Three-layer biomimetic periosteum for promoting periosteal repair and bone tissue regeneration and preparation method thereof

Abstract

This invention discloses a three-layer biomimetic periosteum that promotes periosteal repair and bone tissue regeneration, and its preparation method, belonging to the field of biomedical materials. Polylactic acid is used to prepare a spun fiber membrane to simulate the outer fibrous layer of the natural periosteum, thereby enhancing the adhesion and growth of fibroblasts. A polycaprolactone / mineralized bovine type I collagen (MC) endosteoblastic layer prepared by freeze-drying simulates the bioactive layer of the natural periosteum, promoting the proliferation and osteogenic differentiation of mouse embryonic osteoblast precursor cells. A polycaprolactone barrier layer, prepared by solvent evaporation, serves as an intermediate layer connecting the two layers, acting as a barrier to prevent fibroblast invasion while providing a stable osteogenic microenvironment for the endosteoblastic layer. The three-layer biomimetic periosteum prepared by this invention possesses advantages such as good pore structure, mechanical strength, interlayer adhesion strength, and the ability to promote the proliferation and differentiation of osteoblasts and fibroblasts.

Description

Technical Field

[0001] This invention relates to a three-layer biomimetic periosteum that promotes periosteum repair and bone tissue regeneration, and its preparation method, belonging to the field of biomedical materials. Background Technology

[0002] Currently, severe nonunion of fractures accompanied by periosteal damage or even loss remains a significant challenge. Fractures, as a common accidental injury, can have adverse effects on a person's physical and mental health if not treated aggressively. When a fracture occurs, the injured site is affected by many factors. These factors can interfere with the fracture healing process, ultimately leading to poor healing, delayed healing, or nonunion. Currently, the main materials used for fracture repair include bone, bone cement, metals, and ceramics, but these still have drawbacks such as insufficient donor sites, poor biocompatibility, high stiffness, and susceptibility to localized stress. Increasing evidence suggests that the periosteal membrane, as an important skeletal appendage, plays a crucial role in promoting fracture healing. However, severe fractures are often accompanied by periosteal tears or even defects. Therefore, periosteal repair is considered an important part of treating severe fractures and preventing nonunion.

[0003] Currently, periosteal repair primarily utilizes various natural and synthetic materials, including cell sheets, decellularized mammalian periosteal membranes, hydrogels, and electrospun membranes. However, these materials are limited by preparation requirements and immunogenicity, hindering their practical application. Furthermore, some bioactive factors have been attempted to be directly added to these materials. However, these attempts often result in adverse consequences, such as inflammation and immune responses, excessive or ectopic osteogenesis. Meanwhile, biomimetic periosteums made using the aforementioned materials often focus on single-layer or double-layer structural designs, thus frequently resulting in limited functionality. They unilaterally pursue bone regeneration while neglecting the importance of the natural periosteum in its barrier function and contact with soft tissues (Z. Yang, Z. Yang, L. Ding, P. Zhang, C. Liu, D. Chen, F. Zhao, G. Wang, X. Chen, Self-Adhesive Hydrogel Biomimetic Periosteum to Promote Critical-Size Bone Defect Repair via Synergistic Osteogenesis and Angiogenesis, Acs Applied Materials & Interfaces, 14 (2022) 36395-36410. ;A. Sethi, J. Craig, S. Bartol, W. Chen, M. Jacobsen, C. Coe, R. Vaidya, Radiographic and CT Evaluation of Recombinant Human Bone Morphogenetic Protein-2-Assisted Spinal Interbody Fusion, American Journal of Roentgenology, 197 (2011) 1024.).

[0004] Electrospun polymer membranes, due to their dense fiber arrangement, restrict cell adhesion and growth within the membrane. Furthermore, the poor mechanical properties of individual electrospun fiber membranes make them prone to damage. Secondly, traditional polymer membranes lack sufficient bioactivity, failing to meet the needs for promoting bone regeneration. More importantly, since the natural periosteum does not only contact a single cell, a multidimensional biomimetic periosteum is required to perform different functions for different cells. Summary of the Invention

[0005] The present invention aims to provide a three-layer biomimetic periosteum that promotes periosteal repair and bone tissue regeneration, and its preparation method. The prepared three-layer biomimetic periosteum has excellent mechanical strength and the layers are tightly bonded together. The synergistic effect between different layers enables the periosteum to prevent excessive soft tissue invasion while also having good osteogenic properties, thereby realizing periosteal repair and promoting bone tissue regeneration.

