A biomimetic composite guided bone regeneration membrane, its preparation method and application

Abstract

This invention belongs to the field of surgical dressing technology, specifically disclosing a biomimetic composite guided bone regeneration membrane, its preparation method, and its applications. It comprises a guiding layer, a barrier layer, and an osteogenic layer. The osteogenic layer is a biocomposite hydrogel layer, with one side having a surface structure containing a micro-pit array, and the barrier layer and guiding layer sequentially fixed to the other side from the inside out. The guiding layer is a directionally arranged parallel polycaprolactone fiber structure, and the barrier layer is a randomly arranged coiled polycaprolactone fiber structure. The biomimetic composite guided bone regeneration membrane prepared by this invention has the characteristics of novel structure and excellent biological effects. Its unique hierarchical structure and functional design make it promising for broad applications in dental implant guided bone regeneration barrier membranes and oral repair membranes, and it is expected to play an important role in bone defect repair.

Description

Technical Field

[0001] This invention relates to a surgical dressing, and more particularly to a biomimetic composite guiding bone regeneration membrane, its preparation method, and its application. Background Technology

[0002] Guided Bone Regeneration (GBR) is a crucial technique in modern dental implantology and bone repair, widely applied in various clinical scenarios such as jawbone augmentation, alveolar bone defect repair, periodontitis treatment, and alveolar bone preservation after tooth extraction. The core of this technique lies in using a barrier membrane to isolate soft tissue from the bone defect area, preventing competitive growth of soft tissue cells and thus providing a relatively closed and stable environment for bone regeneration, promoting the healing of bone defects.

[0003] Currently, barrier membranes used in clinical practice can be divided into non-absorbable membranes and absorbable membranes, but both have many limitations. For example, the most commonly used non-absorbable membrane, polytetrafluoroethylene (PTFE) membrane, although it has good biocompatibility and stability, its hardness is not compatible with soft tissue, which may lead to wound dehiscence and require a second surgery for removal, causing additional pain, financial burden, and infection risk to patients. On the other hand, absorbable membranes, such as collagen membranes, although avoiding a second surgery, generally lack the bio-inducibility of cells and cannot effectively promote bone tissue regeneration, limiting their widespread clinical application.

[0004] An ideal barrier membrane should possess both excellent mechanical and biological properties. It needs not only sufficient mechanical strength and flexibility to maintain the spatial structure of the bone defect area, but also good biocompatibility to induce the directional arrangement of gingival soft tissue cells and inhibit their competitive inward growth. Furthermore, excellent osteogenic induction activity is crucial for the regeneration of bone defect areas. How to mimic the microstructure of natural bone tissue and combine it with active ingredients such as chemical ions, growth factors, and drugs to promote osteoblast adhesion, proliferation, and differentiation, thereby providing a more ideal microenvironment for bone tissue regeneration, is a challenging research topic. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a biomimetic composite guided bone regeneration membrane. This membrane not only possesses excellent mechanical and biological properties, but also, by mimicking the microstructure of natural bone tissue and combining active ingredients such as chemical ions, promotes the adhesion, proliferation, and differentiation of osteoblasts while inhibiting the competitive growth of soft tissue cells, thereby providing a more ideal microenvironment for bone tissue regeneration.

[0006] This invention also provides a method for preparing a biomimetic composite guided bone regeneration membrane.

[0007] The technical solution is as follows:

[0008] A method for preparing a biomimetic composite guided bone regeneration membrane includes the following steps:

[0009] Step 1, Preparation of oriented parallel polycaprolactone fibers: The oriented parallel polycaprolactone fibers are prepared by using a melt near-field direct writing process and utilizing the stable state of the jet to prepare the guiding layer;

[0010] Step 2, Preparation of randomly arranged crimped polycaprolactone fibers: Continue to use the melt near-field direct writing process to cover and deposit randomly arranged crimped polycaprolactone fibers on the guide layer using the whipping unstable state of the jet, and prepare a barrier layer;

[0011] Step 3, hydrophilization treatment: The prepared polycaprolactone membrane with the guiding layer and the barrier layer is immersed in sodium hydroxide solution for etching to perform hydrophilization treatment, increase the surface roughness, and obtain a hydrophilized double-layer PCL membrane.

[0012] Step 4, prepare the biocomposite hydrogel: prepare the biocomposite hydrogel corresponding to the osteogenic layer;

[0013] Step 5, mold preparation: Prepare a resin positive mold with the micro-dimple array, and after cleaning and curing, use it to make a polydimethylsiloxane negative mold;

[0014] Step 6, Preparation of composite membrane: Uncrosslinked biocomposite hydrogel is cast onto the surface of the negative mold, covered with a hydrophilic double-layer PCL membrane, and then subjected to low-temperature crosslinking and photocrosslinking in sequence. After separation from the mold, a biomimetic composite guiding bone regeneration membrane is obtained.

[0015] Preferably, the biocomposite hydrogel of the osteogenic layer is composed of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), methacrylamide gelatin-methacrylamide hyaluronic acid (GelMA-HAMA), and magnesium-substituted calcium silicate (CSi-Mg) particles, and has a surface micro-dimple array structure.

[0016] Furthermore, during preparation, based on the volume of the phosphate buffer solution, the added phenyl (2,4,6-trimethylbenzoyl)lithium phosphate is 0.25%–0.3% (g / mL); the added methacrylamide gelatin is 5–15% (g / mL); the added methacrylamide hyaluronic acid is 0.2%–2% (g / mL); and the added magnesium-substituted calcium silicate particles are 1–3% (g / mL).

[0017] Preferably, in step 4, the preparation process and concentration of the bio-composite hydrogel are as follows:

[0018] First, a preliminary solution of phenyl (2,4,6-trimethylbenzoyl)lithium phosphate was prepared using phosphate buffer solution with a mass-volume concentration of 0.25%–0.30% (g / mL).

[0019] Then, methacrylamide gelatin and methacrylamide hyaluronic acid are added. The mass-volume fraction of methacrylamide gelatin added is 5-15% (g / mL) and the mass-volume fraction of methacrylamide hyaluronic acid added is 0.2-2% (g / mL) based on phosphate buffer solution.

[0020] Finally, magnesium-substituted calcium silicate particles are added, with a mass-volume fraction of 1-3% (g / mL).

[0021] More specifically:

[0022] A biomimetic composite guiding bone regeneration membrane and its preparation method, comprising the following steps:

[0023] Step 1: Using a melt near-field direct writing process, oriented parallel polycaprolactone fibers are prepared by utilizing the stable state of the jet to prepare the guiding layer;

[0024] Step 2: Using a melt near-field direct writing process, randomly arranged crimped polycaprolactone fibers are deposited on the surface of the guide layer by utilizing the whipping instability of the jet to prepare a barrier layer.

