Bionic periosteum with double-layer structure as well as preparation method and application of bionic periosteum

Abstract

The invention provides a bionic periosteum with a double-layer structure and a preparation method and application thereof, and belongs to the technical field of biomedical material development. The double-layer membrane design is carried out by imitating the physiological anatomical structure of the natural periosteum, and the problem that the material performance and the structure cannot be matched with the natural periosteum in the prior art is solved by designing the structure that the upper layer is the laminated micro / nano fiber membrane and the lower layer is the HA-PDA-PAM hydrogel. The upper fiber layer can simulate a multi-stage fiber structure of an extracellular matrix, guide behaviors of stem cells, serve as a barrier for enhancing physical strength, improve mechanical properties and provide good biocompatibility and hydrophilicity; macropores between the HA-PDA-PAM hydrogel on the lower layer are beneficial to osteoblast ingrowth, the tissue adhesion and stability are better, the two layers are tightly combined, the structural stability and the functional synergy are ensured, and an innovative solution with transformation potential is provided for clinical bone tissue engineering.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical material development, and in particular to a double-layer bionic periosteum and a preparation method and application thereof. Background Art

[0002] Bone defects secondary to trauma, degenerative diseases, congenital malformations or tumor resection, especially critical-sized bone defects, remain a huge challenge in clinical practice. Currently, the methods for clinical reconstruction of damaged bones mainly include autologous bone transplantation and allogeneic bone transplantation. Although autologous grafts are the gold standard for bone reconstruction, their application is limited by factors such as insufficient sources, donor site morbidity, and difficulty in implanted bone reconstruction. In addition, bone replacement scaffolds used to repair large segmental defects have some limitations, such as incomplete vascular anastomosis and invasion, low osteogenesis efficiency, and slow new bone reconstruction. Therefore, there is an urgent need for an effective and sustainable long-term treatment strategy for bone repair.

[0003] The natural periosteum is a thin, tough, and highly vascularized double-layer membrane of connective tissue that covers the surface of bone tissue. It covers the outer surface of almost all cortical bones, except for joints. The periosteum is composed of specialized connective tissue, fixed to the bone by Sharpey fibers. Structurally, it can be divided into two distinct functional layers: the fibrous layer and the cambium layer. The thicker fibrous layer is located in the outer layer, in contact with the soft tissue, and is primarily composed of fibroblasts, a vascular network, and oriented collagen fibers (along the direction of bone growth). The thinner cambium layer is located in the inner layer, in contact with the bone tissue, and is rich in various bone cells and local growth factors. Although various periosteal biomimetic materials have been studied, they have not fully considered the physiological and anatomical structure of the natural periosteum. Their performance and structure cannot match that of the natural periosteum, resulting in limited application in subsequent repairs. Summary of the Invention

[0004] The purpose of the present invention is to provide a double-layer bionic periosteum and its preparation method and application, which is similar to the anatomical and physiological structure of natural periosteum, more compatible with natural periosteum in structure and performance, and has greater potential in repairing bone defects.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a biomimetic periosteum gel double-layer scaffold, comprising the following steps:

[0007] S1, electrospinning a polycaprolactone solution to obtain an electrospun membrane;

[0008] S2, mixing the electrospun membrane with an alkaline solution of a polysaccharide polymer material for 10 to 20 minutes, then transferring the mixture to an ethanol aqueous solution, mixing and contacting for 0.5 to 2 hours, to obtain a self-assembled product;

[0009] S3, dispersing dopamine in an alkaline buffer solution and performing a prepolymerization treatment to obtain a dopamine dispersion;

[0010] S4, mixing the dopamine dispersion, hydroxyapatite, acrylamide, ammonium persulfate, N,N-methylenebisacrylamide and tetramethylethylenediamine, and curing to obtain a hydrogel;

[0011] S5. Placing the self-assembled product on the upper layer of the hydrogel for combination to obtain a biomimetic periosteum gel double-layer scaffold.

[0012] Preferably, the solute of the alkaline solution of polysaccharide polymer material is selected from one or more of cellulose, chitin, chitosan or sodium alginate.

