Fiber membrane, keratinized gingival repair membrane containing fiber membrane and preparation method of keratinized gingival repair membrane

Abstract

The invention relates to a fibrous membrane, a keratinized gingival repair membrane containing the fibrous membrane and a preparation method of the keratinized gingival repair membrane. The fiber membrane is a recombinant collagen-polycaprolactone fiber membrane containing zinc oxide nanoparticles, and is prepared by the following steps: dissolving recombinant III-type collagen and polycaprolactone in hexafluoroisopropanol, adding the zinc oxide nanoparticles, and uniformly stirring to form a spinning solution; and then carrying out electrostatic spinning. The fiber membrane is good in strength, hydrophilicity, softness, antibacterial performance and the like, when the fiber membrane is applied to the repairing membrane, compared with an existing repairing membrane, the mechanical property, the antibacterial performance and the biological activity are remarkably improved through the bionic three-layer structural design, and a more effective solution is provided for keratogingival repairing.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to a fiber membrane, a keratinized gingival repair membrane containing the fiber membrane, and a preparation method thereof. Background Art

[0002] The field of keratinized gingival repair and regeneration mainly focuses on the regeneration and functional restoration of gingival tissue, especially for gingival recession and defects caused by periodontal disease, trauma or surgery, insufficient width of keratinized gingiva or insufficient thickness of soft tissue, which requires soft tissue augmentation. Traditional treatment methods usually use autologous free gingival transplantation, that is, obtaining keratinized gingival tissue from the patient's palate and transplanting it to the defect site. However, this method has problems such as trauma to the donor site, high surgical complexity, and strong patient discomfort. In addition, the amount of donor tissue is limited, making it difficult to meet the needs of large-area repair. To overcome the above limitations, in recent years, researchers have developed a variety of absorbable repair membranes for keratinized gingival regeneration.

[0003] At present, the absorbable repair membranes for keratinized gingival repair on the market mainly include single-layer or multi-layer biomaterials, such as gelatin, polycaprolactone (PCL) and other natural or synthetic polymer materials. Common repair membrane designs in the prior art are mostly purified collagen membranes, decellularized matrix membranes, single materials or simple composite materials. Although these repair membranes have certain biocompatibility, they have obvious deficiencies in mechanical strength, biological functionality, and antibacterial properties. In particular, the repair membranes of a single material have mechanical properties that are difficult to balance softness and support, and cannot provide sufficient support to help keratinized gingival tissue regeneration, nor are they conducive to surgical operations and stability after implantation. In addition, most of the existing repair membranes lack antibacterial properties. The oral environment is full of a variety of microorganisms, and keratinized gingival defects are easily infected by bacteria, resulting in unsatisfactory tissue regeneration or inflammatory reactions. Although some repair membranes improve their antibacterial properties by adding antibiotics to the materials, this approach has great limitations. Long-term use of antibiotics may cause antibiotic resistance problems, leading to the production of resistant strains of bacteria, which not only reduces the efficacy of the repair membrane, but also may cause potential harm to the overall health of the patient. In addition, the repair membranes in the prior art are also deficient in terms of bioactivity and promotion of tissue regeneration. Although materials such as gelatin and polycaprolactone have certain biocompatibility, they lack sufficient bioactivity to promote the regeneration and healing of gingival tissue. The regeneration of keratinized gingiva requires not only good support and antibacterial environment, but also requires the material to have bioactivity, which can promote cell adhesion, proliferation and differentiation, thereby accelerating the process of tissue repair.

[0004] Therefore, the development of new repair membranes with good mechanical support, antibacterial properties and the ability to promote tissue regeneration is a key need in the current field of keratinized gingival repair. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention develops a fiber membrane, and based on this fiber membrane, designs a novel keratinized gingiva repair membrane with the following characteristics: 1) providing sufficient mechanical support and softness to ensure the stability and easy operation of the repair membrane during implantation; 2) having good antibacterial properties to effectively inhibit oral pathogens and reduce the risk of infection; 3) possessing excellent bioactivity, capable of promoting cell proliferation and tissue regeneration, and accelerating the repair process.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In the first aspect of the present invention, a fiber membrane is provided. The fiber membrane is obtained by dissolving recombinant type III collagen and polycaprolactone in hexafluoroisopropanol, adding zinc oxide nanoparticles, and stirring evenly to form a spinning solution; then electrospinning is carried out.

[0008] The addition of recombinant collagen can improve the hydrophilicity of polycaprolactone and enhance the flexibility of the membrane layer. At the same time, recombinant type III collagen provides good biocompatibility and bioactivity, which can promote cell adhesion, proliferation, and tissue regeneration. In addition, zinc oxide can provide excellent antibacterial properties, effectively inhibit pathogens in the oral cavity, and reduce the risk of infection. Through solvent blending and then electrospinning, in the obtained fibers, zinc oxide particles are embedded in the fiber cavities, which can play a slow-release and dispersion role, improving the antibacterial activity and biocompatibility of zinc oxide, and being more suitable for the application scenario of oral repair.

