Micro-nano fiber composite biomembrane based on electrospinning technology and preparation method thereof

Abstract

The application relates to the technical field of biomedical materials, and discloses a micro-nano fiber composite biomembrane based on electrospinning technology and a preparation method thereof, which comprises the following steps: preparing polycaprolactone, chloroform and dimethyl sulfoxide; mixing the chloroform and the dimethyl sulfoxide to prepare a solvent; dissolving a polymer solution of the polycaprolactone in the solvent and uniformly stirring; continuously collecting fibers by a roller at an ambient temperature of 10 DEG C-25 DEG C and a continuous humidity of 20%RH-70%RH, and performing electrospinning; continuously and gradually changing the fiber membrane structure from order to disorder and from smoothness to porosity in one step by regulating the ambient humidity and the roller rotating speed; and completing the electrospinning to prepare the micro-nano fiber composite biomembrane based on the electrospinning technology. The ambient humidity and the roller rotating speed are regulated to continuously and gradually change the fiber membrane structure from order to disorder and from smoothness to porosity in one step, the interface weak area of a traditional double-layer membrane is avoided, and the mechanical barrier and the biological activity are combined.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular, to a micro / nanofiber composite biomembrane based on electrospinning technology and its preparation method. Background Technology

[0002] In clinical settings such as dental implantology, periodontal treatment, and bone defect repair, guided bone regeneration (GBR) technology is widely used to promote bone tissue regeneration. GBR membranes, as a key barrier material, must simultaneously fulfill two core functions: mechanical barrier function (blocking the invasion of non-osteoblasts such as fibroblasts) and bioactive delivery function (promoting osteoblast migration and nutrient diffusion).

[0003] Currently, most commercially available GBR membranes employ a bilayer heterogeneous structure design, consisting of a dense layer (blocking soft tissue) and a porous layer (promoting bone growth). However, such bilayer membranes suffer from the following technical drawbacks:

[0004] 1. Abrupt change in mechanical properties at the interlayer interface: The mechanical properties of the dense layer and the porous layer are significantly different, resulting in uneven stress distribution inside the material. After implantation, interlayer delamination is easily triggered by mechanical load or cell activity.

[0005] 2. Risk of cell interface accumulation: The interfacial gaps formed after stratification can easily cause cell accumulation, forming a physical isolation zone, which hinders the cross-layer migration of osteoblasts and the diffusion of nutrients such as oxygen and glucose, thus leading to local hypoxia and inhibiting bone regeneration.

[0006] 3. Insufficient structural stability: The compression of the interlayer interface by cell clusters will further accelerate material delamination, eventually leading to the failure of GBR membrane function.

[0007] To address these issues, researchers have attempted to develop hierarchical pore gradient structures to replace traditional bilayer membranes, but existing technologies still have limitations:

[0008] 1. Planar gradient structures (such as the nano / microfiber meshes prepared by Luo et al. through co-electrospinning) cannot achieve three-dimensional bioactivity delivery;

[0009] 2. Natural polymer gradient membranes (such as the periosteum constructed by Lei et al. based on the electrochemical deposition of polysaccharide proteins) have insufficient mechanical strength, making it difficult to meet the mechanical requirements of GBR membranes;

[0010] 3. Non-connected pore gradient membranes (such as porous membranes prepared by Wang et al. using the phase inversion method) cannot achieve transmembrane transport of nutrients due to the closed pores.

[0011] In summary, existing GBR membranes have significant shortcomings in terms of structural continuity, mechanical stability, and synergistic biological functions. Summary of the Invention

[0012] This invention provides a micro / nanofiber composite biomembrane based on electrospinning technology and its preparation method. By controlling the ambient humidity and the rotation speed of the receiving roller, the fiber membrane structure can be continuously and gradually changed from ordered to disordered and from smooth to porous in one step. This enables structural continuity and bidirectional cell regulation function, providing a novel gradient structure that combines mechanical barrier and biological activity, and avoiding the interfacial weakness problem of traditional bilayer membranes.

[0013] According to one aspect of the present invention, a method for preparing a micro / nanofiber composite biomembrane based on electrospinning technology is provided, comprising the following steps: S100, preparing polycaprolactone, chloroform, and dimethyl sulfoxide; S200, mixing chloroform and dimethyl sulfoxide to obtain a solvent; S300, dissolving a polymer solution of polycaprolactone in the solvent and stirring evenly; S400, collecting fibers with a roller at an ambient temperature of 10℃-25℃ and a continuous humidity of 20%RH-70%RH, and performing electrospinning; by controlling the ambient humidity and the roller speed, a continuous gradual change in the fiber membrane structure from ordered to disordered and from smooth to porous is achieved in one step; S500, completing the electrospinning to obtain a micro / nanofiber composite biomembrane based on electrospinning technology.

