Electrostatic spinning nanofiber membrane for promoting jaw defect osteogenesis and preparation method

Abstract

The invention discloses an electrostatic spinning nanofiber membrane for promoting jawbone defect osteogenesis and a preparation method, relates to the technical field of medical tissue engineering materials, and aims to solve the problem that a tissue engineering scaffold applied to periodontal tissue regeneration is mostly soaked in a solution, and the physical loading mode is not beneficial to long-term stable release of growth factors. In the prior art, metal ions are often used for activating osteoblasts, but the activation effect is limited, so that the metal ions are difficult to promote the regeneration of periodontal tissues because the metal ions are difficult to fully mobilize the initiative of the cells. The invention provides an electrostatic spinning nanofiber membrane for promoting jaw defect osteogenesis and a preparation method thereof. The preparation method comprises the following steps: preparing freeze-dried CGF powder; preparing a PVA / SA / nHA solution, and adding the freeze-dried CGF powder to obtain a spinning solution; and collecting the spinning solution on an aluminum foil roller to obtain the PVA / SA / CGF / nHA electrostatic spinning nanofiber membrane. Concentrated growth factors can be continuously and stably released, and osteogenic differentiation of peripheral jawbones is promoted.

Description

Technical Field

[0001] This invention relates to the field of medical tissue engineering materials technology, specifically to an electrospun nanofiber membrane that promotes osteogenic formation in jawbone defects and its preparation method. Background Technology

[0002] In the field of jawbone defect repair, the preparation and functionalization of tissue-engineered electrospun fiber membranes are crucial for achieving efficient bone regeneration. Current research mainly focuses on electrospun nanocomposites, hydrogels, and growth factor loading technologies, but significant technical bottlenecks still exist. 1. Limitations of electrospun nanocomposites Existing research primarily employs electrospinning technology to prepare polymer fiber membranes such as polylactic acid (PLA) and polycaprolactone (PCL) for periodontal bone defect repair and neurovascular regeneration. PCL / gelatin composite fiber membranes prepared by electrospinning have been shown to promote osteoblast adhesion; however, the structural uniformity of the fiber membranes makes it difficult to synergistically regulate their mechanical properties and biological activities. Furthermore, simple polymer scaffolds lack the ability to actively regulate cell behavior, requiring further loading with active ingredients to enhance osteogenic efficacy.

[0003] 2. The Synergistic Challenge of Vascularization and Osteogenesis in Hydrogels Hydrogels are widely used for bone defect repair due to their high water content and biomimetic extracellular matrix properties. For example, gelatin-methacrylamide (GelMA) hydrogels can be loaded with vascular endothelial growth factor (VEGF) to promote angiogenesis. However, the degradation rate of hydrogels does not match the osteogenic process well, and loading a single growth factor is difficult to achieve spatiotemporal synergy between angiogenesis and osteogenic differentiation, thus limiting repair efficiency.

[0004] 3. Defects of growth factor loading methods Current tissue engineering scaffolds often use solution immersion to directly load growth factors (such as BMP-2 and VEGF), but this method suffers from burst release effects and insufficient long-term stability. The physical adsorption loading method makes growth factors susceptible to environmental factors (such as pH and enzymatic hydrolysis), resulting in uncontrollable release curves and an inability to maintain effective concentrations to continuously activate osteogenic signaling pathways.

[0005] 4. Inefficiency of metal ion activation Metal ions (such as Mg²⁺ and Zn²⁺) are used to activate osteoblast function due to their low cost and good biocompatibility. Mg²⁺ can promote osteogenic differentiation by upregulating Runx2 and ALP expression, but its effect depends on the ion release concentration. However, the sustained-release capacity of metal ions in the scaffold is limited, making it difficult to maintain an effective stimulating concentration over a long period. In addition, single metal ions are difficult to simultaneously regulate multicellular behaviors (such as the synergy between osteoblasts and vascular endothelial cells), resulting in insignificant periodontal tissue regeneration effects.

[0006] In summary, there is an urgent need to develop a composite scaffold that combines mechanical adaptability, long-term slow release of growth factors, and osteogenic promotion mechanisms to achieve efficient repair of jawbone defects through multi-mechanism synergistic activation of osteoblasts and vascular endothelial cells. Summary of the Invention

[0007] To address the aforementioned problems, this invention aims to provide an electrospun nanofiber membrane that promotes osteogenic formation in jawbone defects and its preparation method, which can continuously and stably release concentrated growth factors to promote osteogenic differentiation of the surrounding jawbone.