[0025] Step 3: The obtained polycaprolactone membrane with a guiding layer and a barrier layer is hydrophilized using sodium hydroxide solution to obtain a hydrophilized bilayer PCL membrane.

[0026] Step 4: Place the double-layer PCL membrane in a mold with a micro-pit array structure corresponding to the above, pour the prepared bio-composite hydrogel into the mold, and solidify it to obtain a biomimetic composite guiding bone regeneration membrane.

[0027] More specifically, a biomimetic composite guided bone regeneration membrane and its preparation method include the following steps:

[0028] Step 1, Preparation of oriented parallel PCL fibers: Using a high-precision melt near-field direct writing process, oriented parallel PCL fibers are prepared by utilizing the stable state of the jet.

[0029] Step 2, Preparation of randomly arranged crimped PCL fibers: On the parallel PCL fibers prepared in the above steps, a high-precision melt near-field direct writing process is used to cover and deposit randomly arranged crimped PCL fibers by utilizing the whipping instability of the jet.

[0030] Step 3, hydrophilization treatment: The prepared PCL film with the guiding layer and the barrier layer is immersed in sodium hydroxide solution for etching to perform hydrophilization treatment and increase the surface roughness.

[0031] In practice, sodium hydroxide particles are dissolved in deionized water to obtain a sodium hydroxide solution. The PCL membrane with the guide layer and barrier layer prepared above is immersed in the sodium hydroxide solution for etching and hydrophilization treatment to increase surface roughness and facilitate cell adhesion.

[0032] Step 4, prepare the biocomposite hydrogel: first prepare the LAP preparative solution, then add GelMA and HAMA to obtain the hydrogel matrix, and finally add CSi-Mg particles and disperse them evenly to obtain the uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel.

[0033] In practice:

[0034] (4-1) Preparation of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) preparative solution: Add a small amount of LAP photoinitiator to phosphate (PBS) buffer solution and heat in a metal bath to dissolve it completely.

[0035] (4-2) Preparation of methacrylated gelatin-methacrylated hyaluronic acid (GelMA-HAMA) hydrogel matrix: Add appropriate amounts of GelMA and HAMA to the above LAP preparation solution, continue to heat in a metal bath to fully dissolve the two, and filter with a 0.22μm bacterial filter membrane.

[0036] (4-3) Adding UV-sterilized magnesium-substituted calcium silicate (CSi-Mg) particles: Add an appropriate amount of CSi-Mg particles to the above hydrogel matrix and disperse them using an ultrasonic disperser to avoid particle agglomeration. Uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel is obtained.

[0037] Step 5, mold preparation: First, a resin positive mold is prepared using a high-resolution projection-type light curing printer. After cleaning and curing, a polydimethylsiloxane (PDMS) negative mold is prepared using the positive mold.

[0038] In practice:

[0039] (5-1) A resin positive mold with a surface micro-dimple array structure was prepared using a high-resolution projection-type photopolymerization printer. The positive mold was then placed in an ultrasonic cleaner and cleaned with 95% alcohol solution, followed by a second ultrasonic cleaning with fluorinated liquid to remove residual resin from the surface.

[0040] (5-2) Place the resin positive mold in an oven and heat it, then irradiate it with a blue light curing lamp to fully cure it.

[0041] (5-3) Prepare polydimethylsiloxane (PDMS), pour the curing agent into the main body, stir evenly, and obtain uncured PDMS.

[0042] (5-4) Pour the uncured polydimethylsiloxane into the resin positive mold, put it in a vacuum chamber to evacuate the vacuum, and ensure that the internal air bubbles are completely removed. Then place it in an oven to heat and cure, resulting in a PDMS negative mold with surface microstructure.

[0043] Step 6, Preparation of composite membrane: Uncrosslinked biocomposite hydrogel is cast onto the surface of PDMS negative mold, covered with a hydrophilic double-layer PCL membrane, and then subjected to low-temperature crosslinking and photocrosslinking in sequence. After separation from the mold, a biomimetic composite guiding bone regeneration membrane is obtained.

[0044] In practice:

[0045] (6-1) The uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel obtained in step 4 is poured onto the surface of the PDMS negative mold obtained in step 5 and placed on a heating table for heating to prevent the liquid from cooling down too quickly and forming gel.

[0046] (6-2) The hydrophilic double-layer PCL membrane obtained in step 3 is covered on the ungelled biocomposite hydrogel, and low-temperature crosslinking and photocrosslinking are performed in sequence. After separation from the mold, a biomimetic composite guiding bone regeneration membrane is obtained.

[0047] Step 7, Irradiation sterilization: The guiding bone regeneration membrane obtained in step 6 is sterilized by ultraviolet light irradiation.

[0048] Furthermore, in step 3, the concentration of the sodium hydroxide solution is 2-5 mol / L, and even more specifically, the concentration of the sodium hydroxide solution in step 3 is 3 mol / L; the immersion etching time is 2-3 hours, and after completion, the double-layer PCL membrane is rinsed with deionized water (1-5 times). Even more specifically, in step 3, the concentration of the sodium hydroxide solution is 3 mol / L, the immersion etching time is 2-3 hours, and after completion, the double-layer PCL membrane is rinsed with deionized water 3 times.

[0049] Furthermore, in step 4, the concentration of the LAP preparation solution is 0.25% to 0.30% (g / mL, based on the volume of PBS buffer solution in the LAP preparation solution), the mass volume fraction of GelMA added is 10%, and the mass volume fraction of HAMA added is 1%.

[0050] Furthermore, in step 5, the resin positive mold is ultrasonically cleaned for 5 seconds using a 95% alcohol solution and a fluorinated liquid (electronic fluorinated liquid, MX-056, Suzhou Pengrui Nanotechnology Co., Ltd.), and repeated 2-3 times.

[0051] Furthermore, in step 5, PDMS is configured with a curing agent to substrate ratio of 1:10, an oven heating temperature of 80°C, and a curing time of 30 minutes.

[0052] Furthermore, in step 6, the low-temperature crosslinking temperature is 4°C, the duration is 5 minutes, and the photocrosslinking uses 405nm blue light at a frequency of 30mW / cm². 2 Irradiate with the intensity for 2 minutes.

[0053] A biomimetic composite guiding bone regeneration membrane is prepared by any of the preparation methods described above.

[0054] A biomimetic composite guided bone regeneration membrane includes a guiding layer, a barrier layer, and an osteogenic layer; the osteogenic layer is a biocomposite hydrogel layer, one side of which has a surface structure with a micro-pit array, and the barrier layer and the guiding layer are fixed sequentially from the inside to the outside on the other side; the guiding layer is a directionally arranged parallel polycaprolactone fiber structure, and the barrier layer is a randomly arranged coiled polycaprolactone fiber structure.

