Barrier membrane for guided bone regeneration and method for its production

Abstract

This invention discloses a barrier membrane for guiding bone regeneration and its preparation method, relating to the field of materials technology for guiding bone regeneration. The barrier membrane comprises a stacked nanowire functional layer and a barrier layer, wherein the nanowire functional layer includes a Cu-loaded surface. 2+ Sr 2+ The barrier membrane, doped with hydroxyapatite nanowires, comprises polylactic acid as the barrier layer. This invention provides a barrier membrane that combines sequential ion release, directional ion release, immune regulation, angiogenesis promotion, osteoproliferation, and antibacterial adhesion functions, adapting to the stage-specific needs of bone repair and guiding bone regeneration. This invention effectively solves the problems of existing barrier membranes, which only provide physical barrier function, lack biological activity, are difficult to actively promote bone regeneration, have a single mode of active metal ion release, are difficult to achieve sequential regulation, have a high risk of postoperative infection, exhibit disordered diffusion of active components, and have limited functionality.

Description

Technical Field

[0001] This invention relates to the field of materials for guiding bone regeneration, and in particular to a barrier membrane for guiding bone regeneration and its preparation method. Background Technology

[0002] Currently, in the fields of dental implants and bone defect repair, barrier membranes, also known as guided bone regeneration membranes, are commonly used in clinical practice to cover bone defect areas. On the one hand, the barrier membrane prevents epithelial cells and connective tissue from prematurely ingrowing into the bone defect area, maintaining regeneration space; on the other hand, the barrier membrane provides a relatively stable microenvironment for the bone defect area, which is conducive to the migration, attachment, and tissue regeneration of osteoblast-related cells.

[0003] Currently, commonly used barrier membranes in clinical practice include imported BIO-GIDE membranes and domestically produced Hai'ao membranes. These products are primarily collagen-based barrier membranes, typically derived from porcine collagen. Their main functions are physical isolation and space maintenance, which can meet the needs of guided bone regeneration therapy to a certain extent. In addition, some existing studies have constructed barrier membranes with certain osteoinductive capabilities by introducing components such as polylactic acid into the membrane material; other studies have attempted to enhance the angiogenesis, immunomodulation, or osteogenic properties of the material by doping it with metal ions. However, most existing barrier membranes suffer from the following drawbacks: (1) Existing clinical barrier membranes are expensive. Currently, the commonly used imported BIO-GIDE membrane and some domestic collagen membranes are expensive. The material cost for bone augmentation treatment of a single tooth can usually reach 1,000 to 2,000 yuan, which increases the treatment cost for patients and is not conducive to its widespread application.

[0004] (2) Existing barrier membranes mainly function as physical barriers, lacking sufficient biological activity and failing to meet the stage-specific needs of the bone repair process. Most collagen membranes currently used clinically primarily block soft tissue ingrowth and maintain space, lacking the osteogenic induction, immune regulation, and angiogenesis functions required to promote bone regeneration, making it difficult to actively regulate the bone defect microenvironment. Bone repair is a dynamic process with a clear temporal sequence, requiring antibacterial, immune regulation, and angiogenesis in the early stages, and continuous promotion of osteogenic differentiation and bone tissue reconstruction in the middle and later stages. However, most existing materials struggle to achieve the staged and programmed release of different active components, resulting in a mismatch between biological effects and the rhythm of bone repair.

[0005] (3) Insufficient control of infection risk. The surface of implanted materials can easily become a carrier for bacterial adhesion and biofilm formation. Traditional barrier membranes usually do not have significant antibacterial adhesion ability, which can easily increase the risk of postoperative infection. Even if some materials have antibacterial components, they are mostly focused on bactericidal release and are difficult to simultaneously inhibit early bacterial adhesion and subsequent biofilm formation.

[0006] (4) Most existing membrane materials cannot achieve the directional effect of active ingredients on the bone defect side.

[0007] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the present invention aims to provide a barrier membrane for guiding bone regeneration and a method for preparing the same, and to provide a double-layer barrier membrane for guiding bone regeneration that has the functions of sequential ion release, directional ion release, immune regulation, angiogenesis promotion, osteoproliferation and antibacterial adhesion.