[0013] Preferably, in S1, the solvent used in the polycaprolactone solution is hexafluoroisopropanol;

[0014] The concentration of the polycaprolactone solution is 0.1 to 0.2 g / mL;

[0015] The polycaprolactone solution is magnetically stirred for 10 to 14 hours before the electrospinning treatment;

[0016] In the electrospinning process, the spinning voltage is 15 to 20 kV, and the flow rate of the injection pump is 0.001 to 0.005 mm / s.

[0017] Preferably, in S2, the solvent used in the alkaline solution of the polysaccharide polymer material contains LiOH, KOH, urea and water;

[0018] The mass ratio of LiOH, KOH, urea and water is 4-5:6-8:7-9:75-85.

[0019] Preferably, in S2, the temperature of the ethanol aqueous solution is -20°C to 5°C;

[0020] The volume percentage concentration of the ethanol aqueous solution is 70-90%.

[0021] Preferably, the concentration of the dopamine dispersion is 1-2 mg / mL.

[0022] Preferably, in S3, the pH value of the alkaline buffer solution is 8 to 9;

[0023] The alkaline buffer solution is Tris-HCl solution;

[0024] The prepolymerization treatment time is 20 to 40 minutes.

[0025] Preferably, in S4, the volume ratio of the dopamine dispersion, the mass of hydroxyapatite, the mass of acrylamide, ammonium persulfate, the mass of N,N-methylenebisacrylamide and the volume ratio of tetramethylethylenediamine is 5-15 mL: 400-600 mg: 2-3 g: 100-300 mg. : 10~30mg:10~30μL.

[0026] The present invention also provides a bionic periosteum gel double-layer scaffold prepared by the preparation method.

[0027] The present invention also provides the use of the bionic periosteum gel double-layer scaffold in preparing bone defect repair materials.

[0028] Beneficial effects of the present invention:

[0029] The present invention mimics the physiological and anatomical structure of the natural periosteum with a double-layer membrane design. By setting a structure with a stacked micro / nanofibrous membrane as the upper layer and a HA-PDA-PAM hydrogel as the lower layer, a biomimetic periosteum gel double-layer scaffold is obtained. The upper fiber layer of the double-layer scaffold in the present invention is a stacked micro / nano hierarchical structure, which can not only mimic the multi-level fiber structure of the extracellular matrix and guide the behavior of stem cells, but also provide a barrier with enhanced physical strength due to the dense deposition of polysaccharide polymer nanofibers, thereby improving mechanical properties and providing good biocompatibility and hydrophilicity. The large pores between the lower layer HA-PDA-PAM hydrogel of the double-layer scaffold of the present invention are conducive to the growth of osteoblasts, the incorporation of dopamine increases its tissue adhesion, and the addition of nanohydroxyapatite can enhance the stability of the natural polymer hydrogel membrane, resulting in sustained degradation time, biomineralization and long-term ion release, which is beneficial to bone regeneration. The tight bonding between the two layers ensures structural stability and functional synergy, providing an innovative solution with translational potential for clinical bone tissue engineering. The preparation method provided by the present invention has low production cost and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the structural diagram of the double-layer bionic periosteal scaffold;

[0031] Figure 2 is the SEM image of PCL;

[0032] Figure 3 is the SEM image of CS;

[0033] Figure 4 is the SEM image of PCL-CS;

[0034] Figure 5 is the SEM image of HA-PDA-PAM;

[0035] Figure 6is the infrared spectrum of PCL-CS;

[0036] Figure 7 is the rheological diagram of PCL-CS;

[0037] Figure 8 is the mechanical tensile diagram of PCL-CS;

[0038] Figure 9 is the elastic modulus diagram of PCL-CS;

[0039] Figure 10 is the infrared spectrum of HA-PDA-PAM;

[0040] Figure 11 is the XRD pattern of HA-PDA-PAM;

[0041] Figure 12 The results of CCK-8 assay after Huvec cells were cultured for 1, 3, and 5 days;

[0042] Figure 13 These are the FITC-DAPI staining results of MC3T3-E1 cells cultured in the extracts of PP, CP-P, P-HP, and CP-HP materials for 3 days.

