A periosteal material and methods of making and using the same

Abstract

The application discloses a kind of periosteal materials and preparation method and application thereof, belong to the technical field of biomedical materials, including polyvinyl alcohol layer, polycaprolactone layer and methacrylated gelatin layer, polyvinyl alcohol layer, polycaprolactone layer and methacrylated gelatin layer are stacked in turn, and methacrylated gelatin layer is loaded with fracture-derived extracellular vesicles (BFVs).The application uses the above-mentioned periosteal material and its preparation method and application, and the prepared periosteal material has a three-layer structure, can stably release BFVs, improve the aging bone microenvironment through epigenetic reprogramming, significantly promote critical size bone defect repair, especially suitable for elderly individuals.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a periosteum material, its preparation method, and its application. Background Technology

[0002] Critical sized bone defects (CSBDs) are bone defects that cannot heal completely on their own, posing a significant challenge in orthopedics. This is especially true with the accelerating aging of the global population, making the repair of CSBDs in elderly patients even more difficult. The aging bone microenvironment exhibits various pathological changes, including impaired function of key cells, hindered vascular network regeneration, and abnormal regulation of inflammatory factors, leading to delayed healing or even non-healing, severely impacting patients' quality of life.

[0003] Epigenetics refers to mechanisms that directly regulate phenotypes without altering DNA sequences, primarily through transcriptional controls such as DNA methylation. Studies have shown that epigenetic alterations are one of the fourteen major markers of aging. These alterations lead to age-related functional impairments in the bone microenvironment, promoting bone aging. Therefore, treatment strategies targeting epigenetic reprogramming hold promise as a new approach to treating age-related bone damage. 3D printing technology offers possibilities for repairing critical-sized bone defects. This technology can fabricate scaffolds that mimic the layered porous structure and mechanical properties of the natural bone matrix, enabling immediate anatomical reconstruction and long-term biological repair of the defect site. However, clinically applied 3D-printed scaffolds lack bioactive components that improve the aging bone microenvironment and cannot reverse the effects of age-related damage on bone regeneration. For elderly patients, prolonged healing time increases the risk of complications, significantly reduces quality of life, and in severe cases, can even lead to death. Therefore, developing innovative bone repair materials with synergistic therapeutic functions is of significant clinical importance for improving the repair outcomes of large bone defects in elderly patients.

[0004] The periosteum is a dense layer of connective tissue covering the bone surface, playing a crucial role in bone development, growth, remodeling, and repair. Polymer-based periosteum substitutes can mimic the extracellular matrix structure of the natural periosteum and serve as controlled delivery systems for drugs or bioactive molecules. For example, Hao et al. developed a "hamburger-shaped" periosteum material that utilizes silk fibroin hydrogels to control oxygen release during the repair of large bone defects, creating an oxygen-rich environment that effectively combats apoptosis and promotes osteogenic and angiogenesis. Similarly, Zhuang and colleagues designed a periosteum material loaded with zinc oxide and silica nanoparticles, demonstrating significant anti-inflammatory and antibacterial effects through sequential release within the microenvironment of diabetic bone defects.

[0005] In the field of bone therapy for aging, extracellular vesicles (EVs) have attracted widespread attention due to their low immunogenicity and multi-target regulatory effects, representing a promising cell-free therapy. Notably, EVs possess potential epigenetic reprogramming capabilities. Studies have shown that EVs derived from young individuals can delay aging, reduce bone loss, and enhance bone structural stability. Our previous research identified EVs during bone healing, namely fracture-derived extracellular vesicles (BFVs), as an optimized subtype with superior capacity to reverse aging and promote bone regeneration. However, whether this EV subtype (BFVs) can regulate epigenetic function remains unclear. Summary of the Invention

[0006] The purpose of this invention is to provide a periosteal material, its preparation method, and its application. The prepared periosteal material has a three-layer structure, can stably release bone fibrillary vesicles (BFVs), improves the aging bone microenvironment through epigenetic reprogramming, and significantly promotes the repair of critical-size bone defects, making it particularly suitable for elderly individuals.

[0007] To achieve the above objectives, the present invention provides a periosteum material comprising a polyvinyl alcohol (PVA) layer, a polycaprolactone (PCL) layer, and a methacrylamide gelatin (GelMA) layer, wherein the PVA layer, PCL layer, and methacrylamide gelatin layer are stacked sequentially, and the methacrylamide gelatin layer is loaded with fracture-derived extracellular vesicles.