[0014] Further, in step S200, the mixing ratio of chloroform and dimethyl sulfoxide is 7:3-10:1 by volume.

[0015] Furthermore, by volume ratio, the mixing ratio of chloroform to dimethyl sulfoxide is 9:1.

[0016] Furthermore, in step S300, the stirring is carried out using a magnetic stirrer at room temperature for 4-8 hours.

[0017] Furthermore, in step S300, the stirring is performed using a magnetic stirrer at room temperature for 6 hours.

[0018] Furthermore, in step S400, the roller speed is gradually reduced from 1200r-1600r to 300r-600r, and the total time for the roller to collect fibers is 4h-8h, thus completing electrospinning.

[0019] Furthermore, in step S400, the roller speed is gradually reduced from 1400r to 500r, and the total time for the roller to collect fibers is 4h-8h, thus completing electrospinning.

[0020] Furthermore, in step S100, the weight-average molecular weight of polycaprolactone is Mw = 80,000.

[0021] Furthermore, the polycaprolactone selected is polycaprolactone from Sigma Aldrich.

[0022] Furthermore, in step S100, the purity of both chloroform and dimethyl sulfoxide is 99%.

[0023] Furthermore, the chloroform and dimethyl sulfoxide are selected from Sinopharm Group.

[0024] According to another aspect of the present invention, a micro / nanofiber composite biomembrane based on electrospinning technology is also provided, which is prepared by the above-described method for preparing the micro / nanofiber composite biomembrane based on electrospinning technology.

[0025] The present invention has the following beneficial effects:

[0026] 1. Continuous and gradual structural change to avoid the risk of interfacial delamination: By controlling the ambient humidity and roller speed, the fiber membrane achieves a continuous and gradual structural change from ordered to disordered and from smooth to porous in one step, eliminating the delamination problem caused by the abrupt change in the mechanical properties of the interlayer interface of traditional bilayer membranes and improving the overall structural stability of the material.

[0027] 2. Bidirectional cell regulation function: The smooth and dense side (low humidity / low speed spinning zone) can effectively block the invasion of non-target cells such as fibroblasts, meeting the mechanical barrier requirements; the porous and loose side (high humidity / high speed spinning zone) has a pore structure that promotes osteoblast migration and transmembrane diffusion of nutrients (such as oxygen and glucose), avoiding local hypoxia caused by cell interface accumulation.

[0028] 3. Simplified process and controllability: The gradient structure can be controlled by electrospinning in a single step (without multi-layer composite or post-processing), which reduces the risk of batch variation caused by the complexity of traditional multilayer films.

[0029] 4. Synergistic optimization of material properties: Based on the hydrophobicity and biocompatibility of polycaprolactone (PCL), the gradient fiber membrane maintains mechanical strength while also possessing biodegradability, adapting to the dynamic time requirements of bone repair; the continuous gradual change in fiber porosity avoids the stress concentration problem caused by abrupt changes in pore size in traditional porous membranes, further extending the functional stability after implantation.

[0030] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0032] Figure 1 This is a schematic diagram illustrating the preparation method of micro / nanofiber composite biomembrane based on electrospinning technology according to a preferred embodiment of the present invention.

[0033] Figure 2 This is a bar chart showing the tensile strength of 12 wt% and 15 wt% PCL in the preferred embodiment of the present invention under different CF:DMSO ratios.

[0034] Figure 3 This is a comparison chart of the water contact angle of 12 wt% and 15 wt% PCL in the preferred embodiment of the present invention under the conditions of CF:DMSO ratio of 9:1 and 7:3.

[0035] Figure 4 This is a schematic diagram of the fiber membrane under different humidity and rotation speeds according to a preferred embodiment of the present invention, wherein... Figure 4 (a) shows the microstructure of the fiber membrane under different humidity and rotation speed conditions. Figure 4 (b) is a side view of the gradient fiber membrane. Figure 4 (c) is a bar chart showing the effect of humidity on porosity;

[0036] Figure 5 This is a bar chart of the tensile strength test of a preferred embodiment of the present invention, wherein... Figure 5 (a) is a comparison of the average strength of the gradient fiber membrane and the bilayer membrane. Figure 5 (b) shows the stress-strain curves of the bilayer membrane and the gradient membrane. Figure 5 (c) is a comparison of the average peel strength of the gradient fiber membrane and the bilayer membrane in a 90° peel test under humid conditions. Figure 5 (d) is a graph showing the change in contact angle under different humidity levels;