[0055] The membrane obtained in this invention can be divided into a guiding layer, a barrier layer, and an osteogenic layer according to its structure and function. The guiding layer consists of directionally arranged parallel polycaprolactone (PCL) fibers, mimicking the natural collagen fiber structure of gingival soft tissue, guiding the directional growth of gingival fibroblasts. The barrier layer consists of randomly arranged coiled PCL fibers, which can promote soft tissue cell adhesion and effectively block the invasion of soft tissue cells, providing sufficient space and time for bone tissue growth. The osteogenic layer is a biocomposite hydrogel with a surface micro-pit array, mimicking the bone resorption pit structure formed on the surface after bone tissue defects, to provide topological induction for osteogenic differentiation of bone marrow mesenchymal stem cells.

[0056] Preferably, the polycaprolactone can be medical-grade polycaprolactone with an average molecular weight of 70,000-90,000; more specifically, the polycaprolactone has an average molecular weight of 80,000.

[0057] Preferably, the average diameter of the fibers in the guiding layer is 8-15 μm, and the spacing is 80-150 μm. Furthermore, the parallel polycaprolactone (PCL) fibers in the guiding layer are prepared by a high-precision melt near-field direct writing process, and the fibers are oriented, with an average diameter of 10 μm and a spacing of 100 μm.

[0058] Preferably, the average fiber diameter in the barrier layer is 2-5 μm, and the average pore diameter is 10-20 μm. More specifically, the PCL crimped fibers in the barrier layer are also prepared using a high-precision melt near-field direct writing process, but the fibers are randomly arranged, with an average diameter of 3 μm and an average pore diameter of 15 μm.

[0059] Preferably, the guiding layer and the barrier layer are prepared by layer-by-layer printing using a melt near-field direct writing process, and their surfaces are treated with hydrophilicity.

[0060] Preferably, in the surface structure with the micro-pit array: the micro-pits are uniformly distributed, and the diameter of the micro-pits is 80~120μm and the depth is 10~30μm; the pit ratio is 40~60%.

[0061] Preferably, the barrier layer and the guiding layer are embedded and fixed on the other side of the osteogenic layer, and the fixation is achieved by in-situ curing of the biocomposite hydrogel layer.

[0062] Preferably, the thickness of the guiding layer and the barrier layer is 100~200μm. More specifically, the thickness of the guiding layer and the barrier layer is 130~180μm. Even further, the thickness of the guiding layer and the barrier layer is 155~165μm; more specifically, the thickness of the guiding layer and the barrier layer is 158μm.

[0063] Preferably, the osteogenic layer thickness is 200-300 μm; further, the osteogenic layer thickness is 220-280 μm; even further, the osteogenic layer thickness is 200-300 μm; more specifically, the osteogenic layer thickness is 233 μm.

[0064] Preferably, the total thickness of the biomimetic composite guiding bone regeneration is approximately 350-450 μm. Further, the total thickness of the biomimetic composite guiding bone regeneration is approximately 380-410 μm, and more specifically, the total thickness of the biomimetic composite guiding bone regeneration is approximately 391 μm.

[0065] A biomimetic composite guiding bone regeneration membrane prepared by the above method, and its application in dental implant guiding bone regeneration barrier membrane and oral repair membrane.

[0066] The significant advantages of this invention are as follows:

[0067] (1) The bone regeneration membrane prepared by the present invention has a biomimetic three-layer composite structure. Its guiding layer simulates the natural collagen fiber structure of gingival soft tissue and guides the directional growth of gingival fibroblasts; the barrier layer can prevent the invasion of soft tissue cells; the osteogenic layer simulates the topological structure of bone resorption pits formed on the surface after bone tissue defects, and combined with the osteogenic active ions released by the bioactive hydrogel, it induces osteogenic differentiation of bone marrow mesenchymal stem cells.

[0068] (2) The polycaprolactone, methacrylamide gelatin, methacrylamide hyaluronic acid and magnesium-substituted calcium silicate used in this invention are all biodegradable biomaterials with good biocompatibility, and avoid the pain and damage to patients caused by secondary surgical removal.

[0069] In summary, the biomimetic composite guided bone regeneration membrane prepared in this invention possesses novel structure and excellent biological effects. Its unique hierarchical structure and functional design make it promising for broad applications in areas such as dental implant-guided bone regeneration barrier membranes and oral repair membranes, and it is expected to play an important role in bone defect repair. Attached Figure Description

[0070] Figure 1 This is a schematic diagram of the biomimetic composite guiding bone regeneration membrane structure obtained by the present invention;

[0071] Figure 2 This is a schematic diagram illustrating the principle of fabricating micro / nano fibers using the melt near-field direct writing process of this invention;

[0072] Figure 3 This is a flowchart illustrating the preparation process of the GelMA-HAMA@CSi-Mg biocomposite hydrogel in Example 1 of this invention.

[0073] Figure 4 The following are microscopic images of the surface morphology of the biomimetic composite guided bone regeneration membrane prepared in Example 1 of the present invention: (a) is a scanning electron microscope image of a double-layer PCL membrane composed of a guiding layer and a barrier layer, and (b) is an optical microscope image of the osteogenic layer side.

[0074] Figure 5 This is a cross-sectional scanning electron microscope image of the biomimetic composite guided bone regeneration membrane prepared in Example 1 of the present invention.

[0075] Figure 6 The results of biocompatibility testing of the biomimetic composite guided bone regeneration membranes prepared in Example 1 and Comparative Example 1 of this invention are shown.

[0076] Figure 7 The results of the detection of the ability of the guided bone regeneration composite membrane with directionally arranged PCL layers on the surface and the membrane with randomly arranged PCL layers on the surface prepared in Example 1 and Comparative Example 2 of the present invention to promote fibroblast adhesion, migration and differentiation.

[0077] Figure 8 The quantitative results of the osteogenic-associated alkaline phosphatase (ALP) activity promoted by the guided bone regeneration composite membrane with a pitted surface and the guided bone regeneration composite membrane with a smooth surface prepared in Example 1 and Comparative Example 3 of the present invention are as follows:

[0078] Figure 9 The results of the detection of the ability of the guide bone regeneration composite membrane with a pitted surface and the guide bone regeneration composite membrane with a smooth surface to promote the expression of osteogenic genes in cells are shown in Example 1 and Comparative Example 3 of the present invention.

[0079] Figure 10 The results show the detection of the ability of the biomimetic composite guided bone regeneration membrane prepared in Example 1 and Comparative Example 1 of this invention to promote the expression of osteogenic genes. Detailed Implementation

[0080] To make the content of this invention clearer, the technical solutions and effects of this invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining this invention and do not constitute a limitation thereof. All other implementation methods derived by those skilled in the art based on the embodiments of this invention without creative work are within the protection scope of this invention.