[0008] Preferably, the loading of BFVs in the periosteum material is 50-200 μg / mL.

[0009] Preferably, the polyvinyl alcohol layer is prepared by spin coating of 15-25 wt% PVA1788 solution; the polycaprolactone layer is prepared by spin coating of 3-8 wt% PCL solution; and the methacrylamide gelatin layer is prepared by UV curing of 3-8 wt% GelMA solution, using UV light with a wavelength of 365 nm and an intensity of 10 mW / cm². 2 The illumination time is 20-60 seconds.

[0010] Furthermore, HIF-1α stabilizer can be added to the GelMA solution.

[0011] The present invention also provides a method for preparing a periosteal material, comprising the following steps: Step 1: Preparation of BFVs: Establish a fracture model in juvenile animals, collect serum during the fracture repair period, and extract BFVs; Step 2: Preparation of the three-layer structure of the periosteal material: PVA solution and PCL solution are spin-coated sequentially on a silicon wafer to obtain a polyvinyl alcohol layer and a polycaprolactone layer in sequence; after plasma treatment, a GelMA solution containing BFVs is dropped onto the polycaprolactone layer, and after UV curing, a methacrylamide gelatin layer is obtained, followed by freeze drying to obtain the periosteal material loaded with BFVs.

[0012] Preferably, the specific operation of step one is as follows: establish a juvenile animal fracture model, collect serum during the fracture repair period, extract extracellular vesicles using the ExoQuick kit, and obtain BFVs after centrifugation, filtration and freeze-drying; the BFVs have a particle size of 80-100nm and are identified to express Cd9 and Cd63 marker proteins.

[0013] Preferably, in step one, the juvenile animals are 6-8 week old C57BL / 6 mice; the fracture model is a transverse fracture of the mid-shaft of the femur; the serum collection time is 7-14 days after the fracture; the extraction process of the ExoQuick kit includes: standing overnight at 3-5℃, centrifugation at 1000-2000g for 20-40 min, resuspending in PBS and filtering through a 0.22μm membrane.

[0014] Preferably, in step two, the spin coating process parameters are: 800-1000 rpm, time 30-60s; and the plasma treatment power is 30-50W, time 30-60s.

[0015] This invention also provides an application of a periosteal material, which is used in the repair of bone defects at the critical size of aging. Specifically, it includes the following steps: wrapping the periosteal material around the outer surface of a 3D-printed scaffold and fixing it with sutures or bio-adhesive; the periosteal material improves the bone microenvironment of aging through epigenetic reprogramming; implanting the periosteal material and the 3D-printed scaffold together into the bone defect site; and achieving epigenetic reprogramming through the release of bone follicle cells (BFVs) to promote bone regeneration.

[0016] Preferably, the periosteum material improves the aging bone microenvironment through epigenetic reprogramming by: promoting DNA methylation in the gene body region, upregulating the methylation level and expression level of the Foxo3 gene, thereby alleviating cell senescence, enhancing osteogenic differentiation, regulating the M1 / M2 polarization balance of macrophages, and inhibiting osteoclast formation.

[0017] Preferably, the critical size bone defect for aging is a bone defect with a length greater than 2.5 times the bone diameter. The 3D printed scaffold is prepared using stereolithography technology with a printing resolution of 20-30 μm and a layer thickness of 20-30 μm. The 3D printed scaffold adopts a three-period minimal curved surface structure with a porosity of 50-60% and a pore size of 140-150 μm. The 3D printed scaffold material is one or more of hydroxyapatite, β-tricalcium phosphate, calcium silicate, and calcium sulfate.