[0006] In this invention, considering the practical application requirements of the periosteum and the close contact between the periosteum and soft tissue, ultrasonic processing technology was employed to transform the electrospun membrane from a dense structure to a loose, porous structure. Secondly, to promote bone repair, mineralized bovine collagen and polycaprolactone were fused together to construct an endosteoblastic layer. Simultaneously, addressing the interface problem between the inner and outer layers is crucial; that is, preventing epidermal cells and connective tissue cells from the fibrous layer from invading the osteoblastic layer and disrupting the osteogenic process of the inner layer, solvent evaporation technology was used to obtain a polycaprolactone film with a barrier function. Based on improving the dense structure of the electrospun membrane, this invention, combined with freeze-drying and solvent evaporation technologies, constructs a multifunctional three-layer biomimetic periosteum, providing a solution for achieving periosteal repair and promoting the regeneration of surrounding bone tissue.

[0007] This invention provides a three-layer biomimetic periosteum that promotes periosteal repair and bone tissue regeneration, consisting of an outer fibrous layer (UPLA), an intermediate barrier layer (PC), and an endosteoblastic layer (PM). A polylactic acid-based spun fiber membrane (UPLA) is used to simulate the outer fibrous layer of the natural periosteum to enhance the adhesion and growth of fibroblasts. A polycaprolactone / mineralized bovine type I collagen (MC) endosteoblastic layer (PM), prepared by freeze-drying, simulates the bioactive layer of the natural periosteum to promote the proliferation and osteogenic differentiation of mouse embryonic osteoblast precursor cells. A polycaprolactone barrier layer (PC), prepared by solvent evaporation, serves as the intermediate layer, connecting the two layers and acting as a barrier to prevent fibroblast invasion while providing a stable osteogenic microenvironment for the endosteoblastic layer.

[0008] Specifically, the outer fibrous layer is prepared from polylactic acid solution using electrospinning and ultrasonic technology; the intermediate barrier layer is prepared from polycaprolactone solution using solvent evaporation; and the endosteogenic layer (PM) is prepared from a mixture of polycaprolactone and mineralized collagen (MC) using freeze-drying technology. A mixture of 1,4-dioxane and water is coated on both sides of the intermediate layer, resulting in a three-layer composite structure. The minimum adhesive strength of each layer reaches 68.70 ± 1.7 kPa. This ultimately yields a three-layer biomimetic periosteum that achieves periosteal repair and promotes bone tissue regeneration.

[0009] This invention provides a method for preparing the above-mentioned three-layer biomimetic periosteum that promotes periosteal repair and bone tissue regeneration, comprising the following steps: (1) Preparation of outer fiber layer (UPLA): 18 wt% polylactic acid particles were dissolved in dichloromethane and N,N-dimethylformamide in a volume ratio of 7:3. The mixture was stirred at 1000 r / min for 12 h at room temperature to obtain a slightly viscous, transparent initial spinning solution. The initial spinning solution was allowed to stand for 2-3 h to remove air bubbles and obtain an electrospinning solution. The electrospinning solution was placed in a 20 mL syringe, connected to a 21 G spinning needle, and spun at an ambient temperature of 25℃, humidity of 27%, spinning voltage of 5-15 kV, roller speed of 300-3000 r / min, and spinning extrusion speed of 0. Under the conditions of 0.001~0.004 mm / s, the spinning time lasted for 6 hours; the obtained fiber membrane was placed in a vacuum oven at 37°C and left to stand for 24 hours to obtain polylactic acid electrospun fiber membrane; the obtained fiber membrane was ultrasonically treated in a magnetic ultrasonic machine for 5~15 minutes and then dried in a vacuum oven at 37°C for 24 hours to obtain the outer fiber membrane UPLA. (2) Preparation of intermediate barrier layer (PC): Polycaprolactone particles are dissolved in chloroform to form a 0.5wt%~1.5wt% solution. The solution is stirred continuously at 1000r / min for 2~3h at room temperature to obtain a clear mixed solution. The mixture is poured into a glass mold and dried in a fume hood to obtain a polycaprolactone film. The obtained polycaprolactone film is placed in a vacuum oven at 37℃ and left to stand for 24h until the solvent evaporates completely to obtain the intermediate barrier layer PC. (3) Preparation of mineralized bovine type I collagen: Weigh 4.6g of bovine type I collagen gel and dissolve it in 0.5mol / L glacial acetic acid solution; add calcium chloride solution to the collagen solution at a ratio of 0.12mol calcium ions / 1g collagen gel to obtain a mixed solution; add phosphoric acid solution dropwise to the mixed solution at a calcium-to-phosphorus molar ratio of 1.67; stir at room temperature for 2h, use 1mol / L sodium hydroxide solution to maintain the pH at 7.8~8.2, stir for 12h and then let stand for 12h, remove the supernatant to obtain the precipitate; wash the precipitate with deionized water and centrifuge until the pH of the supernatant is 7; freeze the precipitate at -20℃ for 5h and then place it in a freeze dryer at -96℃; after complete drying, grind it through a 100-mesh sieve to obtain MC powder; (4) Preparation of endosteoblastic layer (PM): Weigh 0.5g of polycaprolactone particles and dissolve them in 5mL of 1,4-dioxane. Stir at room temperature for 6h and magnetic stirring at 1000r / min. Add 0.4g of MC to the obtained clear solution and stir at room temperature for 1h. Pour the obtained white suspension into a pre-frozen glass mold and freeze at -20℃ for 12h. Freeze dry in a freeze dryer at -96℃ until the organic solvent evaporates completely to obtain an endosteoblastic layer PM with a mineralized bovine type I collagen concentration of 8wt%. (5) Preparation of three-layer biomimetic periosteum: 1,4-dioxane and water mixture is used as adhesive and applied to both sides of the middle layer PC. Then it is bonded between the outer fibrous layer UPLA and the inner osteogenic layer PM. After the 1,4-dioxane has completely evaporated by freeze drying technology, the two sides of PC are re-cured to complete the assembly of the three-layer biomimetic periosteum.

