A molybdenum-based nanocluster biological tissue-derived composite scaffold, a preparation method and applications thereof

Abstract

This invention relates to the fields of biomedicine and tissue engineering materials, specifically to a molybdenum-based nanocluster bio-tissue-derived composite scaffold, comprising a composite substrate and molybdenum-based nanoclusters loaded on the composite substrate; the composite substrate is composed of bone-derived decellularized extracellular matrix and methacrylamide gelatin, and the molybdenum-based nanoclusters are dispersed in a three-dimensional porous network structure of the composite substrate. This scaffold retains the functional protein components of bone-derived extracellular matrix (BdECM), while the introduction of GelMA improves its mechanical properties and processability, and the loading of MNCs endows the scaffold with efficient ROS scavenging ability, achieving active regulation of the inflammatory microenvironment.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and tissue engineering materials, and more specifically, to a molybdenum-based nanocluster bio-tissue-derived composite scaffold, its preparation method, and its application. Background Technology

[0002] Periodontitis is a chronic inflammatory disease characterized by the destruction of periodontal supporting tissues. Its core pathogenesis is closely related to the imbalance of oxidative-antioxidant homeostasis. Periodontal defect areas are chronically exposed to an open, complex, and dynamically changing inflammatory microenvironment. Excessive accumulation of reactive oxygen species (ROS) not only directly damages tissue cells but also inhibits stem cell adhesion, proliferation, and differentiation, accelerates the degradation and loss of bioactivity of biomaterials, and severely weakens the repair efficacy of tissue-engineered scaffolds. Furthermore, periodontal tissues consist of three distinct soft and hard tissues—alveolar bone, periodontal ligament, and cementum—and their functional reconstruction relies on the organic and synergistic regeneration of multiple tissues, rather than the independent repair of a single tissue.

[0003] Currently, bone-derived decellularized extracellular matrix (BdECM) is widely used for periodontal bone defect repair because it removes cellular immunogenicity while preserving the ultrastructure and functional protein components of natural tissues. However, while a single regeneration strategy focused on promoting bone growth can effectively repair alveolar bone defects, it is difficult to simultaneously achieve the physiological functional reconstruction of the periodontal ligament, ultimately failing to achieve the ideal goal of synergistic regeneration of periodontal soft and hard tissues. Furthermore, existing BdECM scaffolds generally lack the ability to actively regulate the inflammatory microenvironment, making it difficult to effectively remove excessive ROS accumulated in the periodontal inflammatory microenvironment and reshape redox homeostasis.

[0004] In summary, current technologies have not yet solved the problem of how to achieve redox homeostasis and promote the synergistic regeneration of periodontal soft and hard tissues in a complex inflammatory microenvironment. Therefore, in view of the functional limitations of current BdECM-derived materials, it is of great clinical significance and application value to construct a multifunctional repair system that integrates antioxidant stress and stem cell multi-directional differentiation induction to address the challenges of periodontal complex regeneration in an inflammatory microenvironment. Summary of the Invention

[0005] The purpose of this invention is to provide a molybdenum-based nanocluster bio-tissue-derived composite scaffold, addressing the technical shortcomings of existing bone-derived decellularized extracellular matrix materials, such as limited functionality, lack of active regulation of the inflammatory microenvironment, and difficulty in inducing synergistic regeneration of periodontal soft and hard tissues.

[0006] Another objective of this invention is to provide a method for preparing a molybdenum-based nanocluster bio-tissue-derived composite scaffold, which has a stable process, is easy to operate, and has a wide range of raw material sources, and has good prospects for clinical translation.

[0007] The technical problem solved by this invention is achieved by the following technical solution.

[0008] On one hand, embodiments of the present invention provide a molybdenum-based nanocluster biological tissue-derived composite scaffold comprising a composite substrate (ECM / Gel) and molybdenum-based nanoclusters (MNCs) loaded on the composite substrate; the composite substrate is composed of bone-derived decellularized extracellular matrix (BdECM) and methacrylamide gelatin (GelMA), and the molybdenum-based nanoclusters are dispersed in a three-dimensional porous network structure of the composite substrate. The composite scaffold is formed by UV crosslinking and curing, possessing a three-dimensional porous network structure, and the loading of MNCs has no effect on the porosity of the composite scaffold.

[0009] In some embodiments of the present invention, the degree of substitution of the methacrylamide gelatin is 80-90%, and the concentration is 5-15% (w / v); the amount of bone-derived decellularized extracellular matrix added to the composite substrate is 5-10% (w / v); and the concentration of the molybdenum-based nanoclusters in the composite scaffold is 100-500 μg / mL (w / v).