[0018] Therefore, the present invention, by employing the above-mentioned periosteal material, its preparation method, and its application, has the following beneficial effects: (1) Innovatively combining epigenetic reprogramming with biomaterials, multi-target regulation of the aging bone microenvironment is achieved through BFV loading; (2) The three-layer structure design takes into account both mechanical performance and biological function. The PVA layer and PCL layer provide mechanical and biological adaptability, and the GelMA layer realizes the slow release of BFVs. (3) The prepared periosteal material works synergistically with the 3D printed scaffold to maintain structural integrity and provide bioactivity, thus solving the clinical problem of bone defect repair in the elderly. (4) Through the epigenetic regulation of the Foxo3 gene, a new mechanism of bone repair was revealed, providing a theoretical basis for the development of new bone repair materials; (5) The preparation process is simple and controllable, the raw materials are widely available, and it has good prospects for clinical translation.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 These are the structural characterization, exosome sustained-release curves, and tensile mechanical property test results of the periosteal materials prepared in Example 1 and Comparative Example 1 of this invention. Figure 1 (a) shows the structural characterization of the periosteum material prepared in Comparative Example 1 from a surface perspective. Figure 1 Image (b) shows the structural characterization of the periosteum material prepared in Example 1 from a surface perspective. Figure 1 (c) shows the structural characterization of the periosteal material prepared in Example 1 from a side view. Figure 1 In Figure (d), the exosome release curve of the periosteum material prepared in Example 1 is shown. Figure 1 (e) in the figure shows the tensile mechanical properties test results of the periosteal materials prepared in Example 1 and Comparative Example 1; Figure 2 This is a graph showing the experimental results of the effect of the periosteal material extracts prepared in Example 1 and Comparative Example 1 of this invention on the cell viability of primary senescent bone marrow mesenchymal stem cells (BMSCs). Figure 2 (a) in the diagram is the experimental process diagram. Figure 2 (b) shows the cell proliferation and toxicity experiment of co-culturing periosteal material extract with primary senescent BMSCs. Figure 2 (c) in the image shows alkaline phosphatase staining images on days 7 and 14. Figure 3The figure shows the experimental results of the effect of the periosteal material extract prepared in Examples 1-3 of this invention on the polarization of RAW 264.7 macrophages. Figure 4 This is an in vivo experimental result of the combination of the periosteal material prepared in Example 1 of the present invention and a 3D printed scaffold for the treatment of critical-size bone defects in the femur of mice; Figure 5 This figure shows the bioinformatics analysis based on RRBS sequencing of the periosteum material prepared in Example 1 of this invention after co-culturing with primary senescent BMSCs, and the discovery and verification results of the key hypermethylated gene Foxo3. Figure 5 (a) shows the expression of Foxo3 mRNA in primary senescent BMSCs. Figure 5 (b) shows the statistical analysis of the immunofluorescence staining intensity of Foxo3 protein in primary senescent BMSCs. Figure 5 (c) is an immunofluorescence staining image of Foxo3 protein in primary senescent BMSCs (scale bar: 50 μm). Figure 6 The graphs show the flow cytometry results of the periosteal material extract prepared in Example 1 of this invention on the M1 / M2 polarization phenotype transformation of macrophages and the flow cytometry results of the reactive oxygen species (ROS) levels in the cells. Figure 6 (a) shows the flow cytometry results of macrophage M1 / M2 polarization phenotype transformation. Cd86 represents the M1 phenotype, and Cd206 represents the M2 phenotype. Figure 6 (b) in the figure represents the flow cytometry results for ROS levels. Figure 6 (c) shows the quantitative analysis of mRNA expression of the M1-related inflammatory factor Tnfα. Figure 6 (d) in the figure represents the quantitative analysis of mRNA expression of M1-related inflammatory factor Il6. Figure 6 (e) represents the quantitative analysis of mRNA expression of the M1-related inflammatory factor Il1β. Figure 6 (f) in the figure represents the enzyme-linked immunosorbent assay (ELISA) results of Tnfα, an M1 phenotype-associated inflammatory cytokine. Figure 6 (g) represents the result of an enzyme-linked immunosorbent assay (ELISA) for M1 phenotype-associated inflammatory factor Il6. Figure 6 (h) represents the result of enzyme-linked immunosorbent assay (ELISA) for Il1β, an M1 phenotype-associated inflammatory factor. Detailed Implementation

[0021] The present invention provides a periosteal material comprising a polyvinyl alcohol layer, a polycaprolactone layer, and a methacrylamide gelatin layer, wherein the polyvinyl alcohol layer, the polycaprolactone layer, and the methacrylamide gelatin layer are stacked sequentially, and the methacrylamide gelatin layer is loaded with BFVs.

[0022] In this invention, the polyvinyl alcohol (PVA) layer serves as a sacrificial layer to maintain material rigidity, facilitating preoperative suturing and wrapping. The polycaprolactone (PCL) layer, as a mechanical support layer, is located below the PVA layer and is used to enhance the mechanobiocompatibility of the 3D printed scaffold and prevent soft tissue from invading the bone defect site. A methacrylamide gelatin (GelMA) layer, serving as a functional layer, lies beneath the PCL layer.

[0023] In this invention, the loading of BFVs in the periosteum material is 50-200 μg / mL.