[0043] Figure 14 Micro-CT three-dimensional reconstruction images of skull regeneration in the CON group, PP group, CP-P group, P-HP group, and CP-HP group at 4 and 8 weeks after surgery. DETAILED DESCRIPTION

[0044] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Step 1: Preparation of PCL electrospinning membrane by electrospinning

[0047] 1.5 g of polycaprolactone (PCL) was dissolved in 10 mL of hexafluoroisopropanol and magnetically stirred for 12 hours to produce a uniform, transparent PCL spinning solution. The spinning solution was transferred to a 5 mL syringe equipped with a 21G needle for electrospinning. The positive electrode of a high-voltage DC power supply was connected to the syringe needle, and one end of the receiving plate was connected to the negative electrode of the high-voltage DC power supply. The distance between the receiver and the needle was 14 cm. The voltage during spinning was 18 kV, and the flow rate of the syringe pump was 0.003 mm / s.

[0048] Step 2: Preparation of PCL-CS membrane with micro-nanostructure

[0049] LiOH, KOH, urea, and deionized water were mixed in a mass ratio of 4.5:7:8:80.5 to obtain an alkaline solvent; chitosan (CS) was added to the alkaline solvent, stirred evenly, and then frozen at -80°C for 2 hours, then stirred to dissolve, and the freeze-thaw process was repeated three times. The mixture was centrifuged at 7000 rpm at 4°C for 10 minutes to degas, ultimately obtaining a clear and transparent CS alkaline solution with a mass fraction of 1%;

[0050] The dried PCL electrospun fiber membrane was immersed in a 1% alkaline CS solution for 15 minutes to form a layer on the surface. The unassembled CS was then washed with deionized water for 15 minutes. After washing, the PCL electrospun membrane was placed in an 80% ethanol solution at 0°C for 1 hour to allow for the self-assembly process. The sample was then washed three times with deionized water until its pH became neutral, resulting in the designated PCL-CS fiber membrane.

[0051] Step 3: Preparation of HA-PDA-PAM hydrogel

[0052] 15 mg of dopamine (DA) was dispersed in 10 mL of Tris-HCl solution (pH=8.5) and prepolymerized for 30 min. The color changed from colorless and transparent to dark brown, indicating that the prepolymerization was successful.

[0053] 500 mg of hydroxyapatite (HA) was dispersed in 10 mL of dopamine aqueous solution and magnetically stirred at 800 rpm for 30 minutes. 2.5 g of acrylamide, 200 mg of ammonium persulfate, 20 mg of N, N-methylenebisacrylamide, and 20 μL of tetramethylethylenediamine were then added to the HA-PDA solution and stirred for 5 minutes. The resulting homogeneous prepolymer solution was transferred to various reaction molds and gelled at room temperature to obtain HA-PDA-PAM hydrogel. Finally, the resulting hydrogel was soaked in deionized water and 75% ethanol to remove excess monomers and dried at room temperature for later use.

[0054] Step 4: Preparation of double-layer bionic periosteum

[0055] The PCL-CS membrane was covered on the surface of the HA-PDA-PAM-HA hydrogel and allowed to stand for natural bonding (physical bonding was performed based on PDA adhesion, hydrogen bonding, electrostatic interaction, etc., and a small amount of covalent bonds may be formed during the bonding process). After 2 hours, the bonded PCL-CS-HA-PDA-PAM double-layer scaffold was taken out, which is the prepared double-layer bionic periosteum. The structure is shown in the figure. Figure 1 shown.