[0010] Furthermore, a mixture of 1,4-dioxane and water in a volume ratio of 1:2 was used as a binder.

[0011] Furthermore, after ultrasonic treatment (loosening treatment), the outer fiber membrane UPLA exhibits a uniform and continuous array of fibers, with inter-fiber pore sizes reaching 13.50±0.55µm.

[0012] Furthermore, the intermediate barrier layer PC has a uniform porous structure on its surface with an average pore size of 2.82±0.05µm.

[0013] Furthermore, the solid content of type I bovine collagen in mineralized bovine type I collagen is 1.63%.

[0014] This invention utilizes electrospinning, solvent evaporation, and freeze-drying techniques to prepare a three-layer heterogeneous membrane for use as a biomimetic periosteum. A loose outer fibrous layer, prepared with 18 wt% polylactic acid spinning solution, mimics the fibrous layer structure of the natural periosteum, supporting fibroblast proliferation, adhesion, and inward growth. A porous barrier membrane, prepared with 1% polycaprolactone via solvent evaporation, effectively hinders excessive invasion of fibroblasts into the osteoblastic layer, providing a stable microenvironment for osteoblast differentiation. An inner osteoblastic layer, prepared with 0 wt%, 8 wt% mineralized collagen (MC), and 10% polycaprolactone via freeze-drying, mimics the bioactive layer of the natural periosteum, effectively promoting osteoblast proliferation, differentiation, and migration, providing a driving force for surrounding bone tissue regeneration. The composite three-layer structure exhibits tensile strength similar to the natural periosteum, with a tensile strength of 3.55 ± 0.23 MPa, effectively avoiding potential stress concentration problems after implantation.

[0015] The beneficial effects of this invention are: (1) The three-layer biomimetic periosteum of the present invention has excellent biocompatibility, low toxicity, low immunogenicity, and good cell adhesion ability. Under the premise of simultaneous damage to bone and periosteum, it effectively achieves the repair of periosteum and promotes the regeneration of bone tissue. (2) The three-layer biomimetic periosteum of the present invention has good cell migration ability, which is conducive to cell migration and growth, thereby improving the healing rate of the wound at the bone injury site; (3) The three-layer biomimetic periosteum of the present invention effectively promotes the cell differentiation process, thereby driving osteogenic differentiation of cells at the defect site, laying the foundation for bone tissue regeneration; (4) In this invention, after the smooth, bead-free polylactic acid is ultrasonically treated for 5 minutes, the spun membrane has a uniform pore size, which is conducive to the inward growth of fibroblasts; at a concentration of 1 wt%, polycaprolactone exhibits a uniformly distributed array of small pores, which effectively hinders the further invasion of fibroblasts; when the mass ratio of polycaprolactone to mineralized collagen is 5:4, the prepared endosteoblast has a good ability to promote cell proliferation and differentiation. (5) The three-layer biomimetic periosteum of the present invention has good mechanical properties, interlayer adhesion properties, and the ability to promote cell proliferation, migration and differentiation. Attached Figure Description

[0016] Figure 1 Electron micrographs of the surface morphology of the outer fiber membranes after ultrasonic treatment for different durations in comparative examples, Example 1, Example 2 and Example 3.

[0017] Figure 2 Electron micrographs of the surface morphology of the intermediate barrier layer in Examples 1, 2 and 3.

[0018] Figure 3 The images show electron micrographs of the cross-sectional morphology of the endosteoblast. (a) shows the PCL intima in the comparative example, (b) shows the PM intima in Example 1, and (c) shows the pore size distribution of the PCL intima in the comparative example and the PM intima in Example 1.