[0009] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a barrier film, wherein the barrier film comprises a nanowire functional layer and a barrier layer stacked thereon, the nanowire functional layer comprising a Cu-coated surface. 2+ Sr 2+ Doped hydroxyapatite nanowires; The barrier layer comprises polylactic acid.

[0010] A second aspect of the present invention provides a method for preparing the barrier membrane as described above, comprising the following steps: Preparation of Sr 2+ Doped hydroxyapatite nanowires; The Sr 2+ Hydroxyapatite nanowires were doped to form a film, resulting in a nanowire film. A polylactic acid solution was coated on one side of the nanowire film, and after drying and pressing, a bilayer composite film was obtained. The barrier membrane is obtained by immersing the bilayer composite membrane in a copper salt solution for a predetermined time.

[0011] Optionally, the Sr 2+ The preparation method of doped hydroxyapatite nanowires includes the following steps: Sodium oleate, calcium salt, sodium dihydrogen phosphate, strontium salt, and water are mixed to obtain a mixture. The mixture was subjected to a hydrothermal reaction to obtain the Sr. 2+ Doped hydroxyapatite nanowires.

[0012] Optionally, the molar number of the strontium salt accounts for 2% to 10% of the total molar number of the strontium salt and the calcium salt.

[0013] Optionally, the molar ratio of sodium oleate, calcium salt and sodium dihydrogen phosphate is (5~5.5):1:(1~1.5).

[0014] Optionally, the calcium salt includes at least one of calcium chloride and calcium nitrate, and the strontium salt includes at least one of strontium chloride and strontium nitrate.

[0015] Optionally, the hydrothermal reaction temperature is 200 °C, and the hydrothermal reaction time is 40 h.

[0016] Optionally, the solvent for the polylactic acid solution may include dichloroethane.

[0017] Optionally, the concentration of the copper salt solution is 10 μM, and the preset time is 3~5 min.

[0018] Optionally, the copper salt includes at least one of copper chloride and copper nitrate.

[0019] Beneficial effects: The barrier membrane provided by this invention has the following advantages: (1) It has a time-sequential ion release function, which is more in line with the biological laws of bone repair.

[0020] This invention utilizes Cu 2+ Surface load and Sr 2+ Combination design of doping to achieve Cu 2+ Early rapid release and Sr 2+ The sustained release in the later stages allows it to act on the early antibacterial / angiogenic phase of bone repair and the mid-to-late bone-promoting phase, which is superior to existing single-release materials.

[0021] (2) It has a double-layer directional release structure, which can improve the efficiency of local action.

[0022] This invention employs a dual-layer structure of a functional layer and a barrier layer. When applied, the nanowire functional layer faces the bone defect area, while the barrier layer faces the soft tissue side. This allows active ions to be preferentially released towards the bone defect area, while the barrier layer prevents soft tissue invasion and reduces disordered ion leakage, thus enabling a more effective local bone repair promotion effect.

[0023] (3) It has both antibacterial adhesion and bactericidal functions, making infection control more comprehensive.

[0024] Most existing barrier membranes only provide physical barriers and lack anti-infection design. The nanowire surface structure in this invention can reduce bacterial adhesion and biofilm formation, and the early release of Cu... 2+ It can also further kill free bacteria, thereby reducing the risk of implantation infection.

[0025] (4) It can achieve multifunctional synergy of immune regulation, angiogenesis and osteoporosis.

[0026] The barrier membrane provided by this invention not only maintains the regeneration space, but also allows Cu to pass through. 2+ and Sr 2+ It synergistically regulates macrophage polarization, angiogenesis, and osteogenic differentiation, overcoming the limitation of existing membrane materials having only one function.

[0027] (5) It is expected to reduce the overall cost of clinical treatment.

[0028] Compared with expensive imported collagen membranes, this invention has the potential to obtain a novel alternative material with both functionality and cost advantages through controlled synthesis and composite preparation.

[0029] (6) The raw materials are widely available, the preparation process is feasible, and there is potential for conversion.

[0030] This invention mainly uses hydroxyapatite, polylactic acid and common inorganic salt raw materials, and can be completed through processes such as filtration, coating and soaking loading, and has the foundation for further industrial application.