[0056] Example 2

[0057] Step 1: Preparation of PCL electrospinning membrane by electrospinning

[0058] 1.5 g of polycaprolactone (PCL) was dissolved in 10 mL of hexafluoroisopropanol and magnetically stirred for 12 hours to produce a uniform, transparent PCL spinning solution. The spinning solution was transferred to a 5 mL syringe equipped with a 21G needle for electrospinning. The positive electrode of a high-voltage DC power supply was connected to the syringe needle, and one end of the receiving plate was connected to the negative electrode of the high-voltage DC power supply. The distance between the receiver and the needle was 14 cm. The voltage during spinning was 18 kV, and the flow rate of the syringe pump was 0.003 mm / s.

[0059] Step 2: Preparation of PCL-CS membrane with micro-nanostructure

[0060] LiOH, KOH, urea, and deionized water were mixed in a mass ratio of 4.5:7:8:80.5 to obtain an alkaline solvent; CS was added to the alkaline solvent, stirred evenly, and then frozen at -80°C for 2 hours, then stirred to dissolve. The freeze-thaw process was repeated three times, and the solution was centrifuged at 7000 rpm at 4°C for 10 minutes to degas, ultimately obtaining a clear and transparent CS alkaline solution with a mass fraction of 1.5%;

[0061] The dried PCL electrospun fiber membrane was immersed in a 1.5% alkaline CS solution for 20 minutes to form a layer on the surface. The unassembled CS was then washed with deionized water for 15 minutes. After washing, the PCL electrospun membrane was placed in an 80% ethanol solution at 0°C for 1 hour to allow for the self-assembly process. The sample was then washed three times with deionized water until its pH became neutral, resulting in the designated PCL-CS fiber membrane.

[0062] Step 3: Preparation of HA-PDA-PAM hydrogel

[0063] 10 mg of dopamine (DA) was dispersed in 10 mL of Tris-HCl solution (pH=8.5) and prepolymerized for 30 min. The color changed from colorless and transparent to dark brown, indicating that the prepolymerization was successful.

[0064] 500 mg of hydroxyapatite (HA) was dispersed in 10 mL of dopamine aqueous solution and magnetically stirred at 800 rpm for 30 minutes. 2.5 g of acrylamide, 200 mg of ammonium persulfate, 20 mg of N, N-methylenebisacrylamide, and 20 μL of tetramethylethylenediamine were then added to the HA-PDA solution and stirred for 5 minutes. The resulting homogeneous prepolymer solution was transferred to various reaction molds to obtain HA-PDA-PAM hydrogels. Finally, the resulting hydrogels were soaked in deionized water and 75% ethanol to remove excess monomers and dried at room temperature for later use.

[0065] Step 4: Preparation of double-layer bionic periosteum

[0066] The PCL-CS membrane is covered on the surface of the HA-PDA-PAM-HA hydrogel and allowed to stand for natural combination. The combined PCL-CS-HA-PDA-PAM double-layer scaffold is taken out to obtain the prepared double-layer bionic periosteum.

[0067] Example 3

[0068] Step 1: Preparation of PCL electrospinning membrane by electrospinning

[0069] 1.5 g of polycaprolactone (PCL) was dissolved in 10 mL of hexafluoroisopropanol and magnetically stirred for 12 hours to produce a uniform, transparent PCL spinning solution. The spinning solution was transferred to a 5 mL syringe equipped with a 21G needle for electrospinning. The positive electrode of a high-voltage DC power supply was connected to the syringe needle, and one end of the receiving plate was connected to the negative electrode of the high-voltage DC power supply. The distance between the receiver and the needle was 14 cm. The voltage during spinning was 18 kV, and the flow rate of the syringe pump was 0.003 mm / s.

[0070] Step 2: Preparation of PCL-CS membrane with micro-nanostructure

[0071] LiOH, KOH, urea, and deionized water were mixed in a mass ratio of 4.5:7:8:80.5 to obtain an alkaline solvent; CS was added to the alkaline solvent, stirred evenly, and then frozen at -80°C for 2 hours, then stirred to dissolve, and the freeze-thaw process was repeated three times. The solution was centrifuged at 7000 rpm at 4°C for 10 minutes to degas, ultimately obtaining a clear and transparent CS alkaline solution with a mass fraction of 0.5%;

[0072] The dried PCL electrospun fiber membrane was immersed in a 0.5% alkaline CS solution for 15 minutes to form a layer on the surface. The unassembled CS was then washed with deionized water for 15 minutes. After washing, the PCL electrospun membrane was placed in an 80% ethanol solution at 0°C for 1 hour to allow for the self-assembly process. The sample was then washed three times with deionized water until its pH became neutral, resulting in the designated PCL-CS fiber membrane.