[0009] Preferably, according to the mass ratio, recombinant type III collagen: polycaprolactone = 1:1, which has better hydrophilicity and degradation properties while also having good mechanical properties, and is more suitable for oral repair.

[0010] Preferably, in the spinning solution, according to the mass ratio, the content of recombinant type III collagen is 10% - 20%, the content of polycaprolactone is 10% - 20%, and the content of zinc oxide nanoparticles is 1% - 5%. The obtained fiber membrane has good properties such as strength, hydrophilicity, softness, and antibacterial performance.

[0011] Preferably, the electrospinning conditions are: ambient temperature: 25°C, spinning voltage is 15 - 18 kV, solution flow rate is 12 mL / h, and the distance between the receiving plate is 10 - 15 cm. The obtained fibers have uniform diameters, relatively smooth surfaces, and uniform pore distributions between the fibers, and have good stability and repeatability.

[0012] In the second aspect of the present invention, a keratinized gingiva repair membrane is further provided, which comprises the above-mentioned fiber membrane.

[0013] Further, the repair membrane is composed of an inner layer, a middle layer, and an outer layer, where: the outer layer and the inner layer are the above-mentioned recombinant type III collagen-polycaprolactone fiber membrane containing zinc oxide nanoparticles, and the middle layer is a polycaprolactone fiber membrane.

[0014] The polycaprolactone middle layer provides mechanical strength and support for the entire repair membrane, enabling the repair membrane to remain stable during implantation and facilitating easy operation. The inner layer and the outer layer are recombinant type III collagen-polycaprolactone fiber membranes containing zinc oxide particles, with good hydrophilicity and biocompatibility, and having a continuous antibacterial effect, so as to be more conducive to repair and regeneration.

[0015] Preferably, the fiber diameter of the outer layer and the inner layer is 200 - 500 nm; the fiber diameter of the middle layer is 1000 - 1500 nm.

[0016] Preferably, the thickness ratio of the outer layer, the middle layer, and the inner layer is 1:1:1. For example, in a specific embodiment, the thickness of the fiber membranes of the outer layer, the middle layer, and the inner layer is about 5 μm.

[0017] Preferably, the polycaprolactone repair membrane is obtained by completely dissolving polycaprolactone in chloroform to obtain a polycaprolactone spinning solution and performing electrospinning.

[0018] Preferably, in the polycaprolactone spinning solution, according to the mass ratio, the polycaprolactone content is 20% - 30%. Electrospinning conditions: at room temperature, the spinning voltage is 18 kV, the solution flow rate is 1.0 mL / h, and the distance from the receiving plate is 15 cm. The obtained fibers have a uniform diameter, a relatively smooth surface, and a uniform pore distribution between the fibers, and have good stability and repeatability.

[0019] In the third aspect of the present invention, there is also provided a method for preparing the repair membrane, including the following steps:

[0020] Clamp two layers of recombinant collagen-polycaprolactone fiber membranes containing zinc oxide nanoparticles on both sides of the polycaprolactone fiber membrane and press them in a "sandwich" structure.

[0021] In one preferred embodiment, the pressing temperature is 60 - 70 °C, the pressing pressure is 15 - 20 N, and the time is 30 - 60 seconds.

[0022] Compared with the prior art, the present invention has the following beneficial and unique effects:

[0023] By providing a recombinant type III collagen - polycaprolactone fiber membrane with nano - zinc oxide particles embedded in the fiber lumen, the present invention has good hydrophilicity, softness, and antibacterial properties. Further, through a bionic three - layer structure design, it is applied to a repair membrane. The obtained repair membrane has good mechanical support and softness, ensuring stability and easy operation during implantation. Secondly, zinc oxide nanoparticles are introduced into the outer layer, endowing the repair membrane with excellent antibacterial properties, which can effectively inhibit bacteria in the oral cavity and reduce the risk of infection. In addition, the recombinant collagen spinning layer has good biological activity, which can promote cell adhesion, proliferation, and tissue regeneration, accelerating the healing process. The repair membrane of the present invention has achieved significant improvements in mechanical properties, antibacterial properties, and biological activity compared with existing repair membranes, providing a more effective solution for keratinized gingiva repair. Description of the Drawings

[0024] Figure 1 SEM image of the PCL fiber membrane;

[0025] Figure 2 SEM images of the PCL - RHC and PCL - RHC - ZnO fiber membranes;

[0026] Figure 3 TEM image of the PCL - RHC - ZnO fiber membrane;

[0027] Figure 4 Swelling ratio of different types of fiber membranes;

[0028] Figure 5 Contact angle of different types of fiber membranes;

[0029] Figure 6 Porosity of different types of fiber membranes;

[0030] Figure 7 FIRT chromatogram of different types of fiber membranes;

[0031] Figure 8 Zinc ion release curve of the PCL - RHC - ZnO fiber membrane;

[0032] Figure 9 Antibacterial properties of different types of fiber membranes;

[0033] Figure 10 Statistics of the antibacterial rate of different types of fiber membranes;

[0034] Figure 11 Cell viability of different types of fiber membranes. Detailed Embodiments

[0035] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0037] I. Preparation of fiber membranes

[0038] Example 1 Preparation of polycaprolactone (PCL) fiber membrane.