[0037] Figure 6 This is a diagram of the random radial arrangement of the cytoskeleton according to a preferred embodiment of the present invention, wherein... Figure 6 (a) is a diagram of cells spreading out in a random radial arrangement of the cytoskeleton. Figure 6 (b) A diagram showing the cell infiltration depth of a fiber membrane seeded on both sides of a gradient fiber membrane under different humidity conditions; Figure 6 (c) A diagram of cells that have penetrated the gradient structure fibrous membrane and entered the lower chamber of the transwell after 7 days of culture;

[0038] Figure 7 These are SEM images of PCL fiber membranes with different spinning schemes according to a preferred embodiment of the present invention. Detailed Implementation

[0039] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, the present invention can be implemented in many different ways as defined and covered below. Unless otherwise stated, the raw materials and reagents used in the following embodiments are commercially available or can be prepared by known methods.

[0040] like Figure 1 and Figure 2 As shown, the preparation method of the micro / nanofiber composite biomembrane based on electrospinning technology in this embodiment includes the following steps: S100, preparing polycaprolactone (PCL), chloroform (CF), and dimethyl sulfoxide (DMSO); S200, mixing chloroform and DMSO to obtain a solvent; S300, dissolving the polycaprolactone polymer solution in the solvent and stirring evenly; S400, collecting fibers with a roller at an ambient temperature of 10℃-25℃ and a continuous humidity of 20%RH-70%RH, and performing electrospinning; by controlling the ambient humidity and the roller speed, the fiber membrane structure can be continuously and gradually changed from ordered to disordered and from smooth to porous in one step; S500, completing the electrospinning to obtain the micro / nanofiber composite biomembrane based on electrospinning technology. This invention discloses a method for preparing micro / nanofiber composite biomembranes based on electrospinning technology. The method utilizes humidity gradient electrospinning to prepare polycaprolactone (PCL) hierarchical pore gradient fiber membranes. By controlling the ambient humidity and roller speed, a continuous gradient structure from ordered to disordered, and from smooth to porous, is achieved in one step. This eliminates the delamination problem caused by abrupt changes in the interlayer mechanical properties of traditional bilayer membranes, thus improving the overall structural stability of the material. The smooth, dense side (low humidity / low speed spinning zone) effectively blocks the invasion of non-target cells such as fibroblasts, meeting the mechanical barrier requirements. The porous, loose side (high humidity / high speed spinning zone) features interconnected pore structures. This invention promotes osteoblast migration and transmembrane diffusion of nutrients (such as oxygen and glucose), avoiding local hypoxia caused by cell interface accumulation. The gradient structure can be controllably prepared through a single-step electrospinning process (without multilayer composites or post-processing), reducing the batch variation risk caused by the complex processes of traditional multilayer membranes. Based on the hydrophobicity and biocompatibility of polycaprolactone (PCL), the gradient fiber membrane maintains mechanical strength while also possessing biodegradability, adapting to the dynamic time requirements of bone repair. The continuous gradual change in fiber porosity avoids the stress concentration problem caused by abrupt pore size changes in traditional porous membranes, further extending the functional stability after implantation. Through structural design and process innovation, this invention solves the technical defects of traditional GBR membranes in terms of interface layering, cell regulation contradictions, and process complexity, providing an integrated gradient fiber membrane that combines mechanical barrier function with bioactive delivery capabilities.

[0041] In this embodiment, the content of polycaprolactone (PCL) is 12wt%-17wt%. Preferably, the content of polycaprolactone (PCL) is 15wt%. A four-factor, three-level orthogonal experiment (L9(3)) was conducted.4 An orthogonal experimental design was used to optimize key electrospinning process parameters affecting the morphology and structure of PCL fibers, aiming to establish a stable, controllable, and repeatable basic electrospinning process platform. This optimization process aimed to precisely control the basic characteristics of the composite fibers, such as average diameter, distribution uniformity, tensile strength, and water contact angle, to ensure the preparation of a basic fiber membrane with good fiber morphology uniformity and structural stability. The orthogonal experimental factors were pre-screened to ensure spinnability under these conditions. Table 1 shows the viscosity, conductivity, and surface tension of PCL spinning solutions with different concentrations, and Table 2 shows the spinning schemes for the polymer and solvent systems used in electrospinning.