[0081] Example 1: Bionic composite guided bone regeneration membrane: GelMA-HAMA@CSi-Mg (also refer to...) Figure 1 , Figure 2 and Figure 3 )

[0082] Step 1, Preparation of oriented parallel PCL fibers: A high-precision melt near-field direct writing process is employed, the principle of which is as follows: Figure 2 As shown, using molten PCL (average molecular weight 80,000) as raw material, a nozzle with an inner diameter of 0.15 mm is used. The temperature is maintained at 100°C at a distance of 2 mm from the collection substrate. The printing speed is set to 600 mm / min, the voltage is adjusted to 3.43 kV, and the air pressure is 35 kPa. This produces a layer of parallel PCL fibers with an average diameter of 10 μm and a spacing of 100 μm, which is the guide layer.

[0083] Step 2, Preparation of randomly arranged crimped PCL fibers: On the parallel PCL fiber layer obtained in Step 1, randomly arranged crimped PCL fibers are prepared: Other parameters are the same as those for printing parallel fibers. The voltage is set to 6.50 kV, the air pressure to 20 kPa, and the printing speed to 1200 mm / min. Randomly arranged crimped PCL fibers with an average diameter of 3 μm and an average pore diameter of 15 μm can be obtained, resulting in a bilayer PCL film with a guiding layer and a barrier layer.

[0084] Step 3, hydrophilization treatment: Take 12g of sodium hydroxide particles, dissolve them in 100mL of deionized water, stir evenly and fully dissolve to obtain a sodium hydroxide solution. Place the PCL membrane with the guide layer and barrier layer prepared above into the sodium hydroxide solution for immersion and etching for 3 hours. After completion, the PCL membrane is washed with deionized water 3 times to obtain a hydrophilized double-layer PCL membrane.

[0085] Step 4, prepare the biocomposite hydrogel:

[0086] (1) Weigh 0.03g of LAP photoinitiator and add it to 10mL of PBS buffer solution (pH: 7.2-7.4). Heat in a metal bath at 55℃ for 30 minutes to dissolve it completely and obtain LAP preparation solution.

[0087] (2) Add 10g GelMA and 1g HAMA to the above LAP preparation solution, and continue to heat in a metal bath for 3 hours to fully dissolve the two. Then filter with a 0.22μm bacterial filter membrane to obtain the GelMA-HAMA hydrogel matrix.

[0088] (3) Weigh 2g of CSi-Mg particles, sterilize them under ultraviolet light for 30 minutes, add them to the above GelMA-HAMA hydrogel matrix, and disperse them using an ultrasonic disperser for 1 minute to avoid particle aggregation, thus obtaining uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel. Figure 3 This is for the preparation of materials and subsequent cross-linking processes.

[0089] Step 5, Prepare the mold:

[0090] (1) A resin positive mold with a micro-dimpled array surface structure, with a diameter of 100 μm, a depth of 20 μm, and an area ratio of pit area to flat area of ​​1, was prepared using a high-resolution projection-type light curing printer. The positive mold was placed in an ultrasonic cleaner and cleaned with 95% alcohol solution for 5 seconds. It was then removed and ultrasonically cleaned with fluorinated liquid (electronic fluorinated liquid, MX-056, Suzhou Pengrui Nanotechnology Co., Ltd.) for 5 seconds. This process was repeated 3 times to remove residual resin from the surface.

[0091] (2) Place the resin positive mold in an oven and heat it at 80°C for 3 hours. Then, irradiate it with a blue light curing lamp with a wavelength of 405nm and a power of 72W for 10 seconds to fully cure it.

[0092] (3) Weigh 1g of PDMS curing agent and add it to 10g of PDMS body. Stir evenly to obtain uncured PDMS.

[0093] (4) Pour the uncured polydimethylsiloxane into the resin positive mold, put it in a vacuum box to evacuate the vacuum, and then place it in an oven to heat and cure it at 80°C for 30 minutes to obtain a PDMS negative mold with a surface micropillar structure with a diameter of 100μm.

[0094] Step 6, Prepare the composite membrane:

[0095] (1) Use a pipette to take a small amount of the uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel obtained in step 4, drop it onto the surface of the PDMS negative mold obtained in step 5, and slowly spread it evenly with the pipette tip to form a 0.5 mm thick film. Place it on a heating table at a temperature of 50°C to heat it, so as to avoid the liquid cooling down too quickly and forming a gel, and obtain the ungelled biocomposite hydrogel.

[0096] (2) The hydrophilicated bilayer PCL membrane obtained in step 3 was placed on the ungelled biocomposite hydrogel with the guide layer facing outwards, and then placed in a low-temperature environment of 4°C for 5 minutes for crosslinking (low-temperature crosslinking). Then, a wavelength of 405 nm and an intensity of 30 mW / cm were used for crosslinking. 2 Irradiation with a blue light curing lamp for 2 minutes (photocrosslinking) yields a biomimetic composite guiding bone regeneration membrane after separation from the mold. Figure 1 Here is a schematic diagram of its layered structure. Figure 4 Image (a) shows scanning electron microscope images of the guiding layer and barrier layer sides of the obtained guiding bone regeneration membrane. Figure 4 (b) shows a microscopic image of the surface structure of the biocomposite hydrogel osteogenic layer of the guide bone regeneration membrane after separation from the mold. Figure 5 The image shows a cross-sectional scanning electron microscope image of the biomimetic composite guided bone regeneration membrane prepared in this embodiment. As can be seen from the image, the thickness of the PCL guided barrier layer, which consists of a guiding layer and a barrier layer, is approximately 158 μm, and the thickness of the hydrogel osteogenic layer is approximately 233 μm.

[0097] Step 7, Irradiation sterilization: The guiding bone regeneration membrane obtained in Step 6 is subjected to ultraviolet light irradiation sterilization treatment to obtain the final biomimetic composite guiding bone regeneration membrane: GelMA-HAMA@CSi-Mg.

[0098] Figure 3 The preparation methods of GelMA and HAMA are also provided, as well as the preparation method of uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel. In Example 1, Comparative Examples 1, 2 and 3, commercially available GelMA (EFL-GM-30, Suzhou Yongqinquan Intelligent Equipment Co., Ltd.) and HAMA (EFL-HAMA-150K, Suzhou Yongqinquan Intelligent Equipment Co., Ltd.) were used directly.

[0099] Comparative Example 1: GelMA-HAMA pure hydrogel membrane

[0100] Steps 1 to 3 are the same as in Example 1: a hydrophilic bilayer PCL membrane is prepared.

[0101] Step 4, prepare pure hydrogel:

[0102] (1) Weigh 0.03g of LAP photoinitiator and add it to 10mL of PBS buffer solution. Heat in a metal bath at 55℃ for 30 minutes to dissolve it completely and obtain LAP preparation solution.

[0103] (2) Add 10g GelMA and 1g HAMA to the above LAP preparation solution, and continue to heat in a metal bath for 3 hours to fully dissolve the two. Then filter with a 0.22μm bacterial filter membrane to obtain uncrosslinked GelMA-HAMA pure hydrogel.