[0019] Figure 4 The images show the tensile strength of each layer after bonding in Example 1.

[0020] Figure 5 The images show the adhesion strength of the outer fibrous membrane and the inner osteogenic layer after they are bonded to the intermediate barrier layer in Example 1.

[0021] Figure 6 The CCK-8 assay was used to assess the cell proliferation of fibroblasts in the outer fibrous layer and intermediate barrier layer of the comparative and Example 1.

[0022] Figure 7 The cell proliferation of mouse embryonic osteoblast precursor cells in the endosteoblast layer and intermediate barrier layer in the comparative example and Example 1 was assessed using CCK-8 assay.

[0023] Figure 8 The cell scratch assay was used to examine the effect of the endosteoblast and intermediate barrier layer on the cell migration of mouse embryonic osteoblast precursor cells in the comparative example and Example 1.

[0024] Figure 9 The alkaline phosphatase assay was used to assess the cell differentiation of mouse embryonic osteoblast precursor cells in the endosteoblast layer and intermediate barrier layer in the comparative and Example 1. Detailed Implementation

[0025] The present invention will be further illustrated by the following embodiments, but is not limited to the following embodiments.

[0026] Comparative example: The fibrous membrane was not subjected to ultrasonic treatment, and the endosteogenic layer was not supplemented with mineralized bovine type I collagen. The specific preparation method is as follows: Outer fiber layer (PLA): 1.8 g of polylactic acid was dissolved in a mixture of 7 mL dichloromethane (DCM) and 3 mL N,N-dimethylformamide (DMF) and stirred at 1000 r / min for 12 h at room temperature. The resulting solution was allowed to stand for 2 h to remove air bubbles, thus obtaining an electrospinning solution. The spinning solution was placed in a 20 mL syringe, connected to a 21 G spinning needle, and spun for 6 h under the following conditions: ambient temperature 25℃, humidity 27%, spinning voltage 10 kV, roller speed 3000 r / min, and spinning extrusion speed 0.001 mm / s. The resulting fiber membrane was placed in a vacuum oven at 37℃ and allowed to stand for 24 h to obtain polylactic acid electrospun fibers. The resulting fiber membrane was then dried in a vacuum oven at 37℃ for 24 h to obtain the outer fiber membrane PLA.

[0027] Intermediate barrier layer (PC): Weigh 0.05g of polycaprolactone particles and dissolve them in 5mL of chloroform. Stir continuously at 1000r / min for 3h at room temperature to obtain a clear mixed solution. Pour the mixture into a glass mold with a diameter of 90mm and dry it in a fume hood to obtain a polycaprolactone film. Place the obtained polycaprolactone film in a vacuum oven at 37℃ and let it stand for 24h until the solvent evaporates completely to obtain the intermediate barrier layer PC.

[0028] Internal osteoblastic layer (PCL): 0.5 g of polycaprolactone granules were dissolved in 5 mL of 1,4-dioxane and stirred at room temperature for 6 h with a magnetic stirrer at 1000 r / min. The resulting clear solution was poured into a pre-frozen glass mold and frozen at -20 °C for 12 h. The solution was then freeze-dried at -96 °C until the organic solvent was completely evaporated, yielding the internal osteoblastic layer (PCL).

[0029] An outer fiber layer was prepared by spinning 18 wt% polylactic acid; a porous intermediate barrier layer was prepared by 1% polycaprolactone; an endogenous osteogenic layer was prepared by 10% polycaprolactone; a mixture of 1,4-dioxane and water in a volume ratio of 1:2 was used as an adhesive to coat both sides of the intermediate layer, and the outer fiber layer and the endogenous osteogenic layer were respectively attached to the upper and lower parts of the intermediate layer; the three-layer structure after attachment was freeze-dried to obtain a three-layer biomimetic periosteum that realizes periosteal repair and promotes bone tissue regeneration. Example 1

[0030] The method for preparing a three-layer biomimetic periosteum using the method of the present invention is as follows: Outer fiber layer (UPLA): 1.8 g of polylactic acid was dissolved in a mixture of 7 mL dichloromethane (DCM) and 3 mL N,N-dimethylformamide (DMF) and stirred at 1000 r / min for 12 h at room temperature. The resulting solution was allowed to stand for 2 h to remove air bubbles, thus obtaining an electrospinning solution. The spinning solution was placed in a 20 mL syringe and connected to a 21 G spinning needle. Spinning was carried out for 6 h under the following conditions: ambient temperature 25℃, humidity 27%, spinning voltage 10 kV, roller speed 3000 r / min, and spinning extrusion speed 0.001 mm / s. The resulting fiber membrane was placed in a vacuum oven at 37℃ and allowed to stand for 24 h to obtain a polylactic acid electrospun fiber membrane. The obtained fiber membrane was then ultrasonically treated in a magnetic ultrasonic machine for 5 min and dried in a vacuum oven at 37℃ for 24 h to obtain the outer fiber membrane.