[0008] The main idea of ​​the technical solution adopted in this invention is as follows: Fresh CGF is prepared by obtaining fresh human blood, and the activity and efficacy of CGF growth factors are preserved by freeze-drying to better promote osteogenic formation in the jawbone defect area. The addition of nano-hydroxyapatite (nHA) significantly improves the bioactivity and cell proliferation promotion capacity of the PVA / SA electrospun membrane. By coating the nanofiber membrane with concentrated growth factors, nano-hydroxyapatite, polyvinyl alcohol, and sodium alginate, concentrated growth factors can be released continuously and for a long period into the bone defect area of ​​the jawbone, promoting osteogenic differentiation of the surrounding jawbone and exerting a certain anti-inflammatory effect, effectively promoting jawbone tissue regeneration and rebuilding the biological function of the jawbone.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing an electrospun nanofiber membrane that promotes osteogenic formation in jawbone defects includes the following steps: Step 1: Prepare freeze-dried CGF powder; Step 2: Prepare a PVA / SA / nHA solution and add lyophilized CGF powder to obtain a spinning solution; Step 3: Collect the spinning solution on an aluminum foil roller to obtain a PVA / SA / CGF / nHA nanofiber membrane.

[0010] Based on the above technical solution, the specific steps for preparing freeze-dried CGF powder in step one include: centrifuging the venous blood extracted from the human body, separating it, placing the separated CGF in a freeze dryer to freeze-dry for 48 hours, grinding it into powder, and storing it at -80°C.

[0011] Based on the above technical solution, the specific steps for preparing the PVA / SA / nHA solution in step two are as follows: 6 w / v% PVA and 1 w / v% SA mixed powder are added to deionized water and stirred thoroughly in a 60°C water bath for 8 hours; then 3 w / v% nHA are added to the PVA / SA solution, and the mixture is stirred and ultrasonically vibrated for 4 hours.

[0012] Further, based on the above technical solution, the specific preparation method of the spinning solution is as follows: 1 w / v% lyophilized CGF powder is added to a PVA / SA / nHA solution and stirred at 37°C for 3 h to obtain the spinning solution.

[0013] Based on the above technical solution, further, in step three, an electrospinning device is used, and its specific operating parameters are set as follows: The electrospinning device was set to 20kV voltage, 0.5mL / h flow rate, and 50%–55% relative humidity; The spinning solution is placed into a 10mL syringe with an inner diameter of 0.6mm. An aluminum foil roller is provided on the needle tip side of the syringe, and the distance between the needle tip and the aluminum foil is 15cm.

[0014] Further, using the above technical solutions: In step three, after the PVA / SA / CGF / nHA nanofiber membrane is prepared, it is placed in a vacuum drying oven at 37°C for 12 hours to dry.

[0015] An electrospun nanofiber membrane that promotes osteogenic formation in jawbone defects is prepared by any of the above-described preparation methods.

[0016] Furthermore, through the above technical solution, the nanofiber membrane is coated with PVA, SA, CGF and nHA.

[0017] Furthermore, through the above technical solution, the CGF is continuously released during the use of the nanofiber membrane.

[0018] The application of the electrospun nanofiber membrane that promotes osteogenesis in jawbone defects as described above in bone regeneration of jawbone tissue and periodontal tissue.

[0019] The beneficial effects of this invention are: 1. CGF (Concentrated Growth Factor), as a new generation of platelet concentrate, can promote tissue repair, angiogenesis, cell migration, and differentiation, and has been proven to contribute to the regeneration of bone, periodontal, and dental pulp tissues. CGF is readily available in clinical settings and can be used for electrospinning preparations, or its activity can be preserved through lyophilization. Electrospun PVA / SA / CGF / nHA nanofiber membranes can significantly enhance the proliferation, migration, and osteogenic differentiation capabilities of human bone marrow mesenchymal stem cells (hBMSCs).

[0020] 2. The addition of CGF (concentrated growth factor) and nHA (nano-hydroxyapatite) not only improves the physical properties of electrospun nanofiber membranes, but also promotes good cellular biological responses. The synergistic effect of the two demonstrates their potential application in bone tissue engineering repair in regenerative medicine.