[0039] Polycaprolactone was completely dissolved in chloroform to prepare a 25 wt% polycaprolactone spinning solution.

[0040] The prepared polycaprolactone spinning solution was electrospun to obtain a polycaprolactone fiber membrane, denoted as PCL fiber membrane.

[0041] Electrospinning conditions: The spinning voltage was 15 - 18 kV, the solution flow rate was 1.0 mL / h, and the distance between the receiving plate and the spinneret was 10 - 15 cm. This step was carried out at room temperature.

[0042] The obtained PCL fiber membrane had a fiber diameter distribution in the range of 1000 - 1500 nm and a membrane thickness of 5 ± 0.5 mm.

[0043] Example 2 Preparation of recombinant type III collagen - polycaprolactone (PCL - RHC) fiber membrane.

[0044] Recombinant type III collagen and polycaprolactone were dissolved in a mixed solution of hexafluoroisopropanol according to a weight ratio of 1:1, and stirred evenly to form a spinning solution. In the spinning solution, according to the mass ratio, the content of recombinant type III collagen was 15 wt%; the content of polycaprolactone was 15 wt%.

[0045] The prepared recombinant type III collagen - polycaprolactone spinning solution was electrospun to obtain a fiber membrane, denoted as PCL - RHC fiber membrane.

[0046] Electrospinning conditions: It was carried out in a constant temperature environment at 25 °C, the spinning voltage was 15 - 18 kV, the solution flow rate was 12 mL / h, and the distance between the receiving plate and the spinneret was 10 - 15 cm.

[0047] The obtained PCL - RHC fiber membrane had a fiber diameter distribution in the range of 200 - 500 nm and a membrane thickness of 5 ± 0.5 mm.

[0048] Example 3 Preparation of recombinant type III collagen - polycaprolactone (PCL - RHC - ZnO) fiber membrane containing zinc oxide particles.

[0049] Recombinant type III collagen and polycaprolactone in a weight ratio of 1:1 are dissolved in a mixed solution of hexafluoroisopropanol, and zinc oxide nanoparticles are added and stirred evenly to form a spinning solution. In the spinning solution, according to the mass ratio, the content of recombinant type III collagen is 15wt%; the content of polycaprolactone is 15wt%; and the content of zinc oxide nanoparticles is 3%.

[0050] The prepared recombinant type III collagen-polycaprolactone spinning solution was electrospun to obtain a fiber membrane, which was recorded as PCL-RHC-ZnO fiber membrane.

[0051] Electrospinning conditions: carried out in a constant temperature environment of 25°C, the spinning voltage was 15-18 kV, the solution flow rate was 12 mL / h, and the receiving plate distance was 10-15 cm.

[0052] The PCL-RHC-ZnO fiber membrane was obtained, with a fiber diameter distribution of 200-500nm and a membrane thickness of 5±0.5mm.

[0053] Fiber membrane performance test

[0054] 1.1) Fiber morphology detection

[0055] Test method: Use scanning electron microscopy (SEM) and transmission electron microscopy to observe the morphology of the repair film and analyze the fiber morphology, distribution and surface roughness.

[0056] Test results: Figures 1-3 shown.

[0057] The microstructure of PCL fiber membrane was observed by scanning electron microscopy (SEM). Figure 1 As shown, the fibers present a randomly interlaced mesh structure, with a fiber diameter distribution of 1-1.5 μm and a smooth surface without beads. The fibers are interwoven to form a highly porous three-dimensional network. This pore structure is conducive to cell penetration and attachment, while providing the necessary permeability to support the exchange of nutrients and metabolites. The fiber diameter is uniform, the surface is relatively smooth, and the pores between the fibers are evenly distributed, indicating that the electrospinning process has good stability and repeatability. This porous fiber membrane structure provides good support and a suitable microenvironment for tissue engineering and drug delivery applications, which is conducive to cell proliferation and tissue regeneration.

[0058] The microstructure of the PCL-RHC and PCL-RHC-ZnO fiber membranes was observed by scanning electron microscopy (SEM). Figure 2As shown in the figure, the fibers present a uniformly distributed three-dimensional network morphology, with a fiber diameter distribution of 200-500nm, a smooth surface without beads, and a porous structure interwoven with each other. This pore morphology is conducive to cell attachment, proliferation, and the delivery of nutrients. The fiber surface is relatively smooth, showing good processing quality, and there is no obvious breakage or irregular morphology, indicating that the spinning process has good controllability and stability. The overall fiber arrangement and spatial structural characteristics enable it to have good mechanical properties and biocompatibility in tissue engineering applications, and is suitable for biomedical fields such as oral repair membranes.