[0031] Preparation of intermediate barrier layer (PC): Weigh 0.05g of polycaprolactone particles and dissolve them in 5mL of chloroform. Stir continuously at 1000r / min for 3h at room temperature to obtain a clear mixed solution. Pour the mixture into a glass mold with a diameter of 90mm and dry it in a fume hood to obtain a polycaprolactone film. Place the obtained polycaprolactone film in a vacuum oven at 37℃ and let it stand for 24h until the solvent evaporates completely to obtain the intermediate barrier layer PC.

[0032] Mineralized bovine type I collagen (MC): Weigh 4.6g of bovine type I collagen gel and dissolve it in 0.5mol / L glacial acetic acid solution. Add calcium chloride solution to the collagen solution at a ratio of 0.12mol calcium ions / 1g collagen gel to obtain a mixed solution. Add phosphoric acid solution dropwise to the mixed solution according to a calcium-to-phosphorus molar ratio of 1.67. Stir at room temperature for 2h, maintain the pH at around 8 using 1mol / L sodium hydroxide solution, stir for 12h, let stand for 12h, remove the supernatant to obtain a precipitate. Wash the precipitate with deionized water and centrifuge until the pH of the supernatant is 7. Freeze the washed precipitate at -20℃ for 5h, then place it in a freeze dryer at -96℃. After complete drying, grind it through a 100-mesh sieve to obtain MC powder.

[0033] Endogenous osteoblast (PM): 0.5 g of polycaprolactone granules were dissolved in 5 mL of 1,4-dioxane and stirred at room temperature for 6 h with a magnetic stirrer at 1000 r / min. The resulting clear solution was then added to 0.4 g of MC and stirred at room temperature for 1 h. The resulting white suspension was poured into a pre-frozen glass mold and frozen at -20 °C for 12 h. The solution was then freeze-dried at -96 °C until the organic solvent had completely evaporated, yielding the endogenous osteoblast.

[0034] A porous outer fiber layer was prepared by combining 18 wt% polylactic acid spinning solution with ultrasonic technology; a porous intermediate barrier layer was prepared by 1% polycaprolactone; an endogenous osteogenic layer was prepared by combining 8 wt% mineralized collagen (MC) and 10% polycaprolactone; a mixture of 1,4-dioxane and water in a volume ratio of 1:2 was used as an adhesive to coat both sides of the intermediate layer, and the outer fiber layer and the endogenous osteogenic layer were respectively attached to the upper and lower parts of the intermediate layer; the three-layer structure after bonding was freeze-dried to obtain a three-layer biomimetic periosteum that realizes periosteal repair and promotes bone tissue regeneration. Example 2

[0035] The method for preparing a three-layer biomimetic periosteum using the method of the present invention is as follows: Outer fiber layer (UPLA): 1.8 g of polylactic acid was dissolved in a mixture of 7 mL dichloromethane (DCM) and 3 mL N,N-dimethylformamide (DMF) and stirred at 1000 r / min for 12 h at room temperature. The resulting solution was allowed to stand for 2 h to remove air bubbles, thus obtaining an electrospinning solution. The spinning solution was placed in a 20 mL syringe and connected to a 21 G spinning needle. Spinning was carried out for 6 h under the following conditions: ambient temperature 25℃, humidity 27%, spinning voltage 10 kV, roller speed 3000 r / min, and spinning extrusion speed 0.001 mm / s. The resulting fiber membrane was placed in a vacuum oven at 37℃ and allowed to stand for 24 h to obtain a polylactic acid electrospun fiber membrane. The obtained fiber membrane was then ultrasonically treated in a magnetic ultrasonic machine for 10 min and dried in a vacuum oven at 37℃ for 24 h to obtain the outer fiber membrane.

[0036] Intermediate barrier layer (PC): Weigh 0.025g of polycaprolactone particles and dissolve them in 5mL of chloroform. Stir continuously at 1000r / min for 3h at room temperature to obtain a clear mixed solution. Pour the mixture into a glass mold with a diameter of 90mm and dry it in a fume hood to obtain a polycaprolactone film. Place the obtained polycaprolactone film in a vacuum oven at 37℃ and let it stand for 24h until the solvent evaporates completely to obtain the intermediate barrier layer PC.

[0037] The preparation of the endosteoblast layer is the same as in Example 1.