[0073] Step 3: Preparation of HA-PDA-PAM hydrogel

[0074] 20 mg of dopamine (DA) was dispersed in 10 mL of Tris-HCl solution (pH=8.5) and prepolymerized for 30 min. The color changed from colorless and transparent to dark brown, indicating that the prepolymerization was successful.

[0075] 500 mg of hydroxyapatite (HA) was dispersed in 10 mL of dopamine aqueous solution and magnetically stirred at 800 rpm for 30 minutes. 2.5 g of acrylamide, 200 mg of ammonium persulfate, 20 mg of N, N-methylenebisacrylamide, and 20 μL of tetramethylethylenediamine were then added to the HA-PDA solution and stirred for 5 minutes. The resulting homogeneous prepolymer solution was transferred to various reaction molds to obtain HA-PDA-PAM hydrogels. Finally, the resulting hydrogels were soaked in deionized water and 75% ethanol to remove excess monomers and dried at room temperature for later use.

[0076] Step 4: Preparation of double-layer bionic periosteum

[0077] The PCL-CS membrane is covered on the surface of the HA-PDA-PAM-HA hydrogel and allowed to stand for natural combination. The combined PCL-CS-HA-PDA-PAM double-layer scaffold is taken out to obtain the prepared double-layer bionic periosteum.

[0078] Experimental example

[0079] Based on the technical solution of Example 1, relevant performance characterization and evaluation were performed.

[0080] The micromorphology of PCL electrospun membrane, CS membrane, PCL-CS electrospun membrane and HA-PDA-PAM hydrogel was observed using a scanning electron microscope. After gold was sprayed using a gold sputtering instrument, the samples were attached to the sample stage using conductive glue and placed in the electron microscope chamber for observation of their morphology. The acceleration voltage was 10-15 kV. The results are shown in the figure. Figure 2 、 Figure 3 、 Figure 4 and Figure 5 shown.

[0081] Figure 2 The SEM image of PCL electrospun membrane shows that the PCL electrospun membrane has regular fiber distribution, no beads or spindle-shaped structures, smooth surface and uniform diameter. At the same time, the structure of CS fiber membrane was further observed, such as Figure 3 As shown in the figure, it can be seen that the CS fiber membrane is composed of a dense nanofiber network with a fiber diameter of about 30 nm, which is similar to the diameter of natural collagen fibers. Figure 4 The figure shows the SEM image of PCL-CS. Spider-web-like CS nanofibers are deposited on the PCL micron fibers and inside their pores, forming a micro-nano multi-level structure, which provides a suitable microenvironment for cell adhesion, growth and proliferation. Figure 5 This is the SEM image of HA-PDA-PAM hydrogel. It can be seen that HA-PDA-PAM hydrogel presents a uniform porous structure with uniform pore size distribution.

[0082] The infrared spectra of the samples were analyzed using a Fourier transform infrared spectrometer. The components of CS, DA, and AM samples were analyzed by tableting. During the test, potassium bromide and the sample were ground at a mass ratio of 100:1 and tableted using a tablet press. The components of PCL, PCL-CS, PDA-PAM, and HA-PDA-PAM samples were analyzed by total reflection method. The results are shown in Figure 2. Figure 6 and Figure 10 shown.

[0083] Figure 6 This is the infrared spectrum analysis of PCL, CS, and PCL-CS. In the infrared absorption spectrum of PCL, 1743 cm -1 represents the stretching vibration of C=O, 1178 cm -1 Represents the stretching vibration of CO, 2900 cm -1 and 2970cm -1 Represents CH stretching vibration. In the infrared absorption spectrum of CS, 2900cm -1 and 2950cm -1 represents CH stretching vibration, 3470 cm -1 Represents the stretching vibration of OH, 1616 cm -1 The NH bending vibration is represented by the IR spectrum of PCL-CS at 1616 cm -1 A new peak appears at , which is attributed to the bending vibration of NH in CS. This confirms the successful deposition of CS on PCL.