[0024] In this invention, the polyvinyl alcohol layer is prepared by spin coating of 15-25 wt% PVA1788 solution; the polycaprolactone layer is prepared by spin coating of 3-8 wt% PCL solution; and the methacrylamide gelatin layer is prepared by UV curing of 3-8 wt% GelMA solution, using a UV wavelength of 365 nm and a light intensity of 10 mW / cm². 2 The illumination time is 20-60 seconds.

[0025] In this invention, HIF-1α stabilizer can be added to the GelMA solution.

[0026] The present invention also provides a method for preparing a periosteal material, comprising the following steps: Step 1: Preparation of BFVs: Establish a fracture model in juvenile animals, collect serum during the fracture repair period, and extract BFVs; Step 2: Preparation of the three-layer structure of the periosteal material: PVA solution and PCL solution are spin-coated sequentially on a silicon wafer to obtain a polyvinyl alcohol layer and a polycaprolactone layer in sequence; after plasma treatment, a GelMA solution containing BFVs is dropped onto the polycaprolactone layer, cured by ultraviolet light and then freeze-dried to obtain a methacrylamide gelatin layer, which is then freeze-dried to obtain the periosteal material loaded with BFVs.

[0027] In this invention, the release of BFVs is monitored: the periosteal material loaded with BFVs is immersed in simulated body fluid, and the release of BFVs is continuously monitored for 14 days at 37°C and 100 rpm.

[0028] In this invention, the specific operation of step one is as follows: establish a juvenile animal fracture model, collect serum during the fracture repair period, extract extracellular vesicles using the ExoQuick kit, and obtain BFVs after centrifugation, filtration and freeze-drying; the BFVs have a particle size of 80-100nm and are identified to express Cd9 and Cd63 marker proteins.

[0029] In this invention, BFVs can be replaced with artificially synthesized nanoparticles containing similar epigenetic regulatory factors.

[0030] In this invention, in step one, the juvenile animals are 6-8 week old C57BL / 6 mice; the fracture model is a transverse fracture of the mid-shaft of the femur; the serum collection time is 7-14 days after the fracture; the extraction process of the ExoQuick kit includes: standing overnight at 3-5℃, centrifugation at 1000-2000g for 20-40 min, resuspending in PBS and filtering through a 0.22μm membrane.

[0031] In this invention, in step two, the spin coating process parameters are: 800-1000 rpm, time 30-60s; the plasma treatment power is 30-50W, time 30-60s.

[0032] This invention also provides an application of a periosteal material, which is used in the repair of bone defects at the critical size of aging. Specifically, it includes the following steps: wrapping the periosteal material around the outer surface of a 3D-printed scaffold and fixing it with sutures or bio-adhesive; the periosteal material improves the bone microenvironment of aging through epigenetic reprogramming; implanting the periosteal material and the 3D-printed scaffold together into the bone defect site; and achieving epigenetic reprogramming through the release of bone follicle cells (BFVs) to promote bone regeneration.

[0033] In this invention, the periosteum material improves the aging bone microenvironment through epigenetic reprogramming by: promoting DNA methylation in the gene body region, upregulating the methylation level and expression level of the Foxo3 gene, thereby alleviating cell senescence, enhancing osteogenic differentiation, regulating the M1 / M2 polarization balance of macrophages, and inhibiting osteoclast formation.

[0034] In this invention, the critical-size bone defect of aging is a bone defect with a length greater than 2.5 times the bone diameter. The 3D printed scaffold is prepared using stereolithography technology with a printing resolution of 20-30 μm and a layer thickness of 20-30 μm. The 3D printed scaffold adopts a three-period minimal curved surface structure with a porosity of 50-60% and a pore size of 140-150 μm. The 3D printed scaffold material is one or more of hydroxyapatite, β-tricalcium phosphate, calcium silicate, and calcium sulfate.

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims and are all within the protection scope of the present invention.

[0036] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0037] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0038] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0039] Example 1 This invention provides a periosteal material, the preparation method of which includes the following steps: (1) Preparation and characterization of BFVs: A transverse femoral fracture model was established in 6-8 week old C57BL / 6 mice. Serum samples were collected 7-14 days after surgery, and BFVs were extracted according to the ExoQuick kit instructions. The extraction process included: mixing serum and ExoQuick reagent at a volume ratio of 1:5 and incubating overnight at 4℃; centrifuging at 1500g for 30min to collect the precipitate; resuspending the precipitate with PBS; filtering with a 0.22μm filter; and freeze-drying and storing at -80℃.