[0104] Step 5, Prepare the mold:

[0105] (1) Prepare a resin positive mold with a smooth surface using a high-resolution projection-type light curing printer. Place it in an ultrasonic cleaner and clean it with 95% alcohol solution for 5 seconds. Take it out and clean it with fluorinated liquid for 5 seconds. Repeat 3 times to remove the residual resin on the surface.

[0106] (2) Place the resin positive mold in an oven and heat it at 80°C for 3 hours. Then, irradiate it with a blue light curing lamp with a wavelength of 405nm and a power of 72W for 10 seconds to fully cure it.

[0107] (3) Weigh 1g of PDMS curing agent and add it to 10g of PDMS body. Stir evenly to obtain uncured PDMS.

[0108] (4) Pour the uncured polydimethylsiloxane into the resin positive mold, put it in a vacuum box to evacuate the vacuum, and then place it in an oven to heat and cure at 80°C for 30 minutes to obtain a smooth PDMS negative mold.

[0109] Step 6, Prepare the composite membrane:

[0110] (1) Use a pipette to take a small amount of the uncrosslinked GelMA-HAMA pure hydrogel obtained in step 4, drop it onto the surface of the PDMS negative mold obtained in step 5, and slowly spread it evenly with the pipette tip to form a 0.5 mm thick film. Place it on a heating table at a temperature of 50°C to heat it, and avoid the liquid cooling down too quickly to form a gel.

[0111] (2) The hydrophilicated bilayer PCL membrane obtained in step 3 was placed on the ungelled pure hydrogel and crosslinked in a low temperature environment of 4°C for 5 minutes. Then, a wavelength of 405nm and an intensity of 30mW / cm were used for crosslinking. 2 After being irradiated with a blue light curing lamp for 2 minutes, the biomimetic composite guiding bone regeneration membrane was obtained after being separated from the mold.

[0112] Step 7, Irradiation sterilization: The guiding bone regeneration membrane obtained in step 6 is sterilized by ultraviolet light irradiation.

[0113] Comparative Example 2: GelMA-HAMA@CSi-Mg film with randomly arranged PCL layers on the surface

[0114] Step 1, Preparation of randomly arranged crimped PCL fibers: Other parameters are the same as those for printing parallel fibers in Example 1, except that the voltage is set to 6.50 kV, the air pressure to 20 kPa, and the printing speed to 1200 mm / min. This yields randomly arranged crimped PCL fibers with an average diameter of 3 μm and an average pore diameter of 15 μm, resulting in a film with a randomly arranged PCL layer on the surface.

[0115] Step 2, hydrophilization treatment: Take 12g of sodium hydroxide particles, dissolve them in 100mL of deionized water, stir evenly and fully dissolve to obtain a sodium hydroxide solution. Place the membrane with randomly arranged PCL layers on the surface prepared above into the sodium hydroxide solution for immersion and etching for 3 hours. After completion, the PCL membrane is washed with deionized water 3 times to obtain a hydrophilized membrane with randomly arranged PCL layers on the surface.

[0116] Step 3, prepare the biocomposite hydrogel:

[0117] (1) Weigh 0.03g of LAP photoinitiator and add it to 10mL of PBS buffer solution (pH: 7.2-7.4). Heat in a metal bath at 55℃ for 30 minutes to dissolve it completely and obtain LAP preparation solution.

[0118] (2) Add 10g GelMA and 1g HAMA to the above LAP preparation solution, and continue to heat in a metal bath for 3 hours to fully dissolve the two. Then filter with a 0.22μm bacterial filter membrane to obtain the GelMA-HAMA hydrogel matrix.

[0119] (3) Weigh 2g of CSi-Mg particles, sterilize them under ultraviolet light for 30 minutes, add them to the above GelMA-HAMA hydrogel matrix, and disperse them using an ultrasonic disperser for 1 minute to avoid particle aggregation, thus obtaining uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel. Figure 3 This is for the preparation of materials and subsequent cross-linking processes.

[0120] Step 4, Prepare the mold:

[0121] (1) A resin positive mold with a micro-dimpled array surface structure, with a diameter of 100 μm, a depth of 20 μm, and an area ratio of pit area to flat area of ​​1, was prepared using a high-resolution projection-type light curing printer. The positive mold was placed in an ultrasonic cleaner and cleaned with 95% alcohol solution for 5 seconds. It was then removed and ultrasonically cleaned with fluorinated liquid (electronic fluorinated liquid, MX-056, Suzhou Pengrui Nanotechnology Co., Ltd.) for 5 seconds. This process was repeated 3 times to remove residual resin from the surface.

[0122] (2) Place the resin positive mold in an oven and heat it at 80°C for 3 hours. Then, irradiate it with a blue light curing lamp with a wavelength of 405nm and a power of 72W for 10 seconds to fully cure it.

[0123] (3) Weigh 1g of PDMS curing agent and add it to 10g of PDMS body. Stir evenly to obtain uncured PDMS.

[0124] (4) Pour the uncured polydimethylsiloxane into the resin positive mold, put it in a vacuum box to evacuate the vacuum, and then place it in an oven to heat and cure it at 80°C for 30 minutes to obtain a PDMS negative mold with a surface micropillar structure with a diameter of 100μm.

[0125] Step 5, Prepare the composite membrane:

[0126] (1) Use a pipette to take a small amount of the uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel obtained in step 3, drop it onto the surface of the PDMS negative mold obtained in step 4, and slowly spread it evenly with the pipette tip to form a 0.5 mm thick film. Place it on a heating table at a temperature of 50°C to heat it, so as to avoid the liquid cooling down too quickly and forming a gel, and obtain the ungelled biocomposite hydrogel.

[0127] (2) The hydrophilicated PCL membrane obtained in step 2 was randomly arranged and covered on the ungelled biocomposite hydrogel with the guide layer facing outward. It was then placed in a low-temperature environment of 4°C for 5 minutes for crosslinking (low-temperature crosslinking). Then, a wavelength of 405 nm and an intensity of 30 mW / cm were used for crosslinking. 2 Irradiate with a blue light curing lamp for 2 minutes (photocrosslinking), and then separate from the mold to obtain a biomimetic composite guiding bone regeneration membrane.

[0128] Step 6, Irradiation sterilization: The guiding bone regeneration membrane obtained in step 5 is subjected to ultraviolet light irradiation sterilization treatment to obtain a GelMA-HAMA@CSi-Mg membrane with randomly arranged PCL layers on the surface.

[0129] Comparative Example 3: Smooth GelMA-HAMA@CSi-Mg film

[0130] Steps 1 through 4 are the same as in Example 1.

[0131] Step 5, Prepare the mold:

[0132] (1) Prepare a resin positive mold with a smooth surface using a high-resolution projection-type light curing printer. Place it in an ultrasonic cleaner and clean it with 95% alcohol solution for 5 seconds. Take it out and clean it with fluorinated liquid for 5 seconds. Repeat 3 times to remove the residual resin on the surface.