[0038] A loose outer fiber layer was prepared by combining 18wt% polylactic acid spinning solution with ultrasonic technology; a porous intermediate barrier layer was prepared by 0.5% polycaprolactone; an endogenous osteogenic layer was prepared by combining 8wt% mineralized collagen (MC) and 10% polycaprolactone; a mixture of 1,4-dioxane and water in a volume ratio of 1:2 was used as an adhesive to coat both sides of the intermediate layer, and the outer fiber layer and the endogenous osteogenic layer were respectively attached to the upper and lower parts of the intermediate layer; the three-layer structure after bonding was freeze-dried to obtain a three-layer biomimetic periosteum that realizes periosteal repair and promotes bone tissue regeneration. Example 3

[0039] The method for preparing a three-layer biomimetic periosteum using the method of the present invention is as follows: Outer fiber layer (UPLA): 1.8 g of polylactic acid was dissolved in a mixture of 7 mL dichloromethane (DCM) and 3 mL N,N-dimethylformamide (DMF) and stirred at 1000 r / min for 12 h at room temperature. The resulting solution was allowed to stand for 2 h to remove air bubbles, thus obtaining an electrospinning solution. The spinning solution was placed in a 20 mL syringe and connected to a 21 G spinning needle. Spinning was carried out for 6 h under the following conditions: ambient temperature 25℃, humidity 27%, spinning voltage 10 kV, roller speed 3000 r / min, and spinning extrusion speed 0.001 mm / s. The resulting fiber membrane was placed in a vacuum oven at 37℃ and allowed to stand for 24 h to obtain a polylactic acid electrospun fiber membrane. The obtained fiber membrane was then ultrasonically treated in a magnetic ultrasonic machine for 15 min and dried in a vacuum oven at 37℃ for 24 h to obtain the outer fiber membrane.

[0040] Intermediate barrier layer (PC): Weigh 0.075g of polycaprolactone particles and dissolve them in 5mL of chloroform. Stir continuously at 1000r / min for 3h at room temperature to obtain a clear mixed solution. Pour the mixture into a glass mold with a diameter of 90mm and dry it in a fume hood to obtain a polycaprolactone film. Place the obtained polycaprolactone film in a vacuum oven at 37℃ and let it stand for 24h until the solvent evaporates completely to obtain the intermediate barrier layer PC.

[0041] The preparation of the endosteoblast layer is the same as in Example 1.

[0042] A loose outer fiber layer was prepared by combining 18wt% polylactic acid spinning solution with ultrasonic technology; a porous intermediate barrier layer was prepared by 1.5% polycaprolactone; an endogenous osteogenic layer was prepared by combining 8wt% mineralized collagen (MC) and 10% polycaprolactone; a mixture of 1,4-dioxane and water in a volume ratio of 1:2 was used as an adhesive to coat both sides of the intermediate layer, and the outer fiber layer and the endogenous osteogenic layer were respectively attached to the upper and lower parts of the intermediate layer; the three-layer structure after bonding was freeze-dried to obtain a three-layer biomimetic periosteum that realizes periosteal repair and promotes bone tissue regeneration.

[0043] Figure 1 Electron micrographs of the surface fiber morphology of PLA spun membranes after different ultrasonic treatment times are shown. It was observed that after 5 minutes of ultrasonic treatment, the PLA fibers still maintained the uniform and continuous structure of the un-ultrasonicated form, and the pore size tended to increase. When ultrasonic treatment was performed for 10 minutes, the surface fibers gradually curled up and lost their original fiber morphology, indicating that ultrasonic treatment can effectively change the dense network structure on the fiber surface under appropriate treatment time.

[0044] Figure 2These are electron microscopy images of the surface morphology of the intermediate barrier layer in Examples 1, 2, and 3. Numerous small pores and a multi-level microporous structure are visible on the film surface at different concentrations. Comparison of different concentrations shows that the intermediate layer exhibits the most uniform pore size distribution at a PCL concentration of approximately 1%. The average pore size, measured using ImageJ software, is 2.82 ± 0.05 µm, meeting the requirement of being less than 5 µm, indicating that it possesses barrier potential while also satisfying the basic requirements for nutrient permeation.

[0045] Figure 3 (a) and (b) are electron microscope images of the cross-sectional morphology of the PCL inner membrane in the comparative example and the PM inner membrane in Example 1, respectively. After composite mineralized collagen, the pores in the PM film are more robust and rounded compared to the PCL inner membrane. Figure 3 (c) shows the pore size distribution of the PCL inner membrane in the comparative example and the PM inner membrane in Example 1. It can be seen that the pore size of the PCL inner membrane is 12.07~99.05μm, while the pore size of the PM inner membrane is 40.67~99.08μm, and the pore size distribution is more uniform.

