, LAMA4 and CCND1 in HGEs on the bionic full-thickness gingival flap with honeycomb microstructure was significantly enhanced, indicating that the honeycomb microstructure promoted the expression of proteins related to growth factor activity (TGF-α), cell proliferation, migration and extracellular matrix secretion (LAMA4 and CCND1) in HGEs.

[0054] (3) In vitro fibroblast and osteogenic capacity test: Human gingival fibroblast-1 (HGF-1s) were resuspended in 500 μL of a hydrogel precursor solution containing Gel-(1%-2%) PEO and Gel-L, injected into a 24-well plate at 37°C, and cured with 405 nm blue light for 15-20 seconds. HGF-1s were 3D cultured in Gel-L and PorGel for 3 days, and 1 mL of DMEM medium was added. The medium was changed every day. After the culture was completed, the proliferation activity of HGF-1s was detected by CCK8 assay.

[0055] The light-cured Gel-H was placed in culture medium and completely immersed for 24 hours at 37°C and 5% CO2. Human gingival epithelial cells (HGEs) and rat fibroblast-1 (RAT-1s) were then seeded on the surface. After culturing for 3 days, CLSM 3D reconstruction images were used to assess the 3D spatial relationship between HGEs and RAT-1s and the Gel-H.

[0056] Dense collagen hydrogels (Gel-H) and dense collagen hydrogels containing 5%-20% ZIF-8 (Gel-(5-20)Z) were seeded into the upper chamber of a 24-well Transwell chamber and co-cultured with rat bone marrow mesenchymal stem cells (rBMSCs) in the lower chamber. After reaching a cell density of 70%, rBMSCs were cultured in osteogenic differentiation medium. After 7 days of rBMSC mineralization induction, alkaline phosphatase (ALP) secretion was detected by an alkaline phosphatase (ALP) detection kit, observed with a stereo microscope, and semi-quantitatively analyzed using Image J. After 7 and 14 days of osteogenic differentiation induction, early ( col1α1 、 runx2 and bmp2) and mid-late stage ( alp and ocn ) Gene expression of osteogenic markers. RUNX2 and OCN-IF staining were also used to compare the differences in osteogenesis.

[0057] The results are as follows Figure 5 As shown, Figure 5 -A and Figure 5 -B shows that hydrogel materials with different pore sizes have a significant effect on cell proliferation activity. Medium-pore hydrogel (PorGel-M) and large-pore hydrogel (PorGel-L) can better promote the growth of HGF-1s, while small-pore hydrogel (PorGel-S) is not so good. Figure 5 -C confocal 3D reconstruction images show that the two types of cells form a tight layered structure on the surface of the material, indicating that the material can isolate epithelial cells and fibroblasts and act as a membrane barrier; Figure 5 -D and Figure 5 -G ALP staining and semi-quantitative analysis showed the 3D culture of rBMSCs in dense collagen hydrogel (Gel-H), dense collagen hydrogel containing 5% ZIF-8 (Gel-5Z), dense collagen hydrogel containing 10% ZIF-8 (Gel-10Z), dense collagen hydrogel containing 15% ZIF-8 (Gel-15Z), and dense collagen hydrogel containing 20% ​​ZIF-8 (Gel-20Z). As the content of ZIF-8 in the periosteum layer of the bionic gingival flap gradually increased, the osteogenic marker alp The content gradually increases; Figure 5 -E and Figure 5 The results of immunofluorescence detection and gene detection of -I showed that after 7 days of induction of osteogenic differentiation, the content of ZIF-8 in the periosteum of the bionic gingival flap gradually increased, and the early osteogenic marker runx2 The expression of Figure 5 The results of immunofluorescence and qPCR of ZIF-F showed that after 14 days of induction of osteogenic differentiation, the content of ZIF-8 in the periosteum of the biomimetic gingival flap gradually increased, and the late osteogenic marker ocn The expression of ZIF-8 gradually increased, indicating that ZIF-8 could continuously promote osteogenic differentiation.