[0031] In summary, the barrier membrane provided by this invention possesses sequential ion release, targeted ion release, immune regulation, angiogenesis promotion, osteoproliferation, and antibacterial adhesion functions, enabling it to adapt to the stage-specific needs of bone repair and guide bone regeneration. This invention effectively solves the problems of existing barrier membranes, which only provide physical barrier function, lack biological activity, are difficult to actively promote bone regeneration, have a single mode of active metal ion release, are difficult to achieve sequential regulation, have a high risk of postoperative infection, exhibit disordered diffusion of active components, and have limited functionality. Attached Figure Description

[0032] Figure 1 The figures show the morphological characterization results of HAp NWs and Sr-HAp NWs, where a is the SEM image of HAp NWs; b is the TEM image of HAp NWs; c is the HRTEM image of HAp NWs; d is the SEM image of Sr-HAp NWs; e is the TEM image of Sr-HAp NWs; and f is the HRTEM image of Sr-HAp NWs.

[0033] Figure 2 Figures show the composition and infrared spectral results of different nanowires. a) shows the XRD patterns of Hap NWs and Sr-HAp NWs; b) shows the FTIR results of Hap NWs and Sr-HAp NWs; c) shows the EDS scan of Cu & Sr-HAp NWs; d) shows the total XPS spectrum of Cu & Sr-HAp NWs; e) shows the XPS spectrum of phosphorus in Cu & Sr-HAp NWs; f) shows the XPS spectrum of calcium in Cu & Sr-HAp NWs; g) shows the XPS spectrum of strontium in Cu & Sr-HAp NWs; and h) shows the XPS spectrum of copper in Cu & Sr-HAp NWs.

[0034] Figure 3Figures show the morphology, hydrophilicity / hydrophobicity, and composition characterization results of the CSHP barrier membrane. Among them, a is the SEM image of the Cu&Sr-HAp NWs side of the CSHP barrier membrane; b is the SEM image of the PLA side of the CSHP barrier membrane; c is the contact angle image of both sides of the CSHP barrier membrane; d is the quantitative result of the contact angle of both sides of the CSHP barrier membrane; and e is the EDS surface scan result of the Cu&Sr-HAp NWs side of the CSHP barrier membrane.

[0035] Figure 4 Figures show the test results of the mechanical properties and ion release performance of the CSHP barrier membrane. Specifically, a) is the force-displacement curve of the CSHP barrier membrane; b) is the EDS surface scan result of the cross-section of the CSHP barrier membrane; and c) is the Cu content in the CSHP barrier membrane. 2+ Release characteristic curve; d represents Sr in the CSHP barrier membrane. 2+ Release characteristic curve.

[0036] Figure 5 Figure 1 shows the results of cell compatibility testing of the barrier membrane. Figure 2(a) shows the survival of HUVECs after 24 h of co-culturing with different barrier membrane extracts using the live / dead cell staining method; Figure 3(b) shows the survival of BMSCs after 24 h of co-culturing with different barrier membrane extracts using the live / dead cell staining method; Figure 4(c) shows the changes in cell proliferation capacity of HUVECs after 1, 3, and 5 days of co-culturing with different barrier membrane extracts using the CCK-8 assay. Indicates comparison with the Ctrl group P <0.05; d is the result of CCK-8 assay to evaluate the changes in cell proliferation capacity of BMSCs after co-culturing with different barrier membrane extracts for 1, 3 and 5 days. Indicates comparison with the Ctrl group P <0.001.

[0037] Figure 6 The images show the effects of different barrier membranes on antibacterial adhesion and antibiofilm formation. In the images, a is the crystal violet staining result; b is the SEM image of bacteria colonizing and forming biofilms on different barrier membrane surfaces; and c is the three-dimensional image of biofilms on different barrier membrane surfaces.

[0038] Figure 7 The figure shows the results of a study on the mechanism of barrier membrane antibacterial adhesion. Figure a shows a schematic diagram of an experiment using biomechanical atomic force microscopy to evaluate the interaction between a single live bacterium and the barrier membrane surface; figure b shows... S. aureus Force-distance curves of interaction between bacteria and different barrier membrane surfaces; c is a statistical graph of the magnitude of adhesion forces between bacteria and different barrier membrane surfaces. Indicates comparison with glass plate group P<0.001; d is a statistical graph of the average surface roughness values ​​of the HAp plate and Hap NWs membrane; e is a three-dimensional image of the surface roughness of the HAp plate and Hap NWs membrane.