[0042] Table 1 Properties of spinning solutions with different PCL contents

[0043]

[0044] Table 2 Spinning schemes for polymer and solvent systems used in electrospinning

[0045]

[0046] like Figure 7 As shown, under fixed environmental parameters (30℃, 20%RH) and instrument configuration (22G needle, 0.8ml / h injection speed), the optimal process was established through a three-stage orthogonal experiment. SEM observation revealed that when the PCL concentration was 9 wt%, insufficient molecular chain entanglement resulted in beaded structures in the SEM images of the fiber membrane across the entire parameter range. When the PCL concentration was 12 wt% and 15 wt%, the SEM images of the fiber membrane showed no beading defects. However, at a PCL concentration of 12 wt%, while the molecular chain entanglement increased, uneven relaxation led to discrete fiber diameters, resulting in poorer fiber diameter uniformity compared to the PCL fiber membrane with a PCL concentration of 15 wt%.

[0047] In this embodiment, in step S200, the volume ratio of chloroform to dimethyl sulfoxide is 7:3-10:1. A high proportion of chloroform (7-10 parts) serves as a highly efficient solvent for PCL, ensuring complete dissolution of the polymer and forming a uniform spinning solution, thus preventing fiber breakage or uneven diameter due to incomplete dissolution. An appropriate amount of dimethyl sulfoxide (3-1 part) acts as a less efficient solvent, controlling the solvent evaporation rate and promoting phase separation during electrospinning. This induces the formation of a porous structure (high humidity side) or a dense structure (low humidity side) on the fiber surface under a humidity gradient environment, achieving a continuous and gradual structural change. The 7:3-10:1 mixing ratio of chloroform to dimethyl sulfoxide balances the solubility and volatility of the solvent system, preventing nozzle clogging or fiber adhesion during spinning and ensuring the controllable preparation of the gradient structure. Preferably, the volume ratio of chloroform to dimethyl sulfoxide (DMSO) is 9:1. While ensuring PCL solubility, the introduction of DMSO moderately delays solvent evaporation, making the fiber deposition more susceptible to environmental humidity. This enhances the performance difference between the dense side (barrier function) and the porous side (bioactivity), while avoiding structural brittleness caused by excessive solvent evaporation. A 9:1 mixing ratio of chloroform to DMSO provides the optimal synergistic effect of the solvent ratio and humidity gradient, resulting in a stable fiber membrane with a uniform pore size gradient distribution and reducing batch-to-batch variations.

[0048] Tensile strength tests were conducted on six groups of electrospun fiber membranes with PCL concentrations of 12wt% and 15wt% using a universal testing machine. When the solvent ratio CF:DMSO = 8:2, the fiber membrane exhibited the lowest tensile strength (<3.0 MPa), significantly weaker than the other two solvent ratios. When the CF:DMSO ratio was 9:1 and 7:3, the tensile strength of the fiber membranes could be increased to over 4.0 MPa by adjusting the spinning parameters, exceeding the 3.2 MPa~4.0 MPa range of natural human periosteum. Figure 2 As shown. Further characterization of the surface hydrophilicity of the four groups of fiber membranes with CF:DMSO ratios of 9:1 and 7:3 was performed using a water contact angle meter. The results showed that when the PCL concentration in the spinning solution was 15 wt%, the water contact angle of the fiber membrane was smaller than that of the fiber membrane with a PCL concentration of 12 wt%. Figure 3 As shown.

[0049] In this embodiment, the stirring in step S300 is performed using a magnetic stirrer at room temperature for 4-8 hours. Preferably, it is 6 hours. Magnetic stirring, through gentle shear force, avoids air bubbles or localized overheating that might be introduced by mechanical stirring, ensuring that PCL is fully dissolved in the solvent to form a uniform and stable spinning solution. The 4-8 hour stirring time provides sufficient dissolution time for the PCL molecular chains to fully expand, preventing fiber breakage or nozzle clogging during spinning due to undissolved particles. Limiting the stirring to room temperature eliminates the need for heating, avoiding solvent evaporation or polymer thermal degradation, and ensuring consistent performance across different batches of spinning solutions. The 4-8 hour stirring time range covers the complete dissolution threshold of PCL in the solvent system, while the preferred 6 hours further shortens the process cycle and maintains solution stability. The preferred 6-hour stirring time balances dissolution efficiency and process cost, achieving a suitable solution viscosity. This ensures continuous fiber formation during electrospinning, avoids uneven fiber diameter due to excessively low viscosity, and prevents solvent evaporation or excessively high solution viscosity due to excessive stirring, which could affect spinning stability.