[0021] 3. Electrospun PVA / SA / CGF / nHA nanofiber membranes have demonstrated excellent osteogenic differentiation capabilities in both in vitro and in vivo experiments, providing a new treatment option for patients with defects or loss of jawbone and alveolar bone, and can also achieve the regeneration of other tissues.

[0022] 4. Electrospun PVA / SA / CGF / nHA nanofiber membranes utilize the advantages of CGF and nHA to create a sustained-release system that can continuously release platelet concentrates according to different stages of jawbone tissue repair, effectively promoting osteogenic differentiation. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the technical route of the present invention.

[0024] Figure 2 The diagram shows the morphological characteristics and FTIR spectra of nanofibers; (a) is an image of pulverized CGF powder; (b) are representative SEM images of PVA / SA, PVA / SA / CGF and PVA / SA / CGF / nHA nanofibers; (c) is a frequency distribution diagram of nanofiber diameter; (d) is a schematic diagram of the average diameter of nanofibers; (e) is the FTIR spectrum of freeze-dried CGF powder and PVA / SA, PVA / SA / CGF and PVA / SA / CGF / nHA nanofibers; and (f) is a schematic diagram of the interaction between the components of nanofibers studied by FTIR. Statistical significance is defined as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0025] Figure 3This is a schematic diagram illustrating the physical properties of the electrospun nanofibers of the present invention; wherein, (a) is a representative image of the water contact angle of PVA / SA, PVA / SA / CGF, and PVA / SA / CGF / nHA nanofibers at 4s, (b) is an image of the average water contact angle of the nanofibers at 1s, 4s, and 8s, (c) is a schematic diagram of the swelling ratio of the nanofibers in PBS solution at 5min and 24h, (d) is a SEM image of the PVA / SA, PVA / SA / CGF, and PVA / SA / CGF / nHA nanofibers after 14d of degradation, (e) is an image of the weight loss of the nanofibers in PBS solution over 14d, and (f) is an image of the cumulative release of CGF from the nanofibers. Statistical significance is defined as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0026] Figure 4 The diagram below shows the experimental flowchart and soft tissue healing evaluation of the rat tooth extraction model of this invention; (a) is a schematic diagram of the rat tooth extraction model and the treatment flowchart at each time point. (b) is a schematic diagram of the quantitative analysis of the closure of the tooth extraction wound in rats at 3, 7, and 14 days after extraction. (c) is a representative image showing the changes in soft tissue healing of the tooth extraction wound in each group of rats over time. Statistical significance is defined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. M1, M2, and M3 refer to the first molar, second molar, and third molar, respectively. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0028] The inventors discovered that tissue engineering scaffolds used for periodontal tissue regeneration often involve directly loading growth factors onto the scaffold using a solution soaking method. This physical loading method is not conducive to the long-term stable release of growth factors. Existing technologies also frequently use metal ions (such as magnesium and zinc ions) to activate osteoblasts, but the activation effect is limited, making it difficult for metal ions to fully mobilize the cells' initiative and thus hindering the promotion of periodontal tissue regeneration.

[0029] Based on the above findings, this application proposes an electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects and its preparation method. By coating the nanofiber membrane with concentrated growth factors, nano-hydroxyapatite, polyvinyl alcohol, and sodium alginate, concentrated growth factors can be released continuously and over a long period into the bone defect area of ​​the jawbone, promoting osteogenic differentiation of the surrounding jawbone and exerting a certain anti-inflammatory effect, effectively promoting jawbone tissue regeneration and reconstructing the biological function of the jawbone. Example 1

[0030] See Figure 1 - Figure 4 This application discloses an electrospun nanofiber membrane that promotes osteogenic formation in jawbone defects and its preparation method. The nanofiber membrane is coated with concentrated growth factor (CGF), nano-hydroxyapatite (nHA) (Aladdin, Shanghai, China), polyvinyl alcohol (PVA) (Aladdin, Shanghai, China) and sodium alginate (SA) (Aladdin, Shanghai, China).