[0046] Figure 4 The images show the tensile strength of each layer after bonding in Example 1. When two layers are bonded together, their tensile strengths are 0.63±0.04 MPa (UPLA / PM), 1.22±0.02 MPa (UPLA / PC), and 3.08±0.25 MPa (PC / PM), respectively. The tensile strength of the three-layer composite is 3.55±0.23 MPa (UPLA / PC / PM), which is similar to the tensile strength of the natural periosteum, effectively solving the stress concentration problem after natural periosteum implantation.

[0047] Figure 5 Images show the adhesion strength of the outer fibrous membrane and the inner osteoblast layer after bonding to the intermediate barrier layer in Example 1. The results show that the adhesion strength between the outer fibrous membrane and the intermediate barrier layer is 68.70±1.75 kPa, and the adhesion strength between the inner osteoblast layer and the intermediate barrier layer is 80.33±1.39 kPa, indicating that the layers are tightly bonded together.

[0048] Figure 6 The CCK-8 assay was used to assess the cell proliferation of fibroblasts in the outer fiber layer and intermediate barrier layer of the comparative example and Example 1. The absorbance increased with increasing cell culture time. In the comparative example, the cell proliferation rate of the PLA fiber membrane was 85.37 ± 1.05%, while in Example 2, the cell proliferation rate of the UPLA fiber membrane was 94.26 ± 2.36%, indicating that the UPLA fiber membrane obtained after sonication was more conducive to fibroblast proliferation than the PLA fiber membrane. The control group in the figure represents the cell proliferation of the group without material under the same culture conditions.

[0049] Figure 7 The CCK-8 assay was used to detect the cell proliferation of mouse embryonic osteoblast precursor cells in the endosteoblast layer and intermediate barrier layer in the comparative example and Example 1. The absorbance increased with increasing cell culture time. The cell proliferation rate of the PCL intima in the comparative example was 93.83±1.35%, and the cell proliferation rate of the PM intima in Example 2 was 109.57±1.41%, indicating that compared to pure PCL membrane without mineralized collagen, the PM membrane exhibited a good proliferation-promoting effect on mouse embryonic osteoblast precursor cells.

[0050] Figure 8 The cell scratch assay was used to examine the effect of the endosteoblast and intermediate barrier layers in the comparative and Example 1 on promoting cell migration of mouse embryonic osteoblast precursor cells. The cell migration rate in the pure PCL group was 70.88±2.79%, while the cell migration rate in the PM group was as high as 99.88±0.10%. These results indicate that PM can effectively promote cell migration.

[0051] Figure 9 The alkaline phosphatase assay was used to assess the cell differentiation of mouse embryonic osteoblast precursor cells in the inner osteoblast layer and intermediate barrier layer of the comparative example and Example 1. Compared with the intermediate barrier layer and the PCL membrane without MC, the PM membrane showed a higher absorbance value, indicating that PM effectively promoted the cell differentiation process of mouse embryonic osteoblast precursor cells.

[0052] The three-layer biomimetic periosteum prepared by the above method is simple and readily available, and meets the mechanical properties of natural periosteum, has good interlayer adhesion and biocompatibility, and has broad prospects for clinical use.

Claims

1. A three-layered biomimetic periosteum to promote periosteal repair and bone tissue regeneration, characterized in that: It consists of an outer fibrous layer, an intermediate barrier layer, and an endosteoblastic layer. A polylactic acid (PLA) spun fiber membrane is used to simulate the outer fibrous layer of the natural periosteum to promote fibroblast adhesion and growth. A polycaprolactone / mineralized bovine type I collagen endosteoblastic layer, prepared by freeze-drying, simulates the bioactive layer of the natural periosteum to promote the proliferation and osteogenic differentiation of mouse embryonic osteoblast precursor cells. A polycaprolactone barrier layer, prepared by solvent evaporation, serves as the intermediate layer, connecting the outer fibrous layer and the endosteoblastic layer, acting as a barrier to prevent fibroblast invasion while providing a stable osteogenic microenvironment for the endosteoblastic layer. A mixture of 1,4-dioxane and water is coated on both sides of the intermediate layer to connect the outer fibrous layer and the endosteoblastic layer, with a minimum adhesion strength of 68.70 ± 1.7 kPa. The volume ratio of 1,4-dioxane to water is 1:

2.

2. A method for preparing the three-layer biomimetic periosteum for promoting periosteal repair and bone tissue regeneration according to claim 1, characterized in that: The outer fibrous layer is prepared by electrospinning and ultrasonic technology using polylactic acid solution; the intermediate barrier layer is prepared by solvent evaporation method using polycaprolactone solution; and the endosteoblastic layer is prepared by freeze-drying technology using a mixture of polycaprolactone and mineralized collagen. A mixture of 1,4-dioxane and water is coated on both sides of the intermediate layer to connect the outer fibrous layer and the endosteoblastic layer. The minimum adhesion strength of each layer can reach 68.70±1.7 kPa. Finally, a three-layer biomimetic periosteum is obtained to achieve periosteal repair and promote bone tissue regeneration.