[0010] In some embodiments of the present invention, the degree of substitution of the methacrylamide gelatin is 90%, and the concentration is 10% (w / v); the amount of bone-derived decellularized extracellular matrix added to the composite substrate is 7% (w / v); and the concentration of the molybdenum-based nanoclusters in the composite scaffold is 300 μg / mL (w / v).

[0011] On the other hand, embodiments of the present invention provide a method for preparing a molybdenum-based nanocluster bio-tissue-derived composite scaffold, comprising the following steps: (1) Dissolve methacrylamide gelatin in PBS solution containing LAP, heat in a water bath at 60°C until completely dissolved, and cool to room temperature to obtain hydrogel precursor; add bone-derived decellularized extracellular matrix powder at 37°C, stir for 2 h to obtain composite substrate; (2) Add molybdenum-based nanocluster powder to the composite substrate obtained in step (1), stir continuously at 37 °C for 2 h, and then crosslink and cure under ultraviolet light to obtain a composite scaffold.

[0012] In some embodiments of the present invention, the preparation method of the molybdenum-based nanocluster powder includes the following steps: (NH4)6Mo7O 24Molybdenum-based nanocluster powder (MNC) was obtained by dissolving 4H₂O in water, adding NaH₂PO₄ solution and L-ascorbic acid solution, stirring, adding ethanol to precipitate, centrifuging, dialysis purification, freeze-drying, and ball milling. The average particle size of MNC was 10-15 nm, approximately spherical, and well-dispersed. MNC exhibited rapid valence cycling of molybdenum (Mo). 5+ / Mo 6+ It achieves excellent dose-dependent antioxidant stress resistance.

[0013] The molybdenum-based nanocluster powder (MNC) has an average particle size of 10 nm, is approximately spherical, and exhibits good dispersibility. The MNC is produced through rapid valence cycling of molybdenum (Mo). 5+ / Mo 6+ It achieves excellent dose-dependent antioxidant stress resistance.

[0014] In some embodiments of the present invention, the method for preparing the bone-derived decellularized extracellular matrix is ​​derived from the cancellous bone of the metaphysis of porcine long bones and obtained through decellularization treatment; the method includes the following steps: cutting the bone into 4 mm × 4 mm × 2 mm bone blocks, and then subjecting them to repeated freeze-thaw cycles, formic acid decalcification, peracetic acid sterilization, Triton X-100 / EDTA-Na2 defatting, DNase I enzyme digestion, freeze drying, and low-temperature ball milling to obtain bone-derived decellularized extracellular matrix powder.

[0015] In some embodiments of the present invention, the UV curing time is 20-40 seconds.

[0016] Thirdly, embodiments of the present invention provide the application of a molybdenum-based nanocluster bio-tissue-derived composite scaffold in the preparation of drugs or medical devices for treating periodontal soft and hard tissue defects caused by periodontitis.

[0017] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: (1) This invention combines MNC with the ECM / Gel system to construct a MNC / ECM / Gel composite scaffold that combines anti-oxidative stress and stem cell multi-lineage differentiation induction functions. The scaffold retains the functional protein components of BdECM, while the introduction of GelMA improves mechanical properties and processability. The loading of MNC endows the scaffold with efficient ROS scavenging ability, realizing active regulation of the inflammatory microenvironment. At the same time, the introduction of MNC further realizes the functional expansion of BdECM scaffold from single osteogenic to osteogenic / ligamentogenic dual-lineage differentiation induction. Through the functional synergy strategy, the functional limitations of existing BdECM-derived materials, such as lack of active regulation of the inflammatory microenvironment and difficulty in inducing synergistic regeneration of periodontal soft and hard tissues, are solved.

[0018] (2) Through in vivo animal experiments, it was confirmed that after the MNC / ECM / Gel composite scaffold was implanted into the rat periodontal defect model, it could effectively remove the excessive ROS accumulated locally, continuously alleviate the high oxidative stress state, and achieve the synergistic regeneration of periodontal soft and hard tissues, providing a new strategy and material basis for the regeneration of periodontal complex in the inflammatory microenvironment.

[0019] (3) The preparation method of the present invention is stable, easy to operate, and has a wide range of raw material sources, and has good prospects for clinical translation. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The morphology characterization of MNCs provided in this embodiment of the invention is shown in Figure A, which is a SEM image of the MNCs (scale bar: 50 nm); and Figure B is a histogram of particle size distribution.