[0031] Nanofiber membranes can release concentrated growth factors into the bone defect area of ​​the jawbone in a long-term and continuous manner, promote osteogenic differentiation of the surrounding jawbone, and exert a certain anti-inflammatory effect, effectively promoting the regeneration of jawbone tissue and rebuilding the biological function of the jawbone. Example 2

[0032] The preparation method of the nanofiber membrane described in Example 1 is as follows: 1. Venous blood samples were collected from six healthy, non-smoking volunteers aged 20 to 30 years. Eight ml of blood was drawn from a vein in the arm using a CGF-specific vacuum blood collection device (without anticoagulant) with a red cap, and centrifuged according to the preset protocol on a high-speed centrifuge. After centrifugation, the product in the test tube separated into three layers: the top layer was serum, the middle layer was CGF fibrin gel, and the bottom layer consisted of red blood cells and platelets. After separating the middle CGF layer, it was freeze-dried for 48 hours to create a porous CGF structure, then ground into powder and stored at -80°C.

[0033] 2. Add a certain volume of deionized water to a mixture of 6 w / v% PVA and 1 w / v% SA powder, and stir thoroughly in a 60°C water bath for 8 hours. Then, add 3 w / v% nHA to the PVA / SA solution, and stir and sonicate thoroughly for 4 hours. Finally, add 1 w / v% lyophilized CGF powder to the PVA / SA or PVA / SA / nHA solution, and stir at 37°C for 3 hours to prepare the spinning solution.

[0034] 3. Electrospinning was performed using an electrospinning apparatus at a voltage of 20 kV, a flow rate of 0.5 mL / h, and a relative humidity of 50%–55%. The spinning solution was placed in a 10 mL syringe with a needle inner diameter of 0.6 mm, and the distance between the needle tip and the aluminum foil was 15 cm. The fibers were collected on a grounded aluminum foil roller. PVA / SA, PVA / SA / CGF, and PVA / SA / CGF / nHA nanofibers were prepared using this method. The collected nanofiber membranes were dried in a vacuum drying oven at 37 °C for 12 h.

[0035] 4. Before the experiment, the cut nanofiber membrane was placed in a 3 w / v% calcium chloride anhydrous ethanol solution for crosslinking for 4 hours. After crosslinking, the nanofiber membrane was placed in a vacuum drying oven at 37°C for 4 hours. Example 3

[0036] The nanofiber membrane prepared in Example 2 was subjected to material characterization and observation.

[0037] The surface morphology and fiber diameter of the fibers were observed under a scanning electron microscope (SEM-JSM-6390LV, Japan). The nanofiber membranes were cut into uniformly thick squares of 10*10 mm and sputtered with gold onto a conductive plate. SEM images were then captured at a working voltage of 15 kV. The average fiber diameter was measured using Image-J software from at least three images of each sample.

[0038] Intermolecular bonds and functional groups in the samples were investigated using FTIR spectroscopy (Nicolet iS10, USK). Infrared absorption peaks were recorded in the wavelength range of 400–4000 cm⁻¹, and the bond types were determined. This experiment was performed on lyophilized CGF powder, PVA / SA, PVA / SA / CGF, and PVA / SA / CGF / nHA nanofiber membranes, respectively.

[0039] Figure 2 Figure (a) is a SEM image of the freeze-dried CGF powder after it has been ground into particles, showing that the freeze-dried CGF exhibits a porous structure. Figure 2 As can be seen from Figure (b), all three groups of nanofibers exhibit a disordered and interwoven network structure with no obvious beads and a uniform distribution of fiber diameter. Figure 2 Figure (c) shows the average fiber diameters of the PVA / SA, PVA / SA / CGF, and PVA / SA / CGF / nHA nanofiber membranes, which are 269.18±66.87 nm, 255.34±90.50 nm, and 359.07±86.20 nm, respectively. Figure 2 As can be seen from Figure (d), compared with the PVA / SA group, the fiber diameter of the PVA / SA / CGF group is slightly smaller, while the fiber diameter of the PVA / SA / CGF / nHA group is significantly larger, and the fiber surface is not uniform and has slight protrusions.

[0040] Figure 2 Figure (e) shows the interactions between the components of the nanofiber membrane investigated by FTIR. In the PVA / SA spectrum, the broad and strong absorption peak in the 3335 cm⁻¹ range indicates the stretching vibrations of hydroxyl (OH) and amino (NH) groups, the doublets at 2950 and 2911 cm⁻¹ indicate the CH stretching vibration, the shoulder peaks at 1708 and 1631 cm⁻¹ are related to the carbonyl (C=O) stretching vibration, and the doublets at 1091 and 1027 cm⁻¹ are caused by the combined stretching of CO and bending of OH groups.