[0059] The fiber structure of the formed PCL-RHC-ZnO fiber membrane was further observed by transmission electron microscopy (TEM). Figure 3 As shown in the figure, it can be clearly seen that the fiber presents a tubular morphology, and the zinc oxide nanoparticles are evenly distributed and embedded in the fiber lumen and are wrapped by the fiber material. This wrapping structure helps stabilize the zinc oxide nanoparticles and prevent them from aggregating, and provides a sustained-release carrier that can further improve the antibacterial activity and biocompatibility of zinc oxide, which is suitable for oral repair applications. The scale in the figure is 50nm, indicating that the microscopic scale of the fibers and particles is very suitable for interacting with cells and microorganisms.

[0060] 1.2) Swelling rate test

[0061] Test method: Cut the electrospun membrane to be tested into 2cm×2cm square fiber membranes, weigh them and immerse them in 10ml phosphate buffer at 37℃. After 12 hours, take out the samples, absorb the moisture on the sample surface with filter paper and weigh them.

[0062] According to the swelling ratio (SR, g / g) formula: SR = (mm 0 ) / m 0 (m 0 is the mass of the membrane before swelling, and m is the mass of the membrane after swelling in water), and the swelling rate of the spinning membrane was calculated.

[0063] Test results: Figure 4 As shown in Figure 3, there are obvious differences in the swelling properties of PCL fiber membrane, PCL-RHC fiber membrane and PCL-RHC-ZnO fiber membrane.

[0064] The swelling rate of the PCL fiber membrane is relatively low, indicating that its pore structure has limited liquid absorption capacity. In contrast, the swelling rate of the PCL-RHC blended fiber membrane is significantly increased and reaches the highest level, suggesting that the addition of recombinant collagen significantly enhances the hydrophilicity and liquid absorption capacity of the fiber membrane. The swelling rate of the PCL-RHC-ZnO fiber membrane containing zinc oxide nanoparticles is slightly lower than that of PCL-RHC, but still significantly higher than that of PCL, indicating that although the addition of zinc oxide nanoparticles has a certain impact on the swelling rate, it still retains strong hydrophilicity. These data suggest that PCL-RHC and PCL-RHC-ZnO fiber membranes have the potential to have good biocompatibility and suitable swelling characteristics, and are suitable for application in oral repair materials, which helps to improve tissue hydration and accelerate the healing process.

[0065] 1.3) Contact angle measurement

[0066] Test method: The contact angle tester was used for testing. Specifically, the contact angle tester was connected to the computer terminal; the light source was turned on to adjust the lens clarity until the clearest water droplet was seen; a clean glass slide was placed on the test bench, and the upper lifting platform was adjusted to place the needle within the test range of the lens; then the samples of the examples were fixed for testing respectively; the syringe push rod was rotated to empty the air bubbles in the syringe, and 2 μL of liquid drops were extruded according to the standard during the test; the lifting platform was rotated to catch the liquid drops, and the stopwatch was immediately pressed when 5 liquid drops detached from the syringe, and the screen was captured and read immediately after 10 s. Finally, the above experiment was repeated three times and the average value was taken.

[0067] Test results: As Figure 5 shown.

[0068] According to Figure 5 the contact angle test results of the fiber membranes shown in, the contact angle of the PCL fiber membrane is relatively large, about 120°, indicating that its surface has strong hydrophobicity. While the contact angle of the PCL-RHC fiber membrane is significantly reduced, about 30°, suggesting that after blending with recombinant collagen, the hydrophilicity of the fiber membrane is significantly enhanced. This change is mainly attributed to the introduction of collagen, which itself has excellent hydrophilic properties, making the composite material more likely to absorb water and showing better hydrophilicity.

[0069] This significant change in contact angle indicates that the PCL-RHC fiber membrane has excellent surface hydrophilicity, which helps cell adhesion and growth, thereby enhancing the biocompatibility of the material. In oral repair applications, a lower contact angle and higher hydrophilicity are beneficial for the integration of the repair membrane with the surrounding tissues and help improve the efficiency of tissue regeneration.

[0070] 1.4) Porosity determination

[0071] Testing method: The porosity of the repair membrane was determined using the liquid exclusion method. An appropriate liquid (such as ethanol) was selected to soak the repair membrane, and the porosity was calculated by measuring the weight change before and after soaking. Calculation formula: Porosity (%) = (volume of the soaking liquid / total volume of the repair membrane) × 100%.

[0072] The test results are as Figure 6 shown. It can be seen that different material combinations have a certain influence on the porosity of the fiber membrane. The porosity of the PCL fiber membrane is approximately 75%. The porosity of the PCL-RHC fiber membrane slightly increases to approximately 80%, indicating that the introduction of collagen helps to form a more uniform and open pore structure, which may have a positive impact on cell adhesion and nutrient penetration.

[0073] The porosity of the PCL-RHC-ZnO fiber membrane is comparable to that of PCL-RHC, slightly lower than 80%, indicating that the addition of zinc oxide has little effect on the porosity and still retains a good pore structure. These porosity characteristics contribute to cell growth and tissue fluid exchange, further enhancing the application potential of the repair membrane in tissue engineering. This porous structure plays an important role in promoting cell migration, tissue regeneration, and nutrient supply, making it a good candidate material for oral repair and regeneration.