[0022] Figure 2 The HRTEM and EDS diagrams of MNCs provided in this embodiment of the invention are shown. Blue represents Mo, purple represents O, and yellow represents N (scale bar: 10 nm).

[0023] Figure 3 Characterization of the antioxidant capacity of MNCs provided in this embodiment of the invention; A is the peak spectrum of MNCs before and after H2O2 treatment detected by XPS, and the red arrow indicates the Mo5+ after H2O2 treatment. + The relative intensity of the characteristic peaks decreased; B is the MNC absorption peak spectrum before and after H2O2 treatment detected by UV-Vis.

[0024] Figure 4The following are schematic diagrams of the composition and physicochemical properties of the MNC / ECM / Gel composite scaffolds provided in this embodiment of the invention: A is a schematic diagram of the composition of MNC / ECM / Gel; B is a macroscopic image of each composite scaffold, with the upper left being uncured ECM / Gel, the upper right being photocured ECM / Gel, the lower left being uncured MNC / ECM / Gel, and the lower right being photocured MNC / ECM / Gel; C is a SEM image of the surface microstructure of each composite scaffold (top bar: 400 μm; bottom bar: 200 μm); D is a semi-quantitative analysis of the porosity of each composite scaffold (n = 3); E is a representative elemental energy spectrum image of 3MNC / ECM / Gel, with purple representing Mo, red representing C, yellow representing N, and green representing O (scale bar: 200 μm); F is the cumulative release curve of Mo element in 3MNC / ECM / Gel.

[0025] Figure 5 Characterization of the antioxidant capacity of the MNC / ECM / Gel composite scaffold provided in the embodiments of the present invention; A is the DCFH-DA fluorescence staining image and semi-quantitative analysis of PDLSCs on each composite scaffold after H2O2 treatment; B is a statistical graph of apoptosis detection results by flow cytometry.

[0026] Figure 6 Characterization of osteogenic and ligamentogenic bidirectional differentiation of PDLSCs on the MNC / ECM / Gel composite scaffold provided in this embodiment of the invention. A and B are representative immunofluorescence staining images (scale bar: 50 μm) of osteogenic-related proteins RUNX2 and ALP on each composite scaffold and their semi-quantitative analysis, respectively; C and D are representative ALP staining images and ARS staining images (scale bar: 1 mm) of PDLSCs on each composite scaffold and their semi-quantitative analysis, respectively; E is a representative immunofluorescence staining image (scale bar: 50 μm) of ligamentogenic differentiation-related protein COL-1 on each composite scaffold; F is a statistical graph of the quantitative detection results of Sirius red collagen.

[0027] Figure 7 This study characterizes the ability of the MNC / ECM / Gel composite scaffold provided by the present invention to alleviate oxidative stress in vivo and promote the synergistic regeneration of periodontal soft and hard tissues. Figure 7In the middle: A and B are the effects of in vivo imaging dynamic monitoring of the MNC / ECM / Gel composite scaffold on the in vivo oxidative stress state and their semi-quantitative analysis at 7, 14 and 28 days after surgery (n = 6); C is the representative Micro-CT images of the implanted Gel, ECM / Gel, MNC / ECM / Gel hydrogel and blank control group at 28 days after surgery. Includes Micro-CT 3D reconstruction images, coronal section images, and transverse section images (scale bar: 2 mm); D shows semi-quantitative analysis of BPN, Tb.Th, Tb.N, and BV / TV at 28 days post-implantation for Gel, ECM / Gel, MNC / ECM / Gel hydrogel implantation, and the blank control group (n = 6); E shows representative H&E staining images of tissue sections at 28 days post-implantation for Gel, ECM / Gel, MNC / ECM / Gel hydrogel implantation, and the blank control group (Defect represents the modeling area; TR represents the root area, upper scale bar: 200 μm; lower scale bar: 50 μm) (n = 6); F shows representative Masson staining images of tissue sections at 28 days post-implantation for Gel, ECM / Gel, MNC / ECM / Gel hydrogel implantation, and the blank control group (Defect represents the modeling area; TR represents the root area, upper scale bar: 200 μm; lower scale bar: 50 μm) (n = 6). Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0030] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0031] Example 1: Preparation and Characterization of MNCs 1.1 Preparation and Characterization of MNCs MNC was synthesized using a one-pot method. First, 2 mmol (NH4)6Mo7O was added at 25 °C. 24 • Dissolve 4H₂O in 10 mL of double-distilled H₂O, and dissolve 1.17 mmol of NaH₂PO₄ in 5 mL of double-distilled H₂O. Then, quickly add the prepared NaH₂PO₄ solution dropwise to (NH₄)₆Mo₇O₄. 24In a 4H₂O solution, with continuous stirring, 2 mL of saturated L-ascorbic acid solution was added dropwise. After stirring for 20 min, 80 mL of ethanol was added to separate the MNC precipitate. The precipitate was then collected by centrifugation at 10304 g for 15 min. The precipitate was redispersed in ultrapure water, and free L-ascorbic acid was removed by dialysis with continuous stirring for 3 days and frequent changes of the dialysis buffer. The MNC solution was frozen at -80 °C overnight, then freeze-dried in a vacuum freeze dryer for 3 days. The resulting powder was obtained by grinding the powder evenly using a ball mill.