[0058] In summary, this application has developed a tissue-engineered gingival flap through a layered bionic design, aiming to solve the problem of insufficient gingival sealing in the bone grafting area, avoid secondary trauma of autologous flap transplantation, and broaden the scope of indications for alveolar bone grafting. This material combines antimicrobial peptide-collagen hydrogel, porous collagen hydrogel and tough collagen hydrogel, respectively simulating the bacterial isolation function of the epithelial layer, the supporting and stabilizing function of the lamina propria, and the barrier osteogenesis function of the periosteum, effectively addressing clinical problems such as the high incidence of bacterial infection, bone powder loss and displacement, and loss of osteogenesis space. In addition, the bionic full-thickness gingival flap not only mimics the basic functions of each layer of the autologous gingival flap, but also guides the rapid regeneration of defective tissue through its microstructure. For example, the ultra-micro honeycomb structure accelerates the union of epithelial cells and forms a spike structure, the loose lamina propria accommodates the migration of fibroblasts and vascular cells, and the zinc-rich environment activates the osteogenic differentiation of stem cells. Based on an in-depth analysis of the repair process of soft and hard tissues in the surgical area, this project proposed a repair concept that conforms to physiological processes. By utilizing the semi-permeability of collagen hydrogel, the paracrine activation and regeneration effect of fibroblasts is fully utilized to achieve multi-tissue collaborative reconstruction, thus breaking through the traditional model of isolated layer-by-layer repair, simulating the real physiological process in the body, and providing an efficient and innovative clinical solution.

[0059] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects, characterized in that: It includes the following three layers: Bionic gingival flap epithelium, bionic gingival flap lamina propria and bionic gingival flap periosteum; The bionic gingival flap epithelial layer includes collagen hydrogel and antibacterial polypeptide; The bionic gingival flap lamina propria comprises porous collagen hydrogel; The bionic gingival flap periosteum layer includes dense collagen hydrogel and zinc-nuclear zeolite imidazolate skeleton material-8.

2. The bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to claim 1, characterized in that: The collagen hydrogel surface of the bionic gingival flap epithelium has a honeycomb microstructure; And / or, the collagen hydrogel of the biomimetic gingival flap epithelium is a methacrylated polypeptide hydrogel.

3. The bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to claim 1, characterized in that: The bionic gingival flap propria has a loose and porous structure.

4. A method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to any one of claims 1 to 3, characterized in that: The following steps are involved: The gelatin is reacted with glycidyl methacrylate to form a low-substitution light-cured gelatin, which is then mixed with light-cured polylysine and a photoinitiator is added to form a bionic gingival flap epithelial layer material. The gelatin is reacted with glycidyl methacrylate to form a low-substitution light-cured gelatin, which is then mixed with polyethylene oxide solution and a photoinitiator to form a bionic gingival flap lamina propria raw material. The reaction time of gelatin and glycidyl methacrylate was extended to generate high-substitution light-cured gelatin, and zinc-nuclear zeolite imidazolate skeleton material-8 was dispersed in the high-substitution light-cured gelatin to form the bionic periosteum layer raw material; An elastic negative mold with a honeycomb-shaped micro-convex structure was prepared by an indirect printing method. The raw material of the bionic gingival flap epithelium was poured into the model and then frozen into shape. The bionic gingival flap propria material was then covered on the surface of the bionic gingival flap epithelium and then frozen into shape. The bionic periosteum material was covered on the surface of the bionic gingival flap epithelium and the bionic gingival flap propria. The whole was demolded by photocuring and soaked to obtain a complete bionic full-thickness gingival flap.

5. The method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to claim 4, characterized in that: The method for preparing low-substitution light-cured gelatin comprises the following steps: dissolving gelatin under stirring, slowly adding glycidyl methacrylate and sodium hydroxide solution until the reaction system is alkaline, stirring the reaction in the dark, and then dialyzing the solution. After the dialysis is completed, the solution is centrifuged and freeze-dried to prepare the low-substitution light-cured gelatin.

6. The method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to claim 4, characterized in that: The mixing mass ratio of the low-substitution light-curing gelatin and the light-curing polylysine is (2-5):

1.

7. The method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to claim 4, characterized in that: The photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

8. The method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to claim 4, characterized in that: The zinc nucleus zeolite imidazolate framework material-8 is synthesized by a hydrothermal method.

9. The method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to claim 4, characterized in that: The indirect printing method for preparing an elastic negative mold having a honeycomb-shaped micro-protrusion structure comprises the following steps: designing a template having a honeycomb-shaped micro-structure in a modeling software, exporting the template to an ultra-high-precision light-curing printer to obtain a resin positive film, and then replicating the elastic negative mold having the honeycomb-shaped micro-protrusion structure using polydimethylsiloxane.

10. The method for preparing a bionic full-thickness gingival flap for implant restoration of soft and hard tissue defects according to claim 4, characterized in that: After the bionic full-thickness gingival flap is formed, it needs to be soaked in phosphate buffer for at least 24 hours.

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