[0050] In this embodiment, in step S400, the roller speed is gradually reduced from 1200r-1600r to 300r-600r, and the total time for the roller to collect fibers is 4h-8h, completing the electrospinning. Preferably, the roller speed is gradually reduced from 1400r to 500r, and the total time for electrospinning is 6h. During the high-speed range of 1200-1600 rpm, strong stretching force is generated, causing the fibers to be highly oriented along the direction of roller rotation, forming a dense and ordered layer. As the speed gradually decreases to 300-600 rpm, the fiber orientation decreases accordingly, forming a gradually transitioning porous and disordered layer. At high speeds, the fiber deposition rate is fast, forming a low-porosity dense structure. As the speed decreases, the fiber packing density decreases, and the porosity gradually increases. The continuous and gradual change in speed avoids the delamination interface of traditional multilayer films. The continuous transition of the fiber structure from ordered to disorder enhances the overall mechanical continuity. The defined speed variation range ensures batch-to-batch consistency. The total time of 4-8 hours adapts to the needs of different production scales. The preferred gradient range of 1400-500 rpm optimizes the transition gradient between orientation and disorder, balancing gradient formation efficiency and structural stability. The preferred total spinning time of 6 hours ensures sufficient fiber deposition at each speed stage and avoids abrupt transitions between layers, balancing production efficiency and quality stability. Optionally, the voltage for electrospinning is 12kV-20kV. Optionally, the spinning distance for electrospinning is 10cm-20cm.

[0051] In this embodiment, in step S100, the weight-average molecular weight (Mw) of polycaprolactone (PCL) is 80,000. PCL with a weight-average molecular weight of 80,000 has moderate melt viscosity and chain entanglement, ensuring stable stretching and shaping of the fibers during electrospinning. This avoids problems such as uneven fiber diameter or fiber breakage caused by excessively low molecular weight (easy to break) or excessively high molecular weight (difficult to spin). It imparts balanced mechanical strength and flexibility to the fiber membrane, meeting the mechanical requirements of the GBR membrane for barrier function (such as resistance to soft tissue pressure) while avoiding implantation discomfort due to excessively hard materials. The degradation rate of PCL is directly related to its molecular weight. Mw=80,000 provides a degradation cycle of approximately 6-12 months (based on the known degradation characteristics of PCL), matching the typical bone defect repair time window, avoiding premature degradation leading to loss of barrier function or delayed degradation interfering with bone remodeling. The gradual breakage of molecular chains during degradation maintains the phased mechanical support of the fiber structure, while the concentration of released degradation products (caprolactone monomers) is controllable, preventing excessive accumulation of a local acidic environment. PCL with a weight-average molecular weight (Mw) of 80,000 exhibits good solubility in solvent systems (chloroform / dimethyl sulfoxide = 7:3-10:1), forming a homogeneous and stable spinning solution. Combined with the stirring process in step S300, this prevents undissolved clumps from clogging the nozzle or causing fiber defects. The consistent molecular weight ensures uniform rheological properties across different batches of spinning solution, improving process repeatability. As an FDA-approved biomaterial, PCL's molecular weight of 80,000 falls within the low immunogenicity range, and its degradation products can be safely metabolized, meeting the basic biocompatibility requirements of GBR membranes. Optionally, polycaprolactone (PCL) from Sigma Aldrich can be selected. Sigma Aldrich's PCL has high purity (e.g., low residual catalyst, no plasticizers or other impurities), avoiding interference from impurities in the electrospinning process or affecting the biocompatibility of the fiber membrane. The PCL provided by the company can accurately meet the molecular weight requirement of Mw=80,000 in the technical solution (as clearly stated in the product catalog), avoiding fluctuations in fiber mechanical properties caused by differences in molecular weight specifications from different manufacturers. Sigma Aldrich's PCL products typically meet USP / EP grade standards, and their heavy metal content, endotoxin levels, and other indicators meet the requirements for implantable materials, reducing the biological risks of clinical applications.

[0052] In this embodiment, in step S100, both chloroform and dimethyl sulfoxide have a purity of 99%. The 99% high-purity solvent prevents impurities (such as moisture and organic residues) from interfering with the PCL dissolution process, preventing polymer precipitation or gelation due to solvent impurities, and ensuring the uniformity and stability of the spinning solution. Excessive moisture in chloroform may trigger PCL hydrolysis, especially during prolonged stirring; 99% purity effectively suppresses this risk. Impurities (such as low-boiling-point components) may interfere with the solvent evaporation kinetics during electrospinning, leading to fiber surface defects such as beading and uneven pores. The 99% high-purity solvent makes evaporation behavior more controllable, working synergistically with the humidity gradient to precisely control the gradual transition of the fiber structure from dense to porous. If chloroform of insufficient purity contains toxic impurities such as carbon tetrachloride, these impurities may remain in the fiber membrane. A purity of 99% minimizes this risk. Similarly, dimethyl sulfoxide (DMSO), as a cell permeabilizer, may affect cell behavior if it contains impurities (such as DMSO). High purity ensures biocompatibility meets GBR membrane requirements. Solvent purity is a key variable affecting the stability of electrospinning parameters (such as voltage and flow rate). 99% purity reduces batch-to-batch variations, which is especially crucial for precisely controlled processes like gradient structures. Optionally, chloroform and dimethyl sulfoxide (DMSO) from Sinopharm Group can be selected. As a leading chemical supplier in China, Sinopharm Group provides chloroform and DMSO that meet high purity standards (99%), which can avoid impurities (such as moisture and organic residues) from interfering with the dissolution of PCL and the electrospinning process, ensuring the uniformity of fiber morphology and structural integrity, and avoiding fluctuations in solvent evaporation rate or solubility due to differences in purity specifications from different manufacturers, thereby affecting the structural gradient effect of the gradient fiber membrane. Using Sinopharm Group's standard raw materials can eliminate additional solvent purification steps, reducing production costs and process complexity. Sinopharm Group's solvent products usually comply with the Chinese Pharmacopoeia (ChP) or national standards (GB), and the content of heavy metals, residual solvents and other harmful substances is controlled within a safe range, reducing the biological risks caused by fiber membrane implantation.