[0032] 1.2 Characterization of the physicochemical properties and antioxidant properties of MNC (1) Observation of the microstructure and dispersibility of MNC surface by scanning electron microscopy (SEM): MNC powder was dissolved in ultrapure water and ultrasonically dispersed for 20 min. 10 μL of the dispersion was dropped onto the silicon wafer surface, dried at room temperature and then sputtered with gold. The microstructure and dispersibility of MNC were observed by SEM.

[0033] (2) High-resolution transmission electron microscopy (HRTEM) observation of the microstructure and elemental distribution of the MNC surface: Take 10 μL of the above MNC dispersion and drop it onto the ultrathin carbon film. Carefully remove excess liquid from the edge of the copper mesh with filter paper and let it stand and dry at room temperature for 10 min. Fix the completely dried ultrathin carbon film in the HRTEM sample holder and quickly insert the sample holder into the electron microscope sample chamber. After the vacuum reaches the required level, observe and photograph the surface microstructure. Then switch to scanning transmission electron microscopy (STEM) mode and use X-ray energy dispersive spectroscopy (EDS) to perform surface scanning (mapping) on ​​the selected area. Collect characteristic X-ray signals through the detector and accurately identify the element types based on the characteristic peaks (blue represents Mo, purple represents O, and yellow represents N). Perform simultaneous multi-element scanning on the same area to visually display the spatial distribution of each element on the MNC surface.

[0034] 1.3 Results SEM results showed that MNCs were approximately spherical, well-dispersed, and had a particle size of about 10 nm. Figure 1 A and B). HRTEM results showed slight protrusions on the MNC surface, and EDS surface scan results showed a strong Mo element signal (A and B). Figure 2 XPS results showed that after MNC reacted with H2O2, some Mo... 5+ Mo is oxidized to a higher oxidation state. 6+ ( Figure 3 A). UV-Vis spectroscopy further shows that as the H2O2 concentration increases from 1 mM to 4 mM, the absorption peak of MNC in the near-infrared region decreases in a dose-dependent manner. Figure 3B). The above results collectively demonstrate that MNCs can achieve dose-dependent ROS scavenging through reversible changes in the Mo valence state, exhibiting excellent antioxidant stress resistance.

[0035] Example 2: Preparation and characterization of MNC / ECM / Gel 2.1 Preparation of MNC / ECM / Gel 2.1.1 Preparation of ECM / Gel Fresh cancellous bone from the metaphysis of pig long bones (4-6 months old) was cut into 4 mm × 4 mm × 2 mm pieces and rinsed with double-distilled water until clear. The following procedures were performed sequentially: three cycles of freeze-thaw at -80 ℃ and 37 ℃; decalcification with 10% (v / v) formic acid at 37 ℃ for 3 days, followed by rinsing with double-distilled H2O; sterilization with 3% (v / v) peracetic acid at room temperature for 5 hours, followed by rinsing with ddH2O until neutral; defatting with 1% (v / v) Triton X-100 / 2 mMEDTA-Na2 at room temperature for 6 days; enzyme digestion with DNase I (600 U / mL) for 24 hours; freeze-drying followed by low-temperature ball milling in liquid nitrogen to obtain BdECM powder, which was stored at -20 ℃ for later use. Methacrylamide gelatin (GelMA) (90% substitution degree, 10% w / v) was dissolved in PBS solution containing LAP (0.25% w / v), heated in a water bath at 60 °C for 30 min, and shaken every 15 min to mix thoroughly. After complete dissolution, it was cooled to room temperature to obtain the hydrogel precursor. Before use, the precursor was warmed to 37 °C, and bone-derived acellular extracellular matrix (BdECM) powder was added at 7% (w / v). The mixture was stirred at 37 °C for 2 h to obtain ECM / Gel, which was stored in the dark for later use.