[0133] (2) Place the resin positive mold in an oven and heat it at 80°C for 3 hours. Then, irradiate it with a blue light curing lamp with a wavelength of 405nm and a power of 72W for 10 seconds to fully cure it.

[0134] (3) Weigh 1g of PDMS curing agent and add it to 10g of PDMS body. Stir evenly to obtain uncured PDMS.

[0135] (4) Pour the uncured polydimethylsiloxane into the resin positive mold, put it in a vacuum box to evacuate the vacuum, and then place it in an oven to heat and cure at 80°C for 30 minutes to obtain a smooth PDMS negative mold.

[0136] Step 6, Prepare the composite membrane:

[0137] (1) Use a pipette to take a small amount of the uncrosslinked GelMA-HAMA@CSi-Mg biocomposite hydrogel obtained in step 4, drop it onto the surface of the PDMS negative mold obtained in step 5, and slowly spread it evenly with the pipette tip to form a 0.5 mm thick film. Place it on a heating table at a temperature of 50°C to heat it, and avoid the liquid cooling down too quickly to form a gel.

[0138] (2) The hydrophilicated bilayer PCL membrane obtained in step 3 was coated on the ungelled composite hydrogel, and then placed in a low temperature environment of 4°C for 5 minutes for crosslinking. Then, a wavelength of 405nm and an intensity of 30mW / cm were used for crosslinking. 2 After being irradiated with a blue light curing lamp for 2 minutes, the biomimetic composite guiding bone regeneration membrane was obtained after being separated from the mold.

[0139] Step 7, Irradiation sterilization: The smooth-surfaced GelMA-HAMA@CSi-Mg film obtained in Step 6 is subjected to ultraviolet light irradiation sterilization treatment.

[0140] I. Cell viability assay:

[0141] The biomimetic composite guided bone regeneration membranes prepared in Example 1 (GelMA-HAMA@CSi-Mg) and the pure hydrogel membrane prepared in Comparative Example 1 were cut into 15 mm diameter discs and placed in 24-well plates for seeding and culturing bone marrow mesenchymal stem cells (BMSCs). Cell viability was measured using the Alamar Blue assay on days 3 and 5. Alamar Blue CellViability Reagent dye can be reduced by mitochondrial enzymes in living cells, resulting in a fluorescence change. The fluorescence intensity is directly proportional to the number of living cells. The fluorescence density (OD) detected by a microplate reader was used to estimate the number of living cells. The excitation wavelength was 535 nm, and the emission wavelength was 595 nm. The specific method is as follows: The guided bone regeneration composite membranes prepared in Example 1 and Comparative Example 1 were placed in 24-well plates. The BMSC seeding density was 1 × 10⁻⁶. ^4 Cells / well, add 1 mL of culture medium to each well. After culturing for 3 and 7 days, replace 1 mL of fresh culture medium in each well, then add 100 μL of Alamar Blue reagent and incubate at 37°C in the dark for 2 hours. After the reaction, pipette 100 μL of culture medium containing Alamar Blue into each well of a 96-well plate and read the fluorescence intensity using a microplate reader.

[0142] The Alamar Blue assay can rapidly test the effect of composite membranes on the viability of BMSCs. The cell viability of BMSCs cultured on the composite membrane is as follows: Figure 6 As shown. From Figure 6 It can be seen that, with the extension of culture time, the cell activity in Example 1 is significantly higher than that in Comparative Example 1, indicating that the GelMA-HAMA@CSi-Mg system with micro-dimpled array surface structure has better biocompatibility than the pure GelMA-HAMA hydrogel with flat surface, and its toxicity to cells is negligible.

[0143] II. Detection of the ability of oriented PCL layers to promote cell adhesion, migration, and differentiation:

[0144] The guided bone regeneration composite membrane with a directionally arranged PCL layer on its surface prepared in Example 1 and the membrane with a randomly arranged PCL layer on its surface prepared in Comparative Example 2 were cut into 15 mm diameter discs and placed in 24-well plates for inoculation and culture of L929 mouse fibroblast cells. On day 7, the expression of adhesion, migration, and differentiation genes (COL1A-1, Itgb-1, CXCR4, FAK, Fibronectin, Vinculin) was measured using reverse transcription-polymerase chain reaction (RT-PCR). The specific method is as follows: cells were inoculated and cultured on the guided bone regeneration membrane with a directionally arranged PCL layer on its surface prepared in Example 1 and the membrane with a randomly arranged PCL layer on its surface prepared in Comparative Example 2, respectively. The basal medium was replaced with fresh medium every 2 days, and samples were collected on day 7 after inoculation. The sample collection process was as follows: After terminating the culture, the cells were washed twice with PBS. Total RNA was extracted using the RNAeasy kit, and the RNA content (μg / mL) and purity of each sample were determined using a Nanodrop 2000 instrument at the A260 / A280 ratio. RNA of good quality had an A260 / A280 ratio between 1.8 and 2.0. Reverse transcription was performed using the PrimeScript RT MasterMix (Takara) kit. The reverse transcription system was 10 μL, with 500 ng of total RNA as the starting material. Reverse transcription conditions: 37℃, 15 min → 85℃, 5 sec → 4℃.

[0145] PCR amplification process: A 10 μL reaction system was used, and RT-PCR detection was performed using an Applied Biosystems Via7 (ThemoFisher Scientific) real-time PCR instrument and a SYBR Green I kit (Takara). Reaction conditions: 95℃ for 30 sec, 40 cycles (95℃, 5 sec → 60℃, 34 sec). Each sample was set up with 3 technical replicate wells. The Ct value was read after the reaction. Ct represents the number of cycles required for the amplification product to reach the set threshold. The fold increase of the target gene = 2^-ΔCt, which can be used to relatively quantify the expression level of the target gene in the experimental sample.

[0146] RT-PCR can measure the expression levels of adhesion, migration, and differentiation genes (COL1A-1, Itgb-1, CXCR4, FAK, Fibronectin, Vinculin, etc.) in the L929 mouse fibroblast cell line. The expression levels of adhesion, migration, and differentiation genes in fibroblasts cultured on a composite membrane are shown below. Figure 7As shown, the cell adhesion, migration, and differentiation genes on the guided bone regeneration composite membrane with a directionally arranged PCL layer on the surface prepared in Example 1 (the figure shows directional PCL) are significantly higher than those on the membrane with a randomly arranged PCL layer on the surface in Comparative Example 2 (the figure shows random PCL). This indicates that the directionally arranged PCL layer simulates the ordered structure of the extracellular matrix in vivo, which can provide a more favorable microenvironment for fibroblasts, thereby promoting fibroblast adhesion, migration, and differentiation.