[0084] The infrared absorption peak of HA-PDA-PAM hydrogel was analyzed, and the results were as follows: Figure 10 As shown in the infrared absorption peak of DA, at 3200-3500 cm -1 The absorption peak at 3072 cm represents the stretching vibration of OH and NH. -1 The absorption peak at 1515 cm represents the stretching vibration of the benzene ring CH. -1 represents the vibration of the C=C skeleton of the benzene ring, 1299cm -1 represents CN stretching vibration, 1205cm -1 represents the stretching vibration of CO. In the infrared absorption peak of AM, 1690 cm -1 represents the stretching vibration of C=O, 1629cm -1 represents the stretching vibration of C=C, 1446cm -1 The NH bending vibration is represented by the NH in PDA-PAM. In PDA-PAM, due to the introduction of DA, the C=O of the amide group in AM may form a hydrogen bond with the amino or hydroxyl group in DA, resulting in a red shift of the C=O peak to a lower wave number, from 1690 cm -1becomes 1670cm -1 PDA-PAM compares DA and AM at 3200-3500cm -1 The changes in the intensity and morphology of the broad peak reflect the formation of a hydrogen bond network between dopamine and acrylamide. In PDA-PAM, the benzene ring may form a conjugated structure through oxidative self-polymerization during the formation of PDA. -1 The C=C skeleton vibration peak at 1620cm -1 The peak at 1064 cm is the NH deformation peak of the primary amine from PDA, indicating that dopamine is successfully polymerized in the system. -1 The peak at may originate from the CN bond in DA and PAM. Due to the synergistic effect of DA and AM, the vibration of the CN bond may enhance the absorption signal in this band.

[0085] The PCL-CS fiber membrane was subjected to rheological tests and characterized by the frequency sweep test mode under the oscillation mode of the rheometer. First, the hydrogel was made into a disc. In the experiment, the sample was placed at 25°C and a flat plate with a diameter of 30mm and a test spacing of 0.1mm was used to perform the test frequency sweep. The parameters were set to a fixed strain of 1% and a frequency of 0.1-10HZ. The changes in the storage modulus (G′) and loss modulus (G″) with strain were recorded. The results are shown in the figure. Figure 7 shown.

[0086] Figure 7 The rheological diagram of CS assembled with PCL membrane shows that the higher strain dependence test of storage modulus (G′) compared with loss modulus (G″) indicates that the assembled CS forms a gel state.

[0087] To explore the mechanical properties of different membranes, stress-strain measurements were performed on PCL, PCL-0.5% CS, PCL-1% CS, and PCL-2% CS deposited with 0, 0.5%, 1%, and 2% CS concentrations. The fiber membranes were made into strip membranes, where the fiber membranes needed to be sampled along the same direction as the PCL fiber orientation. The mechanical properties of the samples were measured using a Shimadzu EZ-LX universal testing machine with a maximum load cell of 50N and a speed of 5mm / min at room temperature. The results are shown in Figure 2. Figure 8 The elastic modulus is analyzed and the results are shown as follows. Figure 9 As shown in the figure, the elastic moduli of PCL, PCL-0.5% CS, PCL-1% CS and PCL-2% CS membranes are 29.43 MPa, 31.90 MPa, 37.77 MPa and 42.76 MPa, respectively. It can be seen that with the increase of CS deposition amount, the elastic modulus of PCL-CS membrane is gradually enhanced. This enhancement of mechanical properties is attributed to the establishment of micro-nano hierarchical fiber structure and the hydrogen bond interaction between PCL and CS.