[0053] The micro / nanofiber composite biomembrane based on electrospinning technology in this embodiment was prepared using the above-described method for preparing micro / nanofiber composite biomembranes based on electrospinning technology.

[0054] In practice, a hierarchical pore gradient structure fiber membrane is produced by controlling humidity through electrospinning and phase separation technology. The fiber surface of this membrane gradually changes from a smooth surface to wrinkles, then to mesopores, and finally to a porous structure with both macropores and mesopores. The mechanical properties show a continuous change, and the fiber arrangement direction transitions from ordered to disordered, resulting in different adhesion states on both sides of the cells. The ordered low-porosity side inhibits cell invasion, while the disordered high-porosity side promotes cell migration and differentiation. The gradient feature reduces the possibility of material delamination, providing a new approach for the preparation of GBR membranes.

[0055] Example 1:

[0056] Polycaprolactone (PCL, Mw=80,000) was purchased from Sigma Aldrich; 99% chloroform (CF) and 99% dimethyl sulfoxide (DMSO) were purchased from Sinopharm and were used directly without purification.

[0057] A 15 wt% PCL polymer solution was dissolved in a solvent of CF:DMSO = 9:1 (v / v) and stirred at room temperature for 6 h on a magnetic stirrer.

[0058] Then, at an ambient temperature of 20℃ and a continuous humidity of 20%RH-70%RH, the fibers are collected by a roller, with the roller speed gradually reduced from 1400r to 500r, completing the electrospinning process in a total of 6 hours. Figure 1 As shown;

[0059] The resulting fibrous membrane exhibits a gradient structure where the fibers gradually transition from non-porous to porous, and the fiber arrangement changes from ordered to disordered.

[0060] Comparative Example 1:

[0061] The difference from Example 1 is that a double-layer membrane with a humidity of 65%RH, a roller speed of 1400r, and fiber collection for 3 hours was prepared, and then the humidity was adjusted to 20%RH, the roller speed was 500r, and fiber collection for 3 hours was carried out as a control.

[0062] Characterization methods: The microstructure of fiber membranes prepared under different humidity conditions and those prepared under gradient humidity conditions was characterized using a Hitachi S-4800 scanning electron microscope (SEM) to determine the structure and porosity of the fiber membranes. Gradient membranes and control bilayer membranes were cut into 3mm × 30mm pieces and subjected to tensile tests on a universal testing machine to compare the tensile strength of the two groups. Gradient membranes and control bilayer membranes were cut into 10mm × 50mm pieces, soaked in PBS for 1 hour, and then adhered to 20mm × 80mm test paper. T-type peel tests were performed on a universal testing machine to compare the interfacial bonding strength of the two groups and to infer the ease of separation in vivo. To investigate the effect of fiber structure on hydrophilicity, the static water contact angle of fiber membranes prepared under different humidity conditions was tested at room temperature using a contact angle testing system (JC2000X1, China).

[0063] Cell adhesion and cell migration: by using 2 × 10⁻⁶ cells per well 4BMSC cells were evenly seeded on both sides of a UV-sterilized gradient fiber membrane for cell adhesion experiments. DAPI was used to label the cell nuclei, and FITC-phalloidin was used to label the cytoskeleton. Cell morphology and penetration depth were examined using a confocal laser scanning microscope (CLSM, Leica, Germany) to determine the effect of fiber structure on BMSC cell growth. Five 5×10⁶ cells were placed in each well. 4 Cells were evenly spread on the surface of the gradient membrane and bilayer membrane placed in the upper chamber of the transwell well. Serum-free culture medium was used in the upper chamber, and culture medium containing 20% ​​serum was used in the lower chamber to promote cell migration. After 7 days, the number of cells that migrated in the lower chamber was observed to indirectly assess the separation of the fibrous membrane.