[0041] In the infrared spectrum of freeze-dried CGF, the absorption peaks at 1631, 1533, and 1395 cm⁻¹ represent amide I (C=O stretching vibration), amide II (NH bending vibration), and amide III (CN stretching vibration) groups, respectively, which are related to the presence of fibroin in CGF. Furthermore, the absorption peaks at 1708 and 1631 cm⁻¹ are significantly increased in the PVA / SA / CGF and PVA / SA / CGF / nHA spectra. Simultaneously, after electrospinning, the peak at 3281 cm⁻¹ in freeze-dried CGF becomes significantly sharper and broader, shifting towards a higher wavenumber of 3335 cm⁻¹, suggesting that hydroxyl and amino groups in the structure associate to form hydrogen bonds.

[0042] In the PVA / SA / CGF / nHA spectrum, the absorption peaks at 1091 and 1027 cm⁻¹ become stronger, which may be due to the interaction between nHA and other component molecules, which changes the original crystal structure of nHA. Example 4

[0043] The physical properties of the nanofiber membrane prepared in Example 2 were studied. The physical property studies included research on water contact angle, swelling rate, degradation rate, and in vitro release.

[0044] Nanofiber membranes were cut into 15*15mm squares. The initial mass (W0) was weighed using a laboratory balance, and then the membranes were immersed in PBS solution. The nanofiber membranes were then incubated at 37°C for 2 weeks. Samples were removed on days 1, 3, 5, 7, and 14 of incubation, washed with deionized water, dried, and weighed (Wt). Furthermore, SEM images of the 14-day-old samples were taken to observe the morphological changes of the nanofiber membranes during degradation, and the degradation status of the nanofiber membranes was calculated using a formula.

[0045] The formula for calculating the degradation rate of nanofiber membranes is: Degradation rate (%) = (W0 - W) t ) / W0×100 The surface hydrophilicity of biomaterials is an important factor affecting cell proliferation, migration and differentiation. Figure 3 Figure (a) shows the average water contact angles of PVA / SA, PVA / SA / CGF and PVA / SA / CGF / nHA nanofibers at 4 s, which are 75.87°, 72.05° and 61.62°, respectively. Figure 3 As shown in Figure (b), the average water contact angles at 1, 4, and 8 s were significantly lower in the PVA / SA / CGF / nHA group compared to the PVA / SA and PVA / SA / CGF groups, indicating a further increase in hydrophilicity. Furthermore, changes in the surface roughness of the PVA / SA / CGF / nHA nanofibers also led to changes in hydrophilicity.

[0046] Material swelling ratio is an important parameter for verifying the water absorption performance of biomaterials. For example... Figure 3 As shown in Figure (c), the expansion ratios of PVA / SA, PVA / SA / CGF, and PVA / SA / CGF / nHA nanofibers after 24 h of incubation were 6.85, 6.65, and 5.09, respectively. The results indicate that the expansion rate of the PVA / SA / CGF / nHA group was significantly lower than that of the PVA / SA and PVA / SA / CGF groups, which is beneficial for the long-term preservation of the fiber structure.

[0047] Ideally, the degradation of a material should be matched with tissue regeneration. For example... Figure 3 As shown in Figure (e), the degradation rates of nanofibers after 2 weeks of immersion in PBS were 76.94±3.18%, 69.96±1.80%, and 57.83±1.86%, respectively, for PVA / SA, PVA / SA / CGF, and PVA / SA / CGF / nHA nanofibers. Compared with the PVA / SA and PVA / SA / CGF groups, the PVA / SA / CGF / nHA group showed a significantly lower degree of degradation, which is considered to be due to the addition of nHA delaying the degradation of the nanofibers. Figure 3 As shown in the SEM image of (d), the nanofibers expanded and became irregular in shape after two weeks of degradation, exhibited a significant decrease in mechanical strength, an increase in surface roughness, and some fibers showed obvious shrinkage, collapse, and aggregation. Furthermore, precipitated nHA particles were observed in the PVA / SA / CGF / nHA group.