[0036] 2.1.2 Preparation of MNC / ECM / Gel The above-mentioned ECM / Gel was taken and MNC powder (prepared in 1.1) was added to it to make final concentrations of 100, 300, and 500 μg / mL (w / v), respectively. The mixture was stirred continuously at 37 ℃ for 2 h to uniformly physically load MNC into the ECM / Gel. After UV crosslinking for 30 s, MNC / ECM / Gel was obtained and named 1MNC / ECM / Gel, 3MNC / ECM / Gel, and 5MNC / ECM / Gel, respectively. The ECM / Gel scaffold without MNC was used as the control group (named ECM / Gel).

[0037] 2.2 Characterization of the physicochemical properties of MNC / ECM / Gel 2.2.1 SEM observation of the surface microstructure and elemental distribution of the MNC / ECM / Gel composite scaffold ECM / Gel, 1MNC / ECM / Gel, 3MNC / ECM / Gel, and 5MNC / ECM / Gel scaffolds were immersed in sufficient PBS and incubated sequentially at 4 ℃ for 2 h, -20 ℃ for 4–5 h, and -80 ℃ overnight, followed by freeze-drying for 3 days. The samples were cut in half with a sharp, thin blade, and the cut surfaces were fixed to a sample plate with conductive adhesive, followed by gold sputtering for 60 s. The microstructure of the MNC / ECM / Gel composite scaffold surface was observed using SEM, and representative images were captured. Subsequently, EDS was used to perform surface scanning on selected areas, identifying elemental types (Mo, C, O, and N) based on characteristic peaks. The distribution of each element on the scaffold surface was displayed using color coding (purple for Mo, red for C, yellow for N, and green for O), as shown in the attached figure. Figure 4 As shown, A is a schematic diagram of the composition of the MNC / ECM / Gel composite scaffold; B is a macroscopic image of the MNC / ECM / Gel; C is a representative image of the surface microstructure of the MNC / ECM / Gel composite scaffold observed by SEM (top bar: 400 μm; bottom bar: 200 μm); D is a semi-quantitative analysis of the porosity of the MNC / ECM / Gel (n = 3); E is a representative image of the elemental energy spectrum of the MNC / ECM / Gel, where purple represents Mo, red represents C, yellow represents N, and green represents O (scale bar: 200 μm); F is the cumulative release curve of Mo element from the MNC / ECM / Gel.

[0038] 2.2.2 Porosity Detection The SEM images of ECM / Gel, 1MNC / ECM / Gel, 3MNC / ECM / Gel and 5MNC / ECM / Gel scaffolds were converted to grayscale using ImageJ 1.54g software. A threshold was set to distinguish between pores and matrix. The ratio of pore area to total area was calculated as porosity.

[0039] 2.2.3. Detection of Mo release from MNC / ECM / Gel using inductively coupled plasma mass spectrometry (ICP-MS) Prepare 200 μL cylindrical samples of ECM / Gel, 1 MNC / ECM / Gel, 3 MNC / ECM / Gel, and 5 MNC / ECM / Gel, respectively, and place them in 15 mL centrifuge tubes with 2 mL of PBS added. Incubate at 37 °C. Collect 100 μL of extract at the same time points on days 1, 3, and 7, and immediately replenish with 100 μL of PBS. Determine the concentration of Mo at the characteristic spectral lines using ICP-MS.

[0040] 2.3 Characterization of the antioxidant properties of MNC / ECM / Gel 2.3.1 Intracellular ROS Detection PDLSCs at 5×10 4 Individual probes were seeded per well on ECM / Gel, 1MNC / ECM / Gel, 3MNC / ECM / Gel, and 5MNC / ECM / Gel scaffolds, respectively. After 24 h of culture, except for the normal control group, the other groups were treated with 500 μM H2O2 for 2 h to construct an oxidative stress model. The culture medium was discarded, and 10 μM DCFH-DA probe working solution was added. The samples were incubated at 37 ℃ in the dark for 30 min, and observed and images were taken using a confocal microscope.

[0041] 2.3.2 Flow Cytometry Apoptosis Experiment Cell seeding, grouping, and H2O2 treatment were the same as in 2.3.1. After treatment, cells were collected by trypsin digestion, double-stained with Annexin V-FITC / PI, incubated at room temperature in the dark for 15 min, and the apoptosis rate was detected by flow cytometry.