[0040] The extracted BFVs were characterized: Transmission electron microscopy (TEM) showed that the BFVs were spherical or elliptical, with a particle size of approximately 90-100 nm, and possessed a typical bilayer membrane structure; nanoparticle tracking analysis (NTA) showed that the particle size distribution was mainly concentrated at 90 nm; Western blot analysis confirmed that the BFVs expressed the EV marker proteins Cd9 and Cd63. These results indicate that the extracted BFVs conform to the basic characteristics of EVs.

[0041] (2) Preparation of epigenetic reprogrammed periosteum material: PVA1788 (molecular weight 130,000) and PCL (molecular weight 80,000) were dissolved in deionized water to prepare 20wt% PVA1788 solution and 5wt% PCL solution. The silicon wafer was fixed on a spin coater, PVA solution was added dropwise, and spin-coated at 800rpm for 30s; then PCL solution was added dropwise, and spin-coated under the same conditions. The spin-coated sample was placed in a plasma processor and treated at 30W power for 60s. Subsequently, 5wt% GelMA solution (containing 100μg / mL BFVs) was added dropwise to the PCL layer, and after uniform spreading, it was treated with 365 nm ultraviolet light (10mW / cm²). 2 The sample was irradiated for 60 seconds for photocuring. Finally, the sample was freeze-dried to obtain a three-layer periosteum material loaded with BFVs (P-BFVs).

[0042] (3) Characterization of periosteal material: Scanning electron microscopy (SEM) showed that randomly arranged BFVs were distributed on the surface of the GelMA layer of P-BFVs, and the three-layer structure was clearly shown in the oblique view. Confocal microscopy confirmed that BFVs were uniformly distributed in the GelMA functional layer. The thickness of the periosteal material after freeze-drying was about 0.14 mm, and the thickness after swelling was 0.44-0.46 mm. The BFVs release curve showed that there was rapid release in the early stage, and a stable sustained release stage was entered after 4 days. The cumulative release rate reached 80% after 14 days. Tensile test showed that the stress-strain curves of P-BFVs and P-Blank overlapped in the early stage. The first fracture occurred when the strain reached 200%, with an average fracture strength of 0.32 MPa, an average tensile modulus of 0.22 MPa, an average elongation at break of 200%, and an average fracture load of 0.49 N.

[0043] Example 2 The only difference between this embodiment and Embodiment 1 is that the loading of BFVs in this embodiment is 50 μg / mL, while all other conditions are the same.

[0044] Example 3 The only difference between this embodiment and Embodiment 1 is that the loading of BFVs in this embodiment is 200 μg / mL, while all other conditions are the same.

[0045] Comparative Example 1 The only difference between this comparative example and Example 1 is that the periosteal material does not contain BFVs and is denoted as P-Blank; all other conditions are the same.

[0046] Figure 1 The structural and performance characterization results showed that scanning electron microscopy confirmed that the periosteal material prepared in Example 1 exhibited a clear three-layer stacked structure. Figure 1 In (c) of the GelMA functional layer, BFVs are uniformly distributed on the surface. Figure 1(b) of the example, while Comparative Example 1, lacking BFVs, exhibits a relatively smooth surface morphology. Figure 1 (a) of the text. (The rest of the text appears to be incomplete and requires further context.) Figure 1 (d) indicates that the material exhibits typical biphasic release characteristics: approximately 20% of BFVs are rapidly released within the initial 24 hours to initiate repair signals, followed by a sustained and stable slow-release phase, with a cumulative release rate of 80% after 14 days. This release pattern is beneficial for maintaining long-term bioactivity. Mechanical property testing ( Figure 1 (e) shows that the periosteal material loaded with BFVs (P-BFVs) has similar stress-strain behavior to the blank control (P-Blank), with an elongation at break of 200% and a tensile modulus of 0.22 MPa. This indicates that the introduction of BFVs did not significantly impair the flexibility and mechanical integrity of the material, and met the operational requirements for intraoperative suturing and wrapping.