[0074] 1.5) Chemical bond analysis (Fourier transform infrared spectroscopy, FTIR):

[0075] Testing method: A Fourier transform infrared spectrometer (FTIR) was used to analyze the chemical bonds of the repair membrane to confirm the chemical bonding situation between the components. The repair membrane was mixed with KBr and pressed into a tablet. The test wavenumber range was 4000 - 400 cm-1 to analyze the interactions between recombinant collagen, polycaprolactone, and zinc oxide nanoparticles in the fiber material.

[0076] The test results are as Figure 7 shown.

[0077] The FTIR spectrum of PCL shows a C-H stretching vibration peak near 2900 cm-1 and a strong C=O stretching vibration peak at 1720 cm-1, representing the ester group characteristics in polycaprolactone. In addition, a characteristic peak of C-O stretching vibration also appears near 1100 cm-1.

[0078] In the FTIR spectrum of recombinant type III collagen (RHC), an N-H stretching vibration peak is observed at 3300 cm-1, which represents the amino group characteristics in collagen. Another C=O stretching vibration peak at 1650 cm-1 (i.e., amide I band) and an N-H bending vibration peak near 1550 cm-1 (amide II band) are typical characteristics of collagen.

[0079] The spectrum of the PCL-RHC fiber membrane contains the characteristic absorption peaks of both PCL and RHC. The C=O stretching vibration peak of the ester group (1720 cm-1) and the amide I (1650 cm-1) and amide II (1550 cm-1) peaks of collagen are clearly visible, indicating that PCL and RHC are successfully compounded in the blended fiber and maintain their respective characteristic structures between the two.

[0080] In the spectrum of the PCL-RHC-ZnO fiber membrane, in addition to the characteristic absorption peaks of PCL and RHC, a Zn-O vibration peak between 500-700 cm-1 was also observed, indicating that zinc oxide nanoparticles were successfully incorporated into the fiber membrane.

[0081] It can be seen from the FTIR spectrum analysis that PCL and RHC maintain their respective characteristic structures in the blended fiber membrane, and the successful incorporation of zinc oxide nanoparticles is also confirmed by the characteristic peaks. These data indicate that there is good binding between the components of the fiber membrane, and the composite method of the material retains its respective functional properties, laying a foundation for its multifunctional applications in oral repair, including improving antibacterial properties and promoting cell compatibility.

[0082] 1.6) Zinc ion release curve

[0083] Test method: Cut the fiber membrane into a certain size (such as 1×1 cm 2 ), and record the initial mass of the sample. Place the sample in a 96-well plate or a capped centrifuge tube, and add an appropriate amount of PBS buffer solution to ensure that the sample is completely immersed. Place the sample in a constant temperature shaker at 37°C and gently oscillate to simulate the in vivo environment. At preset time points (such as 1, 3, 7, 14, 21 days), take out a certain amount of supernatant (such as 200 μL) for zinc ion concentration determination, and supplement an equal amount of fresh simulated body fluid to maintain a constant volume. According to the instructions of the zinc ion colorimetric kit, prepare a series of zinc ion standard solutions with known concentrations (such as 0, 0.1, 0.5, 1.0, 2.0 μg / mL).

[0084] Standard curve preparation:

[0085] Add the corresponding concentration of standard solution (50-100 μL) to each well of the 96-well plate. Add the colorimetric reagent, mix well, and incubate at room temperature in the dark for a specified time (such as 15-30 minutes). Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value at a specified wavelength (such as 560 nm) and plot the standard curve (concentration-absorbance).

[0086] Sample detection:

[0087] Take 50 - 100 μL from the sampled supernatant, mix it with the colorimetric reagent, and incubate it in the dark for a specified time. Measure the absorbance value and calculate the zinc ion concentration in the sample through the standard curve. Record the zinc ion concentration at each time point and calculate the cumulative release amount based on the solution volume. Calculate the cumulative release amount (μmol / L) of zinc ions according to the initial mass or volume of the fiber membrane. Plot the zinc ion release curve with time on the horizontal axis and the zinc ion release amount or release percentage on the vertical axis to analyze its release pattern.

[0088] Test results: As Figure 8 shown. According to the zinc ion (Zn 2+ ) release curve of the PCL - RHC - ZnO blended fiber membrane shown in the figure, it can be observed that the release of zinc ions exhibits typical sustained - release characteristics. Within the initial 24 hours, the zinc ion concentration rises rapidly, indicating a relatively high release rate in the early stage, which may be related to the rapid dissolution of the surface - adsorbed zinc oxide nanoparticles. After that, the release rate gradually slows down, and the zinc ion concentration reaches approximately 60 μmol / L at around 500 hours, indicating that this material has the ability of stable release over a long period.