[0048] The release of CGF from PVA / SA / CGF and PVA / SA / CGF / nHA nanofiber membranes was quantitatively determined using a BCA protein concentration assay kit. Specifically, PVA / SA / CGF and PVA / SA / CGF / nHA nanofiber membranes containing 0.5 mg of lyophilized CGF were immersed in 10 mL of PBS solution and incubated at 37°C for 14 days. After incubation for 1, 3, 5, 7, 10, and 14 days, 0.5 mL of PBS was collected, and 0.5 mL of fresh PBS was added. The total protein release concentration of the PVA / SA / CGF and PVA / SA / CGF / nHA nanofiber membranes was then calculated using the BCA assay kit, and cumulative release curves were plotted.

[0049] Figure 3 Figure (f) shows the release of CGF from PVA / SA / CGF and PVA / SA / CGF / nHA nanofibers over 2 weeks. The results indicate that CGF release from the fibers involves three phases: rapid release within the first 24 hours, uniform release over the next week, a gradually slowing release rate after one week, and a stable cumulative release after two weeks, demonstrating the sustained-release effect of CGF in the nanofibers. Furthermore, there was no significant difference in drug release between the PVA / SA / CGF group and the PVA / SA / CGF / nHA group, suggesting that both can promote osteogenic activity over a long period. Example 5

[0050] Animal experiments were conducted to test the function of the nanofiber membrane in Example 1.

[0051] 1. Thirty-six male SD rats aged 8 weeks with an average weight of approximately 200 grams were selected. Thirty-two rats were randomly divided into four groups: a blank control group, a gelatin sponge (AGS) group, a PVA / SA / CGF group, and a PVA / SA / CGF / nHA group. Before the experiment, the SD rats were housed at 22±3°C under a 12 / 12 h light / dark cycle for one week. During this period, the rats were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital solution (5 ml / kg). Four ml of blood was collected from the retro-orbital vein of each rat. Samples were collected using a CGF-specific vacuum blood collection device with a red cap (without anticoagulant) and centrifuged according to the preset protocol on a high-speed centrifuge to extract CGF for nanofiber preparation.

[0052] 2. After one week of rearing, rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (5 ml / kg). The first maxillary molar on the right side of the rat was extracted, and the extraction site was flushed with physiological saline. Nanofibers (2 mm wide and 4 mm long) from different groups were placed in the extraction site, and the wound was sutured to fix the nanofiber membrane. The sutures were removed after 3 days. Four rats from each group were euthanized at 7 days and 28 days after extraction, and another four rats were euthanized immediately after extraction. The right maxillary bone was collected for Micro-CT analysis, tissue staining, and immunohistochemical staining.

[0053] 3. Immediately after tooth extraction, and at 3, 7, and 14 days after extraction, digital cameras were used to photograph the healing of the extraction wound. ImageJ software was used to process the images and measure the wound area to quantitatively analyze the wound closure (WC).

[0054] Rats' maxillae were collected immediately after tooth extraction, at 7 days, and at 28 days. The maxillae were fixed with 4% paraformaldehyde solution for 24 h and examined by Micro-CT. The volume fraction of new bone (BV / TV), trabecular thickness (Tb.th), and trabecular spacing (Tb.sp) of the extraction site were statistically analyzed.

[0055] Rats' maxillae were collected 7 and 28 days after tooth extraction for histological staining. The samples were fixed in 4% paraformaldehyde solution for 24 h and decalcified with 10% EDTA. The decalcified samples were embedded in paraffin blocks, and sections were prepared to a thickness of 4 μm. All tissue sections were stained with hematoxylin and eosin (HE). All tissue sections were observed under an optical microscope.

[0056] The full-thickness wound closure of rat tooth extraction wounds treated with nanofiber materials 14 days later is as follows: Figure 4 As shown in (c). The results showed that after 3 days, 7 days, and 14 days, the average wound closure rate of each treatment group was higher than that of the control group. Specifically, at 3 days and 7 days, the average wound closure rate of each treatment group was significantly higher than that of the control group. Figure 4 As shown in (b), after 14 days, the mean wound closure rates of the control group, gelatin group, PVA / SA / CGF group, and PVA / SA / CGF / nHA group were 74.66±1.95, 82.45±5.65, 94.36±2.62, and 95.18±3.99%, respectively. There was no significant difference in the final extraction wound healing rate between the control group and the gelatin group, while the healing rate of the experimental group differed significantly from that of the control group and the gelatin group. In fact, compared with the control group and the gelatin group, the PVA / SA / CGF and PVA / SA / CGF / nHA groups showed faster wound healing rates after 14 days.