[0088] XRD tests were performed on HA particles and HA-PDA-PAM to verify that the crystal structure was intact under the incorporation of HA. X-ray diffraction analysis of HA and HA-PDA-PAM was performed by X-ray diffraction. A Co target was scanned in the range of 10° to 90° at a scanning rate of 5° / min. Figure 11 As shown in the figure, HA has eight different diffraction peaks, corresponding to the (002), (211), (112), (300), (310), (222), (213), and (004) crystal planes of HA. The characteristic peaks are relatively sharp, reflecting its good crystallinity. After HA is doped into the hydrogel, the characteristic peaks of HA crystals are slightly weakened, indicating that the structure of HA is completely retained in the material.

[0089] PCL, PCL-0.5% CS, PCL-1% CS, and PCL-2% CS were cut into 5 mm x 5 mm diameter squares and placed in a 48-well plate. The front and back sides were sterilized by UV irradiation for 2 h. 500 μL PBS was added to each well to moisten the material for 24 h, and then the PBS was discarded. 1×10 4 Huvec cells were cultured and the cell activity was detected by CCK-8 kit on days 1, 3, and 5.

[0090] Before the assay, discard the original culture medium with a pipette and add 200 μL of culture medium containing 10% CCK-8 working solution to each well. Transfer the well plate to a cell culture incubator and incubate for 1 hour. Then, pipette 100 μL of solution into each well of a 96-well plate and measure the OD value of each well at 450 nm with a microplate reader. The results are as follows: Figure 12 shown.

[0091] like Figure 12 The Huvec cell proliferation activity of the PCL-CS fiber membrane with micro-nano multi-level structure is significantly better than that of the PCL fiber membrane. The deposition of CS can significantly promote cell activity, so that the PCL-CS fiber membrane can effectively promote the proliferation of Huvec cells, and PCL-1% CS has the strongest ability to promote proliferation.

[0092] Immunofluorescence staining was performed on cells cultured with extracts of PP, P-HP, CP-P, and CP-HP materials (the PP group had PCL as the upper layer and PDA-PAM as the lower layer; the CP-P group had PCL-CS as the upper layer and PDA-PAM as the lower layer; the P-HP group had PCL as the upper layer and HA-PDA-PAM as the lower layer; the CP-HP group had PCL-CS as the upper layer and HA-PDA-PAM as the lower layer). After being gently rinsed with PBS, the cells were fixed in 4% paraformaldehyde for 30 minutes, and then the cells were permeabilized with 0.1% TritonX-100 solution for 8 minutes. The cytoskeleton and cell nucleus were incubated in the dark with FITC-phalloidin for 1 hour and DAPI solution for 5 minutes, respectively. After washing the unstained reagent with PBS, the cells were imaged and observed under an inverted fluorescence microscope. The results are shown in Figure 2. Figure 13 shown.

[0093] like Figure 13 Immunofluorescence staining was used to observe the morphology and density of cells in the extracts of each sample. The number of MC3T3-E1 cells in the extracts of PP and CP-P materials was very small, and the cell spreading was poor. In the extracts of P-HP and CP-HP materials, the cells exhibited a typical spindle shape and spread well.

[0094] Twelve SD rats (8 weeks old) were randomly divided into 5 groups, with 3 rats in each group: CON group, PP group, CP-P group, P-HP group, and CP-HP group. The skin and fascia were incised to expose the skull, and a 5 mm hole was drilled in the skull with a dental drill and covered with materials. SD rats were euthanized 4 and 8 weeks after surgery. After sacrifice, their skull specimens were collected and fixed with 4% paraformaldehyde for later use. Before scanning, the materials were removed from the 4% paraformaldehyde and the residual paraformaldehyde on the surface was carefully wiped off. The materials were fixed with a Micro-CT holder and then scanned to evaluate the repair of the skull defect area in SD rats.