[0089] This sustained - release characteristic is of great significance for oral repair applications because it can provide a continuous antibacterial environment, effectively inhibit the growth of bacteria, reduce the risk of infection, and at the same time promote tissue regeneration and healing. In addition, zinc ions also have the effect of promoting angiogenesis, which has a positive impact on accelerating tissue repair. Generally speaking, the PCL - RHC - ZnO blended fiber membrane exhibits good zinc ion sustained - release performance and is suitable for biomedical materials that require long - term antibacterial and healing - promoting effects.

[0090] 1.7) Antibacterial properties of the fiber membrane

[0091] Test method: Use Porphyromonas gingivalis and Streptococcus mutans to detect the antibacterial properties of the oral repair membrane through techniques such as plate counting method and live - dead bacteria staining;

[0092] Incubate the repair membrane sample with the bacterial suspension (Porphyromonas gingivalis or Streptococcus mutans) at 37 °C for 24 hours. After the incubation, perform 10 - fold serial dilutions on the bacterial suspension, take an appropriate amount of the diluted solution and spread it on an agar plate, and culture it at 37 °C for 48 hours. Count the number of colonies formed on the plate. Calculate the antibacterial rate of the repair membrane by comparing the number of colonies in the control group and on the plate.

[0093] Antibacterial rate (%) = (Number of colonies in the control group - Number of colonies in the experimental group) / Number of colonies in the control group × 100%.

[0094] Test results: As Figure 9 and Figure 10 shown. Figure 9In the flat panel antibacterial experiment, the antibacterial properties of different fiber membrane materials against two types of bacteria were shown. For the PCL and PCL-RHC fiber membranes without zinc oxide, the growth of bacteria was observed, and it could be seen that the colonies were dense, indicating almost no inhibitory effect. For the PCL-RHC-ZnO fiber membrane, the significantly reduced number of colonies demonstrated strong antibacterial activity. Compared with PCL and PCL-RHC, PCL-RHC-ZnO showed the most significant inhibitory effect against the two types of bacteria.

[0095] Figure 10 The antibacterial rate data further quantitatively evaluated the antibacterial effects of the fiber membrane materials against P. gingivalis and S. mutans. It could be seen that the inhibition rates of the PCL fiber membrane against these two types of bacteria were relatively low, approximately around 20%, and the antibacterial rate of PCL-RHC was even lower, indicating that although the addition of collagen improved biocompatibility, it had an insignificant effect on antibacterial properties. However, for the PCL-RHC-ZnO containing zinc oxide nanoparticles, the antibacterial rates against P. gingivalis and S. mutans reached nearly 100% and approximately 80% respectively, indicating that the addition of zinc oxide significantly improved the antibacterial properties of the material.

[0096] 1.8) Cytotoxicity detection (MTT):

[0097] Test method: The MTT method was used to detect the cytotoxicity of the repair membrane against human umbilical vein endothelial cells (HUVEC) to evaluate the biocompatibility of the material.

[0098] HUVEC cells were inoculated into 24-well plates, and the repair membrane samples were added to each well. After culturing for 24, 48, and 72 hours, MTT solution was added, and incubation was continued for 4 hours. Then, the culture medium was discarded, and DMSO was added to dissolve the formed blue-violet crystals. The absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 570 nm, and the cell survival rate was calculated. The higher the cell survival rate, the lower the cytotoxicity of the material.

[0099] Test results: As Figure 11As shown, according to the cytotoxicity test results shown in the figure, different types of fiber membranes showed significant differences in the cell viability of human umbilical vein endothelial cells (HUVEC). The cell viability in the PCL fiber membrane remained at about 90%, indicating that the PCL material has a certain biocompatibility. However, due to its hydrophobic properties, the adhesion and proliferation effects on cells are not as ideal as expected. The cell viability in PCL-RHC was slightly higher than that in the PCL group, approaching 100%, indicating that the addition of recombinant collagen significantly improved the biocompatibility of the fiber membrane, and the presence of collagen contributed to cell adhesion and proliferation. The cell viability of PCL-RHC-ZnO was about 80%, lower than that of other groups, which may be related to the release of zinc oxide nanoparticles leading to too high local zinc ion concentration, resulting in slight cytotoxicity. The PCL-RHC fiber membrane showed the best biocompatibility, while the PCL-RHC-ZnO fiber membrane, although having significant antibacterial properties, reduced cell viability to a certain extent.

[0100] The above test results indicate that the PCL-RHC-ZnO fiber membrane has good hydrophilicity, biocompatibility, antibacterial properties, etc., and can be used for oral keratinized gingiva repair.

[0101] II. Keratinized Gingiva Repair Membrane

[0102] Example 4

[0103] A bionic three-layer repair membrane for keratinized gingiva repair and regeneration, which consists of an inner layer, a middle layer and an outer layer. Among them: the outer layer and the inner layer are recombinant type III collagen-polycaprolactone fiber membranes, and nano-zinc oxide particles are embedded in the lumen of the recombinant type III collagen-polycaprolactone fibers that form the fiber membrane; the middle layer is a polycaprolactone fiber membrane.