[0047] Test Example 1 The periosteal materials prepared in Examples 1-3 and Comparative Examples 1-4 were subjected to in vitro cell experiments and in vivo animal experiments, and the specific procedures are as follows: (1) Fabrication of 3D-printed scaffolds: Two models were designed using Materialise Magics software: a cylindrical model (height-to-diameter ratio 0.3) for in vitro experiments and a model based on mouse femur dimensions (diameter 21 mm, height 30 mm, with a central cavity for intramedullary nail fixation) for in vivo experiments. Both models were filled with Gyroid-type three-period minimal surfaces with a designed porosity of 55%. Stereolithography (SLA) was used to 3D print the β-tricalcium phosphate scaffold at a resolution of 25 μm and a layer thickness of 25 μm. After sintering, the actual porosity of the scaffold was approximately 54%, with a pore size of approximately 144 μm, and all pores were interconnected.

[0048] (2) In vitro cell experiments: Primary senescent bone marrow mesenchymal stem cells (BMSCs) were isolated from the femurs of 20-month-old C57BL / 6 mice. Flow cytometry analysis showed high expression of CD44 (>90%). The periosteum material was immersed in culture medium and incubated at 37°C for 72 hours to prepare the extract. CCK-8 assay results are shown below. Figure 2 As shown, Figure 2 Cell compatibility and osteogenic differentiation experiments ( Figure 2 (a) further validates the biological function of the material. CCK-8 assay ( Figure 2 (b) indicates that P-BFVs extract has no cytotoxicity to primary senescent bone marrow mesenchymal stem cells and significantly promotes cell proliferation; alkaline phosphatase staining ( Figure 2 (c) shows that osteogenic differentiation activity increased in a time-dependent manner on days 7 and 14, confirming that BFV loading can effectively reverse osteogenic dysfunction in senescent cells.

[0049] qPCR and immunofluorescence staining showed that P-BFVs extract significantly downregulated the expression of aging-related genes (Il6, P16, P21, P53) and proteins (β-gal, γH2A.x), and upregulated the expression of osteogenic-related genes (Runx2, Col1, Alp, Ocn) and proteins.

[0050] Figure 3 Macrophage polarization experiments revealed that P-BFVs extract significantly inhibited LPS-induced M1 polarization (reducing Cd86 positivity from 64.2% to 40.5%), promoted M2 polarization (increasing Cd206 positivity to 34.5%), reduced ROS accumulation, decreased the expression of pro-inflammatory factors (Tnfα, Il6, Il1β), and increased the expression of anti-inflammatory factors (Il10, Tgfβ). Figure 3 It was found that as the BFV loading increased from 50 μg / mL to 200 μg / mL (Examples 2-3), the proportion of M2 macrophages increased in a dose-dependent manner, with 100 μg / mL (Example 1) achieving the best balance between anti-inflammatory effect and cost-effectiveness.

[0051] Osteoclast assays showed that P-BFVs extract significantly inhibited RANKL-induced osteoclast formation and downregulated the expression of osteoclast-related genes (Trap, Nfatc1, Ctsk, Rank).

[0052] Figure 6 Further immunomodulatory analysis clarified that P-BFVs extract could reduce the proportion of LPS-induced M1 macrophages from 64.2% to 40.5%, while increasing the proportion of M2 macrophages to 34.5%. Figure 6 (a) of the above, accompanied by a decrease in ROS levels ( Figure 6 The mRNA and protein expression of pro-inflammatory factors Tnfα, Il6, and Il1β were significantly inhibited (b) in the study. Figure 6 The (c)-(h) in the figure indicate that the material creates favorable conditions for bone regeneration by regulating the immune microenvironment.

[0053] (3) In vivo animal experiments: C57BL / 6 mice with critical femoral bone defect models (defect length 3 mm) were established at 6 months (mature) and 18 months (aged). Experimental groups: 3D printed scaffold implanted alone (MS and SS groups); 3D printed scaffold + blank periosteum material (MSP-Blank and SSP-Blank groups); 3D printed scaffold + BFVs loaded with periosteum material (SSP-BFVs group). Animals were sacrificed 12 weeks after surgery, and samples were obtained for micro-CT and histological analysis.

[0054] Micro-CT reconstruction showed that the implants in the periosteal material combination group maintained good structural integrity and original morphological length, while the single scaffold group showed compression fractures and fragmentation. Quantitative analysis showed that the bone volume (BV), bone volume fraction (BV / TV), and bone surface area (BS) of the SSP-BFVs group were significantly higher than those of the SSP-Blank group, reaching levels comparable to the MSP-Blank group. Histological analysis (HE and Masson staining) confirmed that the SSP-BFVs group had more mature new bone formation. Immunohistochemistry showed that the expression of the aging marker β-gal was decreased and the expression of the osteogenic markers Runx2 and Col1 was increased in the SSP-BFVs group.