[0095] Figure 14Micro-CT 3D reconstructions of skull bone regeneration in the CON, PP, CP-P, P-HP, and CP-HP groups at 4 and 8 weeks postoperatively are shown. Compared to the 4-week results, the defect area covered by newly formed bone tissue gradually increased in all groups at 8 weeks, indicating new bone growth over time. Bone regeneration was superior to that observed in the blank control group in all implanted materials, with the newly formed bone filling the defect uniformly from the defect edge toward the center. In the blank control group, although there was a slight increase in newly formed bone from 4 to 8 weeks, the amount of newly formed bone was limited, occurring only at the defect edge. Bone repair was poor in the PP and CP-P groups, with newly formed bone appearing only at the defect edge. The P-HP and CP-HP groups demonstrated significantly superior bone repair to the blank, PP, and CP-P groups, as demonstrated by greater newly formed bone coverage and more mature bone tissue. At 8 weeks, mature, high-density callus tissue was observed from the defect edge to the near-center in both the P-HP and CP-HP groups, demonstrating excellent skull defect repair.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a biomimetic periosteum gel double-layer scaffold, characterized in that: The following steps are involved: S1, electrospinning a polycaprolactone solution to obtain an electrospun membrane; S2, mixing the electrospun membrane with an alkaline solution of a polysaccharide polymer material for 10 to 20 minutes, then transferring the mixture to an ethanol aqueous solution, mixing and contacting for 0.5 to 2 hours, to obtain a self-assembled product; S3, dispersing dopamine in an alkaline buffer solution and performing a prepolymerization treatment to obtain a dopamine dispersion; S4, mixing the dopamine dispersion, hydroxyapatite, acrylamide, ammonium persulfate, N,N-methylenebisacrylamide and tetramethylethylenediamine, and curing to obtain a hydrogel; S5. Placing the self-assembled product on the upper layer of the hydrogel for combination to obtain a biomimetic periosteum gel double-layer scaffold.

2. The method for preparing the biomimetic periosteum gel double-layer scaffold according to claim 1, characterized in that: The solute of the alkaline solution of polysaccharide polymer material is selected from one or more of cellulose, chitin, chitosan or sodium alginate.

3. The method for preparing the biomimetic periosteum gel double-layer scaffold according to claim 1, characterized in that: In S1, the solvent used in the polycaprolactone solution is hexafluoroisopropanol; The concentration of the polycaprolactone solution is 0.1 to 0.2 g / mL; The polycaprolactone solution is magnetically stirred for 10 to 14 hours before the electrospinning treatment; In the electrospinning process, the spinning voltage is 15 to 20 kV, and the flow rate of the injection pump is 0.001 to 0.005 mm / s.

4. The method for preparing the biomimetic periosteum gel double-layer scaffold according to claim 1, characterized in that: In S2, the solvent used in the alkaline solution of the polysaccharide polymer material contains LiOH, KOH, urea and water; The mass ratio of LiOH, KOH, urea and water is 4-5:6-8:7-9:75-85.

5. The method for preparing the biomimetic periosteum gel double-layer scaffold according to claim 1, characterized in that: In S2, the temperature of the ethanol aqueous solution is -20°C to 5°C; The volume percentage concentration of the ethanol aqueous solution is 70-90%.

6. The method for preparing the biomimetic periosteum gel double-layer scaffold according to claim 1, characterized in that: The concentration of the dopamine dispersion is 1-2 mg / mL.

7. The method for preparing the biomimetic periosteum gel double-layer scaffold according to claim 6, characterized in that: In S3, the pH value of the alkaline buffer solution is 8 to 9; The alkaline buffer solution is Tris-HCl solution; The prepolymerization treatment time is 20 to 40 minutes.

8. The method for preparing the biomimetic periosteum gel double-layer scaffold according to claim 1, characterized in that: In S4, the volume ratio of the dopamine dispersion, the mass of hydroxyapatite, the mass of acrylamide, ammonium persulfate, the mass of N,N-methylenebisacrylamide and the volume of tetramethylethylenediamine is 5-15 mL:400-600 mg:2-3 g:100-300 mg:10-30 mg:10-30 μL.

9. A biomimetic periosteum gel double-layer scaffold prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the biomimetic periosteum gel double-layer scaffold according to claim 9 in preparing bone defect repair materials.

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