[0104] The preparation method is as follows:

[0105] Prepare PCL fiber membrane and PCL-RHC-ZnO fiber membrane respectively according to the methods of Example 1 and Example 3;

[0106] Clamp two layers of PCL-RHC-ZnO fiber membranes on both sides of the PCL fiber membrane and press them according to the "sandwich" structure. The pressing conditions are: the pressing temperature is 65 °C, the pressing pressure is 18 N, and the time is 50 seconds.

[0107] The thickness of the three-layer composite membrane obtained by pressing is 15 ± 0.5 mm.

[0108] Cut the pressed composite membrane into the required specifications and perform ethylene oxide fumigation sterilization treatment.

[0109] Example 5

[0110] A keratinized gingiva repair membrane, which consists of an inner layer and an outer layer, wherein: the inner layer is a recombinant type III collagen-polycaprolactone fiber membrane, and nano-zinc oxide particles are embedded in the lumen of the recombinant type III collagen-polycaprolactone fiber that forms the fiber membrane; the outer layer is a polycaprolactone fiber membrane.

[0111] The preparation method thereof is as follows:

[0112] Prepare a PCL fiber membrane and a PCL-RHC-ZnO fiber membrane respectively according to the methods of Example 1 and Example 3;

[0113] Press a single-layer PCL fiber membrane and a PCL-RHC-ZnO fiber membrane, and the pressing conditions are: the pressing temperature is 65 °C, the pressing pressure is 18 N, and the time is 50 seconds.

[0114] The thickness of the double-layer composite membrane obtained by pressing is 10 ± 0.5 mm.

[0115] Cut the pressed composite membrane into the required specifications and perform ethylene oxide fumigation sterilization treatment.

[0116] Example 6

[0117] A keratinized gingiva repair membrane, which consists of an inner layer and an outer layer, wherein: the inner layer is a recombinant type III collagen-polycaprolactone fiber membrane, and nano-zinc oxide particles are embedded in the lumen of the recombinant type III collagen-polycaprolactone fiber that forms the fiber membrane; the outer layer is a recombinant type III collagen-polycaprolactone fiber membrane.

[0118] The preparation method thereof is as follows:

[0119] Prepare a PCL-RHC fiber membrane and a PCL-RHC-ZnO fiber membrane respectively according to the methods of Example 2 and Example 3;

[0120] Press a single-layer PCL-RHC fiber membrane and a PCL-RHC-ZnO fiber membrane, and the pressing conditions are: the pressing temperature is 65 °C, the pressing pressure is 18 N, and the time is 50 seconds.

[0121] The thickness of the double-layer composite membrane obtained by pressing is 10 ± 0.5 mm.

[0122] Cut the pressed composite membrane into the required specifications and perform ethylene oxide fumigation sterilization treatment.

[0123] Example 7

[0124] A keratinized gingiva repair membrane, which consists of an inner layer and an outer layer, wherein: the inner layer is a recombinant type III collagen-polycaprolactone fiber membrane, and nano-zinc oxide particles are embedded in the lumen of the recombinant type III collagen-polycaprolactone fiber that forms the fiber membrane; the outer layer is a recombinant type III collagen-polycaprolactone fiber membrane.

[0125] The preparation method is as follows:

[0126] Prepare PCL-RHC fiber membranes and PCL-RHC-ZnO fiber membranes respectively according to the methods of Reference Examples 2 and 3; the difference is that in the spinning solution, according to the mass ratio PCL:RHC = 1:2, the content of polycaprolactone is 10 wt% and the content of recombinant type III collagen is 20 wt% according to the mass ratio; the rest is the same.

[0127] Press the single-layer PCL-RHC fiber membrane and PCL-RHC-ZnO fiber membrane together. Pressing conditions: the pressing temperature is 65 °C, the pressing pressure is 18 N, and the time is 50 seconds.

[0128] The thickness of the double-layer composite membrane obtained by pressing is 10 ± 0.5 mm.

[0129] Cut the pressed composite membrane into the required specifications and perform ethylene oxide fumigation sterilization treatment.

[0130] Test the repair membrane samples obtained in Examples 4-7, and the results are shown in Table 1.

[0131] 2.1) Pores: The same method for measuring porosity as in 1.4) above.

[0132] 2.2) Swelling: The same method for testing the swelling rate as in 1.2) above.

[0133] 2.3) Young's modulus and elongation at break: Use a tensile testing machine to test the mechanical properties of the repair membrane, mainly testing the tensile strength and elongation at break. The sample size is a standard dumbbell shape. Clamp the sample on the fixture of the tensile machine, set the stretching speed to 10 mm / min, record the stretching curve, and obtain the tensile strength and elongation at break.

[0134] Cell proliferation: Cell proliferation ability (CCK-8 assay)

[0135] Use CCK-8 reagent to detect the proliferation ability of cells on the surface of the repair membrane. Inoculate HUVEC cells on the repair membrane, add CCK-8 reagent on the 1st, 3rd, 5th, and 7th days of culture respectively, and measure the absorbance at a wavelength of 450 nm using an enzyme-labeled instrument after incubating for 1-2 hours.