[0055] Figure 4 Micro-CT and histological analysis visually demonstrated the in vivo bone damage repair effect: the SSP-BFVs group showed a complete scaffold morphology and abundant new bone tissue at 12 weeks after surgery, with a bone volume fraction significantly higher than the blank periosteum group and close to the repair level of young mice, while the scaffold alone group showed obvious compression deformation and poor bone integration.

[0056] (4) RRBS sequencing and bioinformatics analysis: RRBS sequencing of bone defect healing tissue revealed 174 hypermethylated genes and 24 hypomethylated genes at CpG sites in the SSP-BFVs group compared to the SSP-Blank group. The methylation level in the gene body region was significantly increased, while the promoter region showed no significant change. GO and KEGG analyses showed that hypermethylated genes are mainly involved in the regulation of immune responses, myeloid cell differentiation, and neurogenesis. The Foxo3 gene was identified as a key regulatory factor, with increased methylation level and enhanced expression in the gene body region. In vitro and in vivo validation experiments confirmed that the P-BFVs extract significantly upregulated the mRNA and protein expression of Foxo3 in senescent BMSCs and macrophages, and the nuclear expression of Foxo3 was enhanced in the bone defect healing tissue of the SSP-BFVs group.

[0057] Figure 5 RRBS sequencing and validation experiments revealed that BFVs achieve epigenetic reprogramming by promoting DNA methylation in the genome region, and the methylation level and expression level of the Foxo3 gene are simultaneously upregulated. Figure 5 (a)-(c) of the transcription factor, enhanced nuclear localization of this transcription factor may be a key molecular switch for alleviating cellular senescence and promoting osteogenic differentiation.

[0058] In conclusion, Figures 1 to 6 The complete chain of evidence fully confirms that the three-layer periosteum material of the present invention not only has suitable mechanical properties and sustained-release characteristics, but also improves the aging bone microenvironment in multiple dimensions through BFV-mediated epigenetic reprogramming, providing an innovative solution for the repair of critical-sized bone defects in the elderly.

[0059] In vitro cell experiments in Example 2 showed that this dose could also significantly improve the osteogenic differentiation capacity of senescent BMSCs, but the effect was weaker than that in Example 1.

[0060] In vitro cell experiments in Example 3 showed that this dose had a similar effect on improving senescent BMSCs as in Example 1, but at a higher cost.

[0061] In vitro experiments in Comparative Example 1 demonstrated that the material significantly reduced the improvement effect on aging BMSCs.

[0062] Comparing the test results of Examples 1-3 and Comparative Example 1, it can be seen that Examples 1-3, by designing a three-layer periosteum structure loaded with BFVs, can effectively improve the bone microenvironment in aging and promote the repair of critical-sized bone defects. Furthermore, comparing Examples 1-3 shows that Example 1 achieved the best results with a BFV loading of 100 μg / ml and a Gyroid scaffold structure. Comparative Example 1 lacked the key component BFVs, resulting in a significant decrease in its bone repair-promoting performance.

[0063] In summary, the periosteum material of the present invention exhibits excellent biological functions: (1) In vitro experiments have shown that the extract of this material can significantly reduce aging markers (β-gal, γH2A.x, p16, p21) in senescent bone marrow mesenchymal stem cells (BMSCs), enhance osteogenic differentiation capacity, and upregulate the expression of osteogenic-related genes (Runx2, Col1, Alp, Ocn); (2) This material can regulate macrophage polarization, inhibit M1 macrophages (high expression of Cd86), promote M2 macrophages (high expression of Cd206), reduce the accumulation of reactive oxygen species (ROS), reduce the expression of pro-inflammatory factors (Tnfα, Il6, Il1β), and increase the expression of macrophages. (3) The material can inhibit osteoclast formation and downregulate the expression of osteoclast-related genes (Trap, Nfatc1, Ctsk, Rank); (4) In vivo experiments have shown that the material, when used in combination with a 3D printed scaffold, can maintain structural integrity, significantly promote the repair of critical-sized bone defects in aged mice, and improve bone regeneration capacity to the level of young mice; (5) RRBS sequencing analysis revealed that the material promotes epigenetic reprogramming by promoting DNA methylation in the gene body region, especially upregulating the methylation level and expression of the Foxo3 gene, which is a key transcription factor that regulates cell senescence, osteogenic differentiation and immune balance.