[0147] III. Quantitative analysis of the activity of osteogenic-associated alkaline phosphatase (ALP) promoted by the pit structure:

[0148] The pitted surface guide bone regeneration composite membrane prepared in Example 1 and the smooth surface guide bone regeneration composite membrane prepared in Comparative Example 3 were cut into 15 mm diameter circular slices and placed in 24-well plates for seeding and culture of rat bone marrow mesenchymal stem cells (rBMSCs). The specific method is as follows: the cells were cultured on the pitted surface guide bone regeneration composite membrane and the smooth surface guide bone regeneration composite membrane prepared in Example 1 and Comparative Example 3, respectively, for 1 day. Then, the osteogenic induction medium (composed of basal medium α-MEM, 10% fetal bovine serum, 1% penicillin-streptomycin solution, and osteogenic induction factor 50 μg / mL ascorbic acid, 10 mM β-glycerol phosphate and 100 nM dexamethasone) was replaced with fresh osteogenic induction medium every 2 days during the subsequent culture process. After 3 and 7 days of culture, the culture was terminated, the cells were washed twice with PBS, and 150 μL of cell lysis buffer (CellLytic™, Sigma) was added. The cells were placed in an ice box on a slow shaker for 15 minutes, mixing several times with a pipette tip during the process. The lysis buffer was collected into a 1.5 mL Eppendorf tube. The cells were centrifuged at 12,000 rpm at 4°C for 15 minutes to precipitate cell debris. The cell supernatant was then transferred to a new 0.2 mL Eppendorf tube for later use.

[0149] ALP activity was detected using the LabAssay ALP (WAKO, Japan) alkaline phosphatase assay kit.

[0150] The specific steps are as follows:

[0151] 1) Prepare working solution (1 matrix tablet dissolved in 5 mL matrix dissolving solution) and graded dilution standard solutions;

[0152] 2) Add 100 μL of processing solution to each well of the 96-well plate, then add 20 μL each of sample, standard solution and deionized water (blank well), mix well and incubate at 37°C for 15 minutes.

[0153] 3) Add 80 μL of reaction stop solution to each well, mix well, and place in a microplate reader to read the OD value at 405 nm. Zero the reading using a blank well.

[0154] A standard curve was plotted based on the OD values ​​of the serially diluted standard solutions, and a calculation formula (R²>0.99) was derived. Then, the corresponding ALP activity was calculated based on the calculation formula and the OD values ​​of the samples.

[0155] Alkaline phosphatase (ALP) activity assays can assess the osteogenic differentiation capacity of rat bone marrow mesenchymal stem cells (rBMSCs). ALP is an early marker of osteoblast function, and increased activity is generally associated with osteoblast maturation and bone matrix mineralization. Figure 8 As shown, the ALP activity of cells on the guided bone regeneration composite membrane prepared in Example 1 (micro-pit array membrane in the figure) was significantly higher than that on Comparative Example 3 (flat surface membrane in the figure). This indicates that the GelMA-HAMA@CSi-Mg biomimetic composite hydrogel with a micro-pit array surface structure can more effectively promote osteogenic differentiation of cells compared to hydrogels with flat surfaces.

[0156] IV. Detection of the ability of pitted structures to promote osteogenic gene expression:

[0157] The guided bone regeneration composite membrane with a pitted surface prepared in Example 1 and the guided bone regeneration composite membrane with a smooth surface prepared in Comparative Example 3 were cut into 15 mm diameter circular slices and placed in 24-well plates for seeding and culture of rat bone marrow mesenchymal stem cells (rBMSCs). On day 7, the expression of osteogenic genes (ALP, BMP-2, BSP, COL1-A, Runx2) in the cells was measured by reverse transcription-polymerase chain reaction (RT-PCR). The specific method is as follows: Cells were cultured for 1 day on the pitted and smooth guided bone regeneration composite membranes prepared in Example 1 and Comparative Example 3, respectively. The culture medium was then replaced with osteogenic induction medium (composed of basal medium α-MEM, 10% fetal bovine serum, 1% penicillin-streptomycin solution, and osteogenic induction factors 50 μg / mL ascorbic acid, 10 mM β-glycerol phosphate, and 100 nM dexamethasone). Fresh osteogenic induction medium was used every 2 days during the subsequent culture period, and samples were harvested on day 7 after osteogenic induction. The sample harvesting process was as follows: After termination of culture, the cells were washed twice with PBS, and total RNA was extracted using the RNAeasy kit. The RNA content (μg / mL) and purity of each sample were determined using a Nanodrop 2000 instrument at the A260 / A280 ratio. RNA of good quality had an A260 / A280 ratio between 1.8 and 2.0. Reverse transcription was performed using the PrimeScript RTMasterMix (Takara) kit. The reverse transcription system consisted of 10 μL of 500 ng total RNA as the starting material. Reverse transcription conditions: 37℃, 15 min → 85℃, 5 sec → 4℃.

[0158] PCR amplification process: A 10 μL reaction system was used, and RT-PCR detection was performed using an Applied Biosystems Via7 (ThemoFisher Scientific) real-time PCR instrument and a SYBR Green I kit (Takara). Reaction conditions: 95℃ for 30 sec, 40 cycles (95℃, 5 sec → 60℃, 34 sec). Each sample was set up with 3 technical replicate wells. The Ct value was read after the reaction. Ct represents the number of cycles required for the amplification product to reach the set threshold. The fold increase of the target gene = 2^-ΔCt, which can be used to relatively quantify the expression level of the target gene in the experimental sample.

[0159] RT-PCR can measure the expression levels of osteogenic genes (ALP, BMP-2, BSP, COL1-A, Runx2, etc.) in rat bone marrow mesenchymal stem cells (rBMSCs). The expression of osteogenic genes in BMSCs cultured on a composite membrane is shown below. Figure 9 As shown, the osteogenic genes on the pitted surface of the guided bone regeneration composite membrane prepared in Example 1 (the micro-pit array membrane in the figure) are significantly higher than those on the smooth surface of the guided bone regeneration composite membrane prepared in Comparative Example 3 (the smooth surface membrane in the figure). This indicates that the GelMA-HAMA@CSi-Mg biomimetic composite hydrogel with a micro-pit array surface structure has better osteogenic induction ability compared to hydrogels with a flat surface.