[0136] 2.4) Degradation period

[0137] Perform an in vitro degradation experiment in simulated body fluid (SBF), monitor the weight change, and record the mass loss of the sample during degradation to evaluate the degradation performance of the fiber membrane under physiological conditions.

[0138] Cut the fiber membrane according to a certain size (such as circular or rectangular), and record the initial dry weight. Prepare to immerse the fiber membrane samples in SBF or enzymatic hydrolysis solution respectively, and place them in a constant temperature shaking incubator at 37 °C for cultivation. Simulated body fluid (SBF), the components include NaCl, KCl, NaHCO 3 , CaCl 2 , MgCl 2 etc., the pH is adjusted to 7.4, and the temperature is 37 °C to simulate the human internal environment.

[0139] Take out the samples at preset time intervals (1 day, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, etc.), wash them with distilled water to remove the surface attachments. Sample drying and weighing: Dry the taken-out samples to a constant weight (vacuum drying or drying in a constant temperature oven can be used), weigh the weight of the dried samples, and calculate the degradation rate.

[0140] Degradation rate (%) = (Initial weight - Weight after each weighing) ÷ Initial weight × 100%

[0141] Obtain its degradation period by plotting the change trend graph of the sample degradation rate over time.

[0142] Table 1 Performance test of each repair membrane sample

[0143]

[0144] According to the results in Table 1, it can be seen that due to the introduction of RHC increasing the porosity and swelling performance, the porosity and swelling rate of the repair membrane containing PCL-RHC are both good. However, when using PCL-RHC fiber membrane composites, the strength is poor and it cannot support well. Moreover, when more RHC is introduced, the strength decreases and the degradation is too fast. The repair membrane in Example 7 degrades in 7 days and is not suitable for application. When using a two-layer design of PCL / PCL-RHC-ZnO, the degradation rate is too slow and the ability to promote repair is weak, which is not conducive to application. In the scheme of Example 4, a three-layer design of PCL-RHC-ZnO / PCL / PCL-RHC-ZnO, with PCL-RHC-ZnO in both the inner and outer layers and a PCL-RHC ratio of 1:1, has a degradation period of 28 days, which is relatively moderate, has good controllable degradation performance, is suitable for tissue repair, and its cell proliferation rate is also relatively high, reaching 120%, indicating good mechanical properties and being suitable as a scaffold for cell growth.

[0145] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as within the scope described in this specification. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A fiber membrane, characterized in that: The fiber membrane is a recombinant collagen-polycaprolactone fiber membrane containing zinc oxide nanoparticles, which is obtained by dissolving recombinant type III collagen and polycaprolactone in hexafluoroisopropanol, adding zinc oxide nanoparticles, stirring evenly to form a spinning solution, and then performing electrostatic spinning.

2. The fiber membrane according to claim 1, characterized in that According to the mass ratio, recombinant type III collagen: polycaprolactone = 1:

1.

3. The fiber membrane according to claim 1, characterized in that In the spinning solution, according to the mass ratio, the content of recombinant type III collagen is 10% to 20%, the content of polycaprolactone is 10% to 20%, and the content of zinc oxide nanoparticles is 1% to 5%.

4. The fiber membrane according to claim 1, characterized in that The electrospinning conditions are as follows: ambient temperature: 25° C., spinning voltage: 15-18 kV, solution flow rate: 12 mL / h, and receiving plate distance: 10-15 cm.

5. A keratinized gingival repair membrane, characterized in that: The keratinized gingival repair membrane comprises the fiber membrane according to any one of claims 1 to 4.

6. The repair film according to claim 5, characterized in that: The repair film is composed of an inner layer, a middle layer and an outer layer, wherein: The outer layer and the inner layer are the fiber membranes described in any one of claims 1 to 4, and the middle layer is a polycaprolactone fiber membrane.

7. The repair film according to claim 6, characterized in that: The fiber diameters of the outer layer and the inner layer are between 200-500 nm; the fiber diameter of the middle layer is between 1000-1500 nm.

8. The repair film according to claim 7, characterized in that: The polycaprolactone repair membrane is obtained by completely dissolving polycaprolactone in chloroform to obtain a polycaprolactone spinning solution and then performing electrostatic spinning.

9. The repair film according to claim 8, characterized in that: In the polycaprolactone spinning solution, the polycaprolactone content is 20% to 30% by mass ratio, and the electrospinning conditions are: room temperature, spinning voltage of 18 kV, solution flow rate of 1.0 mL / h, and receiving plate distance of 15 cm.

10. The method for preparing a repair film according to any one of claims 6 to 9, characterized in that: The steps include: Two layers of recombinant collagen-polycaprolactone fiber membrane containing zinc oxide nanoparticles are sandwiched on both sides of the polycaprolactone fiber membrane and pressed together in a "sandwich" structure.

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