[0042] 2.4 Characterization of the multi-lineage differentiation potential of stem cells promoted by MNC / ECM / Gel 2.4.1 Characterization of the osteogenic differentiation potential of stem cells promoted by MNC / ECM / Gel (1) ALP staining and ARS staining: PDLSCs were stained with 5×10 4 Each well was seeded onto the scaffolds of each group. After 7 and 21 days of osteogenic induction, ALP staining and Alizarin Red S (ARS) staining were performed, and the images were observed and captured under an optical microscope.

[0043] (2) Immunofluorescence staining of osteogenic-related proteins: After 7 days of osteogenic induction, the cells were fixed with 4% paraformaldehyde and blocked with immunofluorescence blocking solution for 1 h. Rabbit anti-ALP antibody (1:200) and Rabbit anti-RUNX2 antibody (1:200) were added and incubated overnight at 4°C. Fluorescent secondary antibody IgG (1:200) was added and incubated at room temperature in the dark for 1 h. Phalloidin (1:200) was incubated at room temperature in the dark for 30 min to label the cytoskeleton. DAPI was used to stain the nucleus for 10 min. The expression of ALP and RUNX2 proteins was observed and images were acquired using a confocal microscope.

[0044] 2.4.2 Characterization of the ligament-forming differentiation potential of stem cells promoted by MNC / ECM / Gel (1) Immunofluorescence staining to detect the expression of ligament-related proteins PDLSCs at 5×10 4Seeds were seeded per well on ECM / Gel, 1MNC / ECM / Gel, 3MNC / ECM / Gel, and 5MNC / ECM / Gel scaffolds, respectively. After ligament induction for 7 days, the cells were fixed with 4% paraformaldehyde, blocked with immunofluorescence blocking solution for 1 h, and then incubated overnight at 4 °C with Rabbit anti-COL-1 antibody (1:300). Then, Alexa Fluor 488-labeled goat anti-rabbit IgG (1:200) was added and incubated at room temperature in the dark for 1 h. Nuclei were stained with DAPI for 10 min. COL-1 protein expression was observed and images were acquired using confocal microscopy.

[0045] (2) Quantitative detection of Sirius red collagen PDLSCs were seeded onto scaffolds in each group, and after ligament induction for 7 days, the culture supernatant was collected. 1 mL of culture supernatant was added to 250 μL of concentrated buffer, vortexed, and incubated at 4 °C for 16–24 h. The mixture was then centrifuged at 10,000 rpm for 3 min, and the supernatant was discarded. The precipitate was dissolved in 100 μL of glacial acetic acid, and 500 μL of Sirius red solution was added. The mixture was incubated at room temperature for 20 min, centrifuged at 10,000 rpm for 3 min, and the supernatant was discarded. The precipitate was resuspended in 500 μL of washing buffer and washed twice. 250 μL of extraction buffer was added to dissolve the precipitate, and the OD value at 520 nm was measured using a microplate reader. The sample concentration was calculated based on the standard curve and multiplied by the dilution factor to obtain the actual concentration.

[0046] 2.5 Results 2.5.1 Characterization of the physical and chemical properties of MNC / ECM / Gel General observation results are as follows Figure 4 As shown in Figure B, the load MNC has no effect on the photocuring performance. SEM observation shows that all groups of scaffolds exhibit a three-dimensional porous network structure. Figure 4 C). Porosity testing showed that the MNC load did not affect the three-dimensional porous structure of the scaffold. Figure 4 D). EDS surface scanning confirmed that Mo, C, O, and N elements were uniformly distributed. Figure 4 E). ICP-MS results showed that Mo release in MNC / ECM / Gel was rapid on day 1, and gradually slowed down in the following 2-7 days. Figure 4 F).

[0047] 2.5.2 Characterization of the antioxidant properties of MNC / ECM / Gel DCFH-DA staining and flow cytometry apoptosis detection showed that MNC / ECM / Gel dose-dependently reduced total ROS levels in cells and effectively inhibited apoptosis induced by oxidative stress. The cell state in both the 3MNC / ECM / Gel and 5MNC / ECM / Gel scaffold groups recovered to near-normal control levels. Figure 5 A and 5B).