[0160] V. Detection of the ability of biomimetic composite structures to promote osteogenic gene expression:

[0161] The GelMA-HAMA@CSi-Mg guided bone regeneration composite membrane prepared in Example 1 and the pure hydrogel membrane prepared in Comparative Example 1 were cut into 15 mm diameter round slices and placed in 24-well plates for rat bone marrow mesenchymal stem cell (rBMSCs) seeding and culture. The expression of osteogenic genes (BMP-2, ALP, BSP, OCN, COL1-A) in the cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) on days 7 and 14, respectively. The specific method is as follows: Cells were cultured for 1 day on the GelMA-HAMA@CSi-Mg guided bone regeneration composite membrane prepared in Example 1 and the pure hydrogel composite membrane prepared in Comparative Example 1, respectively. The culture medium was then replaced with osteogenic induction medium (composed of basal medium α-MEM, 10% fetal bovine serum, 1% penicillin-streptomycin solution, and osteogenic induction factors 50 μg / mL ascorbic acid, 10 mM β-glycerol phosphate, and 100 nM dexamethasone). Fresh osteogenic induction medium was used every 2 days during the subsequent culture period, and samples were harvested on days 7 and 14 after osteogenic induction. The sample harvesting process was as follows: After termination of culture, the cells were washed twice with PBS, and total RNA was extracted using the RNAeasy kit. The RNA content (μg / mL) and purity of each sample were determined using a Nanodrop 2000 instrument at the A260 / A280 ratio. RNA of good quality had an A260 / A280 ratio between 1.8 and 2.0. Reverse transcription was performed using the PrimeScript RT MasterMix (Takara) kit. The reverse transcription system consisted of 10 μL of 500 ng total RNA as the starting material. Reverse transcription conditions: 37℃, 15 min → 85℃, 5 sec → 4℃.

[0162] PCR amplification process: A 10 μL reaction system was used, and RT-PCR detection was performed using an Applied Biosystems Via7 (ThemoFisher Scientific) real-time PCR instrument and a SYBR Green I kit (Takara). Reaction conditions: 95℃ for 30 sec, 40 cycles (95℃, 5 sec → 60℃, 34 sec). Each sample was set up with 3 technical replicate wells. The Ct value was read after the reaction. Ct represents the number of cycles required for the amplification product to reach the set threshold. The fold increase of the target gene = 2^-ΔCt, which can be used to relatively quantify the expression level of the target gene in the experimental sample.

[0163] Depend on Figure 10 As shown: The osteogenic genes on the biomimetic composite guided bone regeneration composite membrane prepared in Example 1 (GelMA-HAMA@CSi-Mg in the figure) are significantly higher than those in Comparative Example 1 (pure hydrogel in the figure). This indicates that the GelMA-HAMA@CSi-Mg biomimetic composite hydrogel with micro-pit array surface structure, compared with the pure hydrogel system, can simulate the structure and function of natural bone tissue by combining the physical properties of the micro-pit array and the chemical signals of bioactive components, thereby providing a more suitable microenvironment for bone marrow mesenchymal stem cells and promoting the expression of osteogenic-related genes and osteogenic differentiation of cells.

[0164] Furthermore, while preferred embodiments of the present invention have been shown and described above, those skilled in the art should understand that the present invention is not limited to the above embodiments, and various changes and modifications can be made thereto without departing from the spirit and scope of the invention, all of which fall within the scope of the claimed invention. The present invention is not limited to the specific embodiments disclosed herein, but includes the full scope of the appended claims and their equivalents.

Claims

1. A biomimetic composite guiding bone regeneration membrane, characterized in that, It includes a guiding layer, a barrier layer, and an osteogenic layer; the osteogenic layer is a biocomposite hydrogel layer, one side of which has a surface structure with a micro-pit array, and the barrier layer and the guiding layer are fixed sequentially from the inside to the outside on the other side; the guiding layer is a directionally arranged parallel polycaprolactone fiber structure, and the barrier layer is a randomly arranged curly polycaprolactone fiber structure.

2. The biomimetic composite guiding bone regeneration membrane according to claim 1, characterized in that, The average fiber diameter in the guiding layer is 8-15 μm, and the spacing is 80-150 μm; the average fiber diameter in the barrier layer is 2-5 μm, and the average pore diameter is 10-20 μm; the total thickness of the guiding layer and the barrier layer is 100-200 μm.

3. The biomimetic composite guiding bone regeneration membrane according to claim 1, characterized in that, The guiding layer and barrier layer are prepared by layer-by-layer printing using a melt near-field direct writing process, and their surfaces are treated with hydrophilicity.

4. The biomimetic composite guiding bone regeneration membrane according to claim 1, characterized in that, The biocomposite hydrogel of the osteogenic layer is composed of lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, methacrylamide gelatin-methacrylamide hyaluronic acid, and magnesium-substituted calcium silicate particles.

5. The biomimetic composite guiding bone regeneration membrane according to claim 4, characterized in that, The biocomposite hydrogel includes a phosphate buffer solution, wherein, based on the volume of the phosphate buffer solution, the added phenyl (2,4,6-trimethylbenzoyl)lithium phosphate is 0.25% to 0.3% by mass; the added methacrylamide gelatin is 5% to 15% by mass; the added methacrylamide hyaluronic acid is 0.2% to 2% by mass; and the added magnesium-substituted calcium silicate particles are 1% to 3% by mass.

6. The biomimetic composite guiding bone regeneration membrane according to claim 1, characterized in that, The surface structure with a micro-pit array has micro-pits that are uniformly arranged: the diameter of the micro-pits is 80~120μm and the depth is 10~30μm; the pit ratio is 40~60%; and the thickness of the osteogenic layer is 200~300μm.

7. The biomimetic composite guiding bone regeneration membrane according to claim 1, characterized in that, The barrier layer and the guiding layer are embedded and fixed on the other side of the osteogenic layer, and are fixed by in-situ curing of the biocomposite hydrogel layer; the total thickness of the guiding bone regeneration membrane is 350~450μm.

8. A method for preparing the biomimetic composite guiding bone regeneration membrane according to any one of claims 1 to 7, characterized in that, Includes the following steps: Step 1: Using a melt near-field direct writing process, oriented parallel polycaprolactone fibers are prepared by utilizing the stable state of the jet to prepare the guiding layer; Step 2: Using a melt near-field direct writing process, randomly arranged crimped polycaprolactone fibers are deposited on the surface of the guide layer by utilizing the whipping instability of the jet to prepare a barrier layer; Step 3: The obtained polycaprolactone membrane with a guiding layer and a barrier layer is hydrophilized using sodium hydroxide solution to obtain a hydrophilized bilayer PCL membrane. Step 4: Place the double-layer PCL membrane in a mold with a micro-pit array structure corresponding to the above, pour the prepared bio-composite hydrogel into the mold, and solidify it to obtain a biomimetic composite guiding bone regeneration membrane.

9. The method for preparing the biomimetic composite guiding bone regeneration membrane according to claim 8, characterized in that, In step 4, the preparation process of the biocomposite hydrogel is as follows: First, a phenyl (2,4,6-trimethylbenzoyl)lithium phosphate preparative solution is prepared using a phosphate buffer solution; then, methacrylamide gelatin and methacrylamide hyaluronic acid are added and dissolved; finally, magnesium-substituted calcium silicate particles are added to prepare the biocomposite hydrogel.

10. The application of the biomimetic composite guiding bone regeneration membrane according to any one of claims 1 to 7 as a guiding bone regeneration barrier membrane for dental implants and an oral repair membrane.

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