[0064] Therefore, the present invention uses the above-mentioned periosteal material, its preparation method and application. The prepared periosteal material has a three-layer structure, can stably release BFVs, improve the aging bone microenvironment through epigenetic reprogramming, and significantly promote the repair of critical-size bone defects, which is especially suitable for elderly individuals.

[0065] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A periosteum material, characterized in that: It includes a polyvinyl alcohol layer, a polycaprolactone layer, and a methacrylamide gelatin layer, which are stacked sequentially, and the methacrylamide gelatin layer is loaded with fracture-derived extracellular vesicles.

2. The periosteal material according to claim 1, characterized in that: The loading of BFVs in the periosteal material is 50-200 μg / mL.

3. The periosteal material according to claim 1, characterized in that: The polyvinyl alcohol layer was prepared by spin coating with a 15-25 wt% PVA1788 solution; the polycaprolactone layer was prepared by spin coating with a 3-8 wt% PCL solution; and the methacrylamide gelatin layer was prepared by UV curing with a 3-8 wt% GelMA solution at a wavelength of 365 nm and an intensity of 10 mW / cm². 2 The illumination time is 20-60 seconds.

4. A method for preparing a periosteal material according to any one of claims 1-3, characterized in that: Includes the following steps: Step 1: Preparation of BFVs: Establish a fracture model in juvenile animals, collect serum during the fracture repair period, and extract BFVs; Step 2: Preparation of the three-layer structure of the periosteal material: PVA solution and PCL solution are spin-coated sequentially on a silicon wafer to obtain a polyvinyl alcohol layer and a polycaprolactone layer in sequence; after plasma treatment, a GelMA solution containing BFVs is dropped onto the polycaprolactone layer, and after UV curing, a methacrylamide gelatin layer is obtained, followed by freeze drying to obtain the periosteal material loaded with BFVs.

5. The method for preparing a periosteal material according to claim 4, characterized in that: The specific operation of step one is as follows: establish a juvenile animal fracture model, collect serum during the fracture repair period, extract extracellular vesicles using the ExoQuick kit, and obtain BFVs after centrifugation, filtration and freeze-drying; the particle size of BFVs is 80-100nm, and the expression of Cd9 and Cd63 marker proteins is identified.

6. The method for preparing a periosteal material according to claim 5, characterized in that: In step one, the juvenile animals were 6-8 week old C57BL / 6 mice; the fracture model was a transverse fracture of the midshaft of the femur; the serum was collected 7-14 days after the fracture; the extraction process of the ExoQuick kit included: overnight incubation at 3-5℃, centrifugation at 1000-2000g for 20-40 min, resuspending in PBS, and filtration through a 0.22μm membrane.

7. The method for preparing a periosteal material according to claim 4, characterized in that: In step two, the spin coating process parameters are: 800-1000 rpm, time 30-60s; the plasma treatment power is 30-50W, time 30-60s.

8. An application of a periosteal material, characterized in that: Applying the periosteal material according to any one of claims 1-3 to the repair of age-critical bone defects specifically includes the following steps: wrapping the periosteal material around the outer surface of a 3D-printed scaffold and fixing it with sutures or bio-adhesive; the periosteal material improves the bone microenvironment of aging through epigenetic reprogramming; implanting the periosteal material and the 3D-printed scaffold together into the bone defect site; and achieving epigenetic reprogramming through the release of bone follicle cells (BFVs) to promote bone regeneration.

9. The application of the periosteal material according to claim 8, characterized in that: Periosteal materials improve the aging bone microenvironment through epigenetic reprogramming, specifically by promoting DNA methylation in the gene body region, upregulating the methylation and expression levels of the Foxo3 gene, thereby alleviating cell senescence, enhancing osteogenic differentiation, regulating the M1 / M2 polarization balance of macrophages, and inhibiting osteoclast formation.

10. The application of the periosteal material according to claim 8, characterized in that: The critical size of bone defects in aging is defined as bone defects with a length greater than 2.5 times the bone diameter. The 3D printed scaffold is prepared using stereolithography technology with a printing resolution of 20-30 μm and a layer thickness of 20-30 μm. The 3D printed scaffold adopts a three-period minimal curved surface structure with a porosity of 50-60% and a pore size of 140-150 μm. The 3D printed scaffold material is one or more of hydroxyapatite, β-tricalcium phosphate, calcium silicate, and calcium sulfate.

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