[0048] 2.5.3 Characterization of the ability of MNC / ECM / Gel to promote multi-lineage differentiation of stem cells Immunofluorescence staining showed that the fluorescence intensity of ALP and RUNX2 proteins in the MNC / ECM / Gel group was significantly higher than that in the ECM / Gel group, with the most significant expression in the 3MNC / ECM / Gel group. Figure 6 A and 6B). ALP and ARS staining showed that the 3MNC / ECM / Gel and 5MNC / ECM / Gel groups had better staining depth and more mineralized nodules than the ECM / Gel group. Figure 6 C and 6D); ligament differentiation assays showed that the COL-1 immunofluorescence intensity gradually increased with increasing MNC concentration, with the strongest fluorescence signal in the 3MNC / ECM / Gel group (C and 6D). Figure 6 E). Quantitative analysis of Sirius red showed that the collagen secretion level in the 5MNC / ECM / Gel group was significantly higher than that in other groups (E). Figure 6 F). The above results indicate that MNC / ECM / Gel has the ability to induce osteogenic and ligamentogenic differentiation of PDLSCs, with the 3MNC / ECM / Gel group showing the most significant induction effect.

[0049] Example 3: The MNC / ECM / Gel scaffold obtained in Example 2 was used to conduct a rat periodontal defect repair experiment.

[0050] The animal experiment was approved by the Animal Experiment Ethics Committee of the Fourth Military Medical University, and six 6-8 week old Sprague animals were used. Dawley rats (purchased from the Animal Experiment Center of the Fourth Military Medical University) were used in this experiment. The surgical procedure was as follows: Rats were anesthetized with isoflurane in a small animal anesthesia machine. The isoflurane concentration was 3% during induction anesthesia and 2% during maintenance anesthesia. A full-thickness incision was made along the corner of the mouth to the ear to expose the masseter muscle. The facial nerve and parotid duct were located, and a 1 cm incision parallel to the facial nerve was made between them. A bone defect measuring 3 mm (length) × 2 mm (height) × 1 mm (depth) was prepared on the surface of the mandibular body using a 0.8 mm ball bur. Periodontal ligament tissue was removed from the distal root of the first molar and the mesial root of the second molar in the defect area. 10 μL of MNC / ECM / Gel was injected into the defect, and the area was cross-linked under UV light for 30 s.

[0051] 3.1 Detection of the ability of MNC / ECM / Gel to alleviate oxidative stress in the body On days 7, 14, and 28 after MNC / ECM / Gel scaffold implantation, ROS Brite 700 fluorescent probes were injected into the defect site. After incubation for 30 min, fluorescence signals were acquired using an in vivo imaging system (IVIS Spectrum). Parameter settings: excitation wavelength 680 nm, emission wavelength 720 nm, exposure time 2 s. The defect area was designated as the region of interest (ROI), and the average fluorescence intensity was semi-quantitatively analyzed to dynamically assess changes in local ROS levels in each group of periodontal defects.

[0052] 3.2 Evaluation of the efficacy of MNC / ECM / Gel in promoting the synergistic regeneration of periodontal soft and hard tissues On the 28th day after surgery, the animals were euthanized by carbon dioxide asphyxiation, and the mandibular bone specimens were separated for analysis.

[0053] (1) Micro-CT analysis of the effect of MNC / ECM / Gel composite framework on periodontal bone regeneration. The mandibular bone specimen was fixed in 4% paraformaldehyde for 24 h. After fixation, micro-CT was used for scanning. After scanning, VG Studio Max 3.5.1 software was used to reconstruct and analyze the raw data in three dimensions. The region of interest (ROI) was the bone defect area (3 mm long, 2 mm high, and 1 mm deep) prepared before surgery. The new bone formation was quantitatively evaluated. The analysis indicators included bone volume fraction (BV / TV), number of trabeculae (Tb.N), trabecular bone thickness (Tb.Th), and buccal bone plate thickness (BPN) to comprehensively evaluate the periodontal bone regeneration effect.

[0054] (2) Histological staining analysis of the efficacy of MNC / ECM / Gel composite scaffold in promoting the synergistic regeneration of periodontal soft and hard tissues. H&E staining was used to observe new bone formation and the arrangement of periodontal ligament-like fibrous tissue. Mandibular bone specimens were decalcified with 10% EDTA for 4-6 weeks, dehydrated with graded ethanol, embedded in paraffin, and sectioned continuously along the coronal plane of the defect (5 μm thickness). After dewaxing and rehydration, sections were stained with hematoxylin for 5-10 min, differentiated with hydrochloric acid and ethanol, and then blued with running water; eosin was stained for 1-3 min, and the sections were dehydrated, cleared, and mounted. Masson staining was used to observe collagen fiber deposition and maturity. After dewaxing and rehydration, sections were pre-stained with Bouin's solution at 56 ℃ for 1 h, stained with Weigert hematoxylin for 5-10 min, stained with Ponceau S-acid fuchsin for 5-10 min, differentiated with 1% phosphomolybdic acid, counterstained with aniline blue, dehydrated, cleared, and mounted.