3. The method of claim 2, wherein the three-layered biomimetic periosteum is prepared by the steps of: a) providing a first layer of a collagen-based material; b) providing a second layer of a collagen-based material; and c) providing a third layer of a collagen-based material. Includes the following steps: (1) Preparation of outer fiber layer: 18 wt% polylactic acid particles were dissolved in dichloromethane and N,N-dimethylformamide in a volume ratio of 7:

3. The mixture was stirred at 1000 r / min for 12 h at room temperature to obtain a slightly viscous, transparent initial spinning solution. The initial spinning solution was allowed to stand for 2-3 h to remove air bubbles and obtain an electrospinning solution. The electrospinning solution was placed in a 20 mL syringe, connected to a 21 G spinning needle, and spun at an ambient temperature of 25℃, humidity of 27%, spinning voltage of 5-15 kV, roller speed of 300-3000 r / min, and spinning extrusion speed of 0. Under the conditions of 0.001~0.004 mm / s, the spinning time lasted for 6 hours; the obtained fiber membrane was placed in a vacuum oven at 37°C and left to stand for 24 hours to obtain polylactic acid electrospun fiber membrane; the obtained fiber membrane was ultrasonically treated in a magnetic ultrasonic machine for 5~15 minutes and then dried in a vacuum oven at 37°C for 24 hours to obtain the outer fiber membrane UPLA. (2) Preparation of intermediate barrier layer: Polycaprolactone particles are dissolved in chloroform to form a 0.5wt%~1.5wt% solution. The solution is stirred continuously at 1000r / min for 2~3h at room temperature to obtain a clear mixed solution. The mixture is poured into a glass mold and dried in a fume hood to obtain a polycaprolactone film. The obtained polycaprolactone film is placed in a vacuum oven at 37℃ and left to stand for 24h until the solvent evaporates completely to obtain the intermediate barrier layer PC. (3) Preparation of mineralized bovine type I collagen: Weigh 4.6g of bovine type I collagen gel and dissolve it in 0.5mol / L glacial acetic acid solution; add calcium chloride solution to the collagen solution at a ratio of 0.12mol calcium ions / 1g collagen gel to obtain a mixed solution; Phosphoric acid solution was added dropwise to the mixed solution according to a calcium-to-phosphorus molar ratio of 1.

67. The mixture was stirred at room temperature for 2 hours, and the pH was maintained at 7.8-8.2 using a 1 mol / L sodium hydroxide solution. After stirring for 12 hours, the mixture was allowed to stand for another 12 hours. The supernatant was removed to obtain a precipitate. The precipitate was washed with deionized water and centrifuged until the pH of the supernatant was 7. The washed precipitate was then frozen at -20°C for 5 hours and placed in a freeze dryer at -96°C. After complete drying, the precipitate was ground through a 100-mesh sieve to obtain MC powder. (4) Preparation of endosteoblastic layer: Weigh 0.5g of polycaprolactone particles and dissolve them in 5mL of 1,4-dioxane. Stir at room temperature for 6h and magnetic stirring at 1000r / min. Add 0.4g of MC to the obtained clear solution and stir at room temperature for 1h. Pour the obtained white suspension into a pre-frozen glass mold and freeze at -20℃ for 12h. Freeze dry in a freeze dryer at -96℃ until the organic solvent evaporates completely to obtain an endosteoblastic layer PM containing 8wt% mineralized bovine type I collagen. (5) Preparation of three-layer biomimetic periosteum: 1,4-dioxane and water mixture is used as adhesive and applied to both sides of the middle layer PC. Then it is bonded between the outer fibrous layer UPLA and the inner osteogenic layer PM. After the 1,4-dioxane has completely evaporated by freeze drying technology, the two sides of PC are re-cured to complete the assembly of the three-layer biomimetic periosteum.

4. The method for preparing the three-layer biomimetic periosteum that promotes periosteal repair and bone tissue regeneration according to claim 3, characterized in that: The outer fiber membrane UPLA, after ultrasonic treatment, exhibits a uniform and continuous array of fibers with a pore size of 13.50±0.55µm between fibers.

5. The method for preparing the three-layer biomimetic periosteum that promotes periosteal repair and bone tissue regeneration according to claim 3, characterized in that: The intermediate barrier layer PC has a uniform porous structure on its surface with an average pore size of 2.82±0.05µm.

6. The method for preparing the three-layer biomimetic periosteum that promotes periosteal repair and bone tissue regeneration according to claim 3, characterized in that: The solid content of type I bovine collagen in mineralized bovine type I collagen is 1.63%.

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