[0057] The above experiments demonstrate that CGF (concentrated growth factor), as a new generation of platelet concentrate, can promote tissue repair, angiogenesis, cell migration, and differentiation, and has been proven to contribute to the regeneration of bone, periodontal, and dental pulp tissues. CGF is readily available in clinical settings and can be used for electrospinning preparations, or its activity can be preserved through lyophilization. Electrospun PVA / SA / CGF / nHA nanofiber membranes can significantly enhance the proliferation, migration, and osteogenic differentiation capabilities of human bone marrow mesenchymal stem cells (hBMSCs).

[0058] The addition of CGF (concentrated growth factor) and nHA (nano-hydroxyapatite) not only improves the physical properties of electrospun fiber membranes but also promotes favorable cellular biological responses. Their synergistic effect demonstrates their potential application in bone tissue engineering repair in regenerative medicine.

[0059] Electrospun PVA / SA / CGF / nHA nanofiber membranes have demonstrated excellent osteogenic differentiation capabilities in both in vitro and in vivo experiments, providing a new treatment option for patients with defects or loss of jawbone and alveolar bone, and can also achieve the regeneration of other tissues.

[0060] Electrospun PVA / SA / CGF / nHA nanofiber membranes utilize the advantages of CGF and nHA to create a sustained-release system that can continuously release platelet concentrates according to different stages of jawbone tissue repair, effectively promoting osteogenic differentiation.

[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an electrospun nanofiber membrane that promotes osteogenic formation in jawbone defects, characterized in that, Includes the following steps: Step 1: Prepare freeze-dried CGF powder; Step 2: Prepare a PVA / SA / nHA solution and add lyophilized CGF powder to obtain a spinning solution; Step 3: Collect the spinning solution on an aluminum foil roller to obtain a PVA / SA / CGF / nHA nanofiber membrane.

2. The method for preparing an electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects according to claim 1, characterized in that, The specific steps for preparing freeze-dried CGF powder in step one include: centrifuging the venous blood extracted from the human body, separating it, placing the separated CGF in a freeze dryer to freeze-dry for 48 hours, grinding it into powder, and storing it at -80℃.

3. The method for preparing an electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects according to claim 2, characterized in that, The specific steps for preparing the PVA / SA / nHA solution in step two are as follows: 6 w / v% PVA and 1 w / v% SA mixed powder are added to deionized water and stirred thoroughly in a 60℃ water bath for 8 hours; then 3 w / v% nHA are added to the PVA / SA solution, and the mixture is stirred and ultrasonically vibrated for 4 hours.

4. The method for preparing an electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects according to claim 3, characterized in that, The specific preparation method of the spinning solution in step two is as follows: 1 w / v% lyophilized CGF powder is added to the PVA / SA / nHA solution and stirred at 37℃ for 3 h to obtain the spinning solution.

5. The method for preparing an electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects according to claim 4, characterized in that, In step three, an electrospinning device is used, and its specific operating parameters are set as follows: The electrospinning device was set to 20kV voltage, 0.5mL / h flow rate, and 50%–55% relative humidity; The spinning solution is placed into a 10mL syringe with an inner diameter of 0.6mm. An aluminum foil roller is provided on the needle tip side of the syringe, and the distance between the needle tip and the aluminum foil is 15cm.

6. The method for preparing an electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects according to claim 5, characterized in that: In step three, after the PVA / SA / CGF / nHA nanofiber membrane is prepared, it is placed in a vacuum drying oven at 37°C for 12 hours to dry.

7. An electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects, characterized in that: Prepared by the preparation method according to any one of claims 1-6.

8. The electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects according to claim 7, characterized in that: The nanofiber membrane is coated with PVA, SA, CGF and nHA.

9. The electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects according to claim 8, characterized in that: The CGF is continuously released during the use of the nanofiber membrane.

10. The application of an electrospun nanofiber membrane for promoting osteogenic formation in jawbone defects according to any one of claims 7-9 in bone regeneration of jawbone or periodontal tissues.

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