[0055] 3.3 Results In vivo imaging monitoring results showed that with the extension of restoration time, the ROS levels in the defect areas of each group gradually decreased. Among them, the MNC / ECM / Gel group had the lowest oxidative stress levels at all time points, which can significantly alleviate the oxidative stress level during periodontal regeneration in vivo. Figure 7 A and 7B).

[0056] In terms of alveolar bone regeneration, the ECM / Gel group, due to the loading of BdECM, showed a certain osteogenic effect, with its bone volume fraction, trabecular bone number, and thickness all superior to the blank control group and the Gel group. The MNC / ECM / Gel group performed best among all alveolar bone regeneration indicators, indicating a synergistic enhancement effect between the osteogenic induction capabilities of MNC and BdECM. Figure 7 C and 7D). H&E staining and Masson staining results showed that although more new bone formation was observed in the ECM / Gel group, the periodontal fibers were sparsely arranged, indicating that it was more inclined to promote bone tissue regeneration. In contrast, the surface of the newly formed alveolar bone in the MNC / ECM / Gel group was continuous and smooth, and the periodontal fibers were arranged regularly and densely, showing excellent efficacy in promoting the synergistic regeneration of periodontal soft and hard tissues. Figure 7 E and 7F).

[0057] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A molybdenum-based nanocluster bio-tissue-derived composite scaffold, characterized in that, The invention includes a composite substrate and molybdenum-based nanoclusters loaded on the composite substrate; the composite substrate is composed of bone-derived decellularized extracellular matrix and methacrylamide gelatin, and the molybdenum-based nanoclusters are dispersed in the three-dimensional porous network structure of the composite substrate.

2. The molybdenum-based nanocluster bio-tissue-derived composite scaffold according to claim 1, characterized in that, The degree of substitution of the methacrylamide gelatin is 80-90%, and the concentration is 5-15% (w / v); the amount of bone-derived decellularized extracellular matrix added to the composite substrate is 5-10% (w / v); and the concentration of the molybdenum-based nanoclusters in the composite scaffold is 100-500 μg / mL (w / v).

3. The molybdenum-based nanocluster bio-tissue-derived composite scaffold according to claim 2, characterized in that, The degree of substitution of the methacrylamide gelatin is 90%, and the concentration is 10% (w / v); the amount of bone-derived decellularized extracellular matrix added to the composite substrate is 7% (w / v); and the concentration of the molybdenum-based nanoclusters in the composite scaffold is 300 μg / mL (w / v).

4. A method for preparing a molybdenum-based nanocluster bio-tissue-derived composite scaffold as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Dissolve methacrylamide gelatin in PBS solution containing LAP, heat in a water bath at 60°C until completely dissolved, and cool to room temperature to obtain hydrogel precursor; add bone-derived decellularized extracellular matrix powder at 37°C, stir for 2 h to obtain composite substrate; (2) Add molybdenum-based nanocluster powder to the composite substrate obtained in step (1), stir continuously at 37 °C for 2 h, and then crosslink and cure under ultraviolet light to obtain a composite scaffold.

5. The method for preparing the molybdenum-based nanocluster bio-tissue-derived composite scaffold according to claim 4, characterized in that, The preparation method of the molybdenum-based nanocluster powder includes the following steps: (NH4)6Mo7O 24 ·4H2O was dissolved in water, and NaH2PO4 solution and L-ascorbic acid solution were added. After stirring, ethanol was added to precipitate the mixture. The powder was then purified by centrifugation, dialysis, freeze-drying, and ball milling to obtain molybdenum-based nanoclusters.

6. The method for preparing the molybdenum-based nanocluster bio-tissue-derived composite scaffold according to claim 4, characterized in that, The method for preparing the bone-derived decellularized extracellular matrix includes the following steps: repeatedly freezing and thawing bone blocks, decalcifying with formic acid, sterilizing with peracetic acid, defatting, digesting with DNase I enzyme, lyophilizing, and ball milling at low temperature to obtain bone-derived decellularized extracellular matrix powder.

7. The method for preparing the molybdenum-based nanocluster bio-tissue-derived composite scaffold according to claim 4, characterized in that, The UV curing time is 20-40 seconds.

8. The use of a molybdenum-based nanocluster bio-tissue-derived composite scaffold as described in any one of claims 1-3 in the preparation of a medicament or medical device for treating periodontal soft and hard tissue defects caused by periodontitis.

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