[0064] This invention relates to a method for preparing micro / nanofiber composite biomembranes based on electrospinning technology. The method employs electrospinning combined with phase separation to prepare PCL fiber membranes with a hierarchical pore gradient structure, such as… Figure 1 The diagram shows cell adhesion morphologies, illustrating cell adhesion and growth patterns on fibers with different surface structures. The left image, corresponding to macroporous fibers prepared in a high-humidity region, shows cells that are widely spread out with fully extended pseudopodia, exhibiting a good spindle or polygonal shape. This indicates that cells can effectively recognize the material surface and interact strongly with it, a sign of healthy and active cells. The right image, corresponding to dense / non-porous fibers prepared in a low-humidity region, shows cells that are more circular in shape with a smaller spread area. This indicates weaker interaction between cells and the material surface, resulting in less than ideal adhesion, which usually implies that cell activity and function may be limited. Fibers with porous surfaces prepared in a high-humidity environment are more conducive to cell adhesion and spread, providing a superior growth environment for cells. The hierarchical pore gradient membrane prepared in this invention can guide cells to migrate inward and distribute evenly, successfully constructing a three-dimensional cell-material complex, which traditional simple laminated membranes cannot achieve. This demonstrates that the biomembrane prepared by this invention not only has good biocompatibility, but more importantly, its unique three-dimensional structure can simulate the properties of the natural extracellular matrix, and has great application potential in tissue engineering (such as skin, bone, and blood vessel regeneration).

[0065] The surface roughness of fibers and the porosity of fiber membranes have a significant impact on the biological properties of GBR membranes. The membrane sequence within the gradient structure results in different porosities and roughnesses. Figure 4 (a) illustrates the microstructure of the fiber membrane under different humidity and rotation speeds. With increasing humidity, the fibers gradually transition from a smooth surface to wrinkled surfaces, then mesopores appear, and finally a porous structure (20 nm-700 nm) exhibiting both macropores and mesopores. Viewed from the side of the gradient fiber membrane, the transition from porous to non-porous fibers is evident, with fibers interwoven and without clear boundaries. Figure 4As described in (b), with increasing humidity and decreasing receiving drum speed, the fiber membrane changes from an ordered arrangement to a disordered arrangement, and the non-porous smooth fibers become porous and rough fibers. Statistical analysis using the ImagageJ model shows that the porosity of the fiber membrane increases from 7% to 24%. Figure 4 As shown in (c), the gradient structure avoids stress concentration and cell rejection caused by the abrupt interface of the traditional bilayer structure by providing a smoother structural change.

[0066] For mechanical properties, tensile strength tests were performed on five samples using a universal testing machine. The average strength of the gradient fiber membrane (11.9 ± 2.3 MPa) was observed to be significantly higher than that of the bilayer membrane (4.9 ± 1.0 MPa). Figure 5 As shown in (a), five samples were subjected to a 90° peel test under humid conditions using the same instrument. The average peel strength of the gradient fiber membrane was 0.019±0.004 N / mm, significantly higher than that of the bilayer membrane (0.007±0.003 N / mm). Figure 5 As shown in (b), the stress value of the gradient membrane is consistently higher than that of the double-layer membrane throughout the tensile process, exhibiting superior tensile deformation resistance. This curve further confirms that the gradient membrane possesses higher strength and toughness, and its structural continuity effectively avoids stress concentration at the interface, thereby achieving a significant improvement in mechanical properties; Figure 5 As shown in (c), this is likely due to the continuous structure of the gradient fiber membrane suppressing stress concentration and eliminating weak interfacial regions. This also provides strong evidence for the interpenetration of gradient structure fibers. Furthermore, this result initially reflects that gradient structure fiber membranes can reduce the possibility of intracellular fiber membrane delamination. Figure 5 As shown in (d), the water contact angle decreases with the increase of porosity on the surface of the fiber membrane. The decrease in contact angle indicates an increase in hydrophilicity, which is beneficial to cell adhesion.

[0067] Experiments were conducted using bone marrow mesenchymal stem cells (BMSCs) to assess cell adhesion under different humidity conditions, aiming to determine how fiber surface morphology affects cell morphology. During a 3-day culture period, cell adhesion morphology differed significantly under different humidity conditions. Due to the directional arrangement of the fiber layer at 20% RH, the cell adhesion morphology was spindle-shaped with a significantly increased aspect ratio (up to 7:1 or higher). The porous morphology of the fiber layer at 65% RH further increased the cell contact sites, with the cytoskeleton arranged randomly in a radial pattern, resulting in a significantly increased cell spreading area, with an average increase of 138.42%. Figure 6 As shown in (a), the random radial arrangement of the cytoskeleton promotes osteogenic differentiation and matrix mineralization. When cells are seeded on both sides of a gradient fibrous membrane, the cell infiltration depth at 65% RH on the inner side is significantly higher than at 20% RH (more than 5 times). Figure 6As shown in (b), this indicates that the non-porous layer effectively prevents cells from penetrating deeper into the material. After 7 days of culture, the number of cells penetrating the gradient-structured fibrous membrane into the lower chamber of the transwell was significantly greater than that of the layered fibrous membrane, which indirectly reflects that the gradient structure reduces cell accumulation, such as... Figure 6 As shown in (c).

[0068] By combining humidity-mediated phase separation with electrospinning, a "one-step manufacturing strategy" is provided for preparing polycaprolactone (PCL) membranes with a continuous hierarchical porosity structure and a fiber arrangement transition (ordered → random). This structure solves the problems inherent in traditional bilayer membranes. The seamless gradient structure eliminates stress concentration at the bilayer interface, resulting in a peel strength 257% higher than that of bilayer membranes, thereby reducing the likelihood of periosteal detachment in vivo. Simultaneously, the synergistic effect between low-porosity ordered fibers and high-porosity disordered regions generates "bidirectional cell regulation," where the outer layer inhibits cell invasion while the inner layer promotes cell migration. This hierarchical porosity gradient structure provides a new approach for preparing bone repair membranes.

[0069] Matters not covered in this invention are common knowledge.

[0070] The technical features of the above embodiments can be combined in any way. For the sake of brevity, 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, they should be considered to be within the scope of this specification.

[0071] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a micro-nanofiber composite biomembrane based on electrospinning technology, characterized in that, The method comprises the following steps: S100, preparing polycaprolactone, chloroform and dimethyl sulfoxide; S200, mixing chloroform and dimethyl sulfoxide to prepare a solvent; S300, dissolving a polycaprolactone polymer solution in the solvent and stirring uniformly; S400, electrospinning at an ambient temperature of 10-25 DEG C and a continuous humidity of 20-70% RH, collecting fibers by a roller; by adjusting the ambient humidity and the roller speed, the fiber membrane structure is continuously and gradually changed from order to disorder and from smooth to porous in one step; S500, completing electrospinning to prepare a micro-nano fiber composite biomembrane based on electrospinning technology.

2. The method of claim 1, wherein the electrospinning technique-based preparation of a micro-nanofiber composite biomembrane is characterized by, In step S200, the mixing ratio of chloroform and dimethyl sulfoxide is 7:3-10:1 by volume.

3. The method of claim 2, wherein the electrospinning technique-based preparation of a micro-nanofiber composite biomembrane is characterized by, The mixing ratio of chloroform and dimethyl sulfoxide is 9:1 by volume.

4. The method of claim 1, wherein the electrospinning technique-based preparation of a micro-nanofiber composite biomembrane is characterized by, In step S300, the stirring is performed by a magnetic stirrer at room temperature for 4-8 hours.

5. The method of claim 4, wherein the electrospinning technique-based preparation of a micro-nanofiber composite biomembrane is characterized by, In step S300, the stirring is performed by a magnetic stirrer at room temperature for 6 hours.

6. The method of claim 1, wherein the electrospinning technique-based preparation of a micro-nanofiber composite biomembrane is characterized by, In step S400, the roller speed is gradually reduced from 1200r-1600r to 300r-600r, and the total time for collecting fibers by the roller is 4-8 hours, and the electrospinning is completed.

7. The method of claim 6, wherein the electrospinning technique-based preparation of the micro-nanofiber composite biomembrane is characterized by, In step S400, the roller speed is gradually reduced from 1400r to 500r, and the total time for collecting fibers by the roller is 6 hours, and the electrospinning is completed.

8. The method of claim 1 to 7, wherein the method of preparation of micro- and nanofiber composite biomembranes based on electrospinning technology is characterized by, In step S100, the weight average molecular weight Mw of the polycaprolactone is 80,000.

9. The method of claim 1 to 7, wherein the method of preparation of micro- and nanofiber composite biomembranes based on electrospinning technique is characterized by, In step S100, the purity of chloroform and dimethyl sulfoxide is 99%.

10. A micro-nanofiber composite biomembrane based on electrospinning technology, characterized in that, The micro-nano fiber composite biomembrane based on electrospinning technology is prepared by the method of any one of claims 1-9.

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