[0039] Figure 8 This is a diagram illustrating the bactericidal effect of the barrier membrane, where 'a' represents the effect against... S. aureus Figure a shows the results of the plate coating experiment; Figure b is a statistical graph of the colony data corresponding to Figure a. Indicates comparison with HP group P <0.01; c is for E. coli Figure 1 shows the results of the plate coating experiment; Figure 2 (d) is the colony data statistics corresponding to Figure 3 (c). Indicates comparison with HP group P <0.05.

[0040] Figure 9 The images show the effect of the barrier membrane promoting bone regeneration. Image a is a Micro-CT reconstructed image of the mandibular bone defect; image b is a quantitative analysis of BV / TV and Tb.Th from the Micro-CT results. Indicates comparison with the Ctrl group P <0.01, Indicates comparison with the Ctrl group P <0.001.

[0041] Figure 10 The results show the effects of the barrier membrane on early bone healing and osteoclastosis. In this diagram, a is the H&E staining result of the bone defect area; and b is the TRAP staining result of the bone defect area.

[0042] Figure 11 This image shows the in vivo results of a study on the regulation of macrophage polarization by the bone barrier membrane. Image a shows immunohistochemical staining of iNOS expression in the bone defect region; image b shows immunohistochemical staining of TNF-α expression in the bone defect region; and image c shows the semi-quantitative analysis of iNOS and TNF-α expression levels using immunohistochemical staining. Indicates comparison with the Ctrl group P <0.01, Indicates comparison with the Ctrl group P <0.001; d is an IF staining map of CD86 and CD206 expression in the bone defect area.

[0043] Figure 12 The figures show the in vivo results of barrier membrane angiogenesis studies. Figure a shows the immunohistochemical staining of PDGF expression in the bone defect area; figure b shows the immunohistochemical staining of VEGF expression in the bone defect area; and figure c shows the semi-quantitative analysis of PDGF and VEGF expression levels based on the immunohistochemical staining results. Indicates comparison with the Ctrl groupP <0.05, Indicates comparison with the Ctrl group P <0.001; d is an IF staining map of CD31 expression in the bone defect area.

[0044] Figure 13 The figures show the in vivo assessment results of the biocompatibility of the barrier membrane. Figure a shows the H&E staining of the heart, liver, spleen, lungs, and kidneys; figure b shows the absorbance values ​​of the hemolysis test supernatant. Indicates comparison with the negative control group P <0.001; c is the blood image after the hemolysis experiment. Detailed Implementation

[0045] This invention provides a barrier membrane for guiding bone regeneration and a method for preparing the same. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0047] This invention provides a barrier film comprising a stacked nanowire functional layer and a barrier layer, wherein the nanowire functional layer includes Cu loaded on its surface (i.e., on the surface and in the surface layer). 2+ Sr 2+ Doped hydroxyapatite nanowires; The barrier layer comprises polylactic acid.

[0048] The barrier membrane provided by this invention has the following advantages: (1) It has a time-sequential ion release function, which is more in line with the biological laws of bone repair.

[0049] This invention utilizes Cu 2+ Surface load and Sr 2+ Combination design of doping to achieve Cu 2+ Early rapid release and Sr 2+ The sustained release in the later stages allows it to act on the early antibacterial / angiogenic phase of bone repair and the mid-to-late bone-promoting phase, which is superior to existing single-release materials.

[0050] (2) It has a double-layer directional release structure, which can improve the efficiency of local action.

[0051] This invention employs a dual-layer structure of a functional layer and a barrier layer. When applied, the nanowire functional layer faces the bone defect area, while the barrier layer faces the soft tissue side. This allows active ions to be preferentially released towards the bone defect area, while the barrier layer prevents soft tissue invasion and reduces disordered ion leakage, thus enabling a more effective local bone repair promotion effect.

[0052] (3) It has both antibacterial adhesion and bactericidal functions, making infection control more comprehensive.

[0053] Most existing barrier membranes only provide physical barriers and lack anti-infection design. The nanowire surface structure in this invention can reduce bacterial adhesion and biofilm formation, and the early release of Cu... 2+ It can also further kill free bacteria, thereby reducing the risk of implantation infection.

[0054] (4) It can achieve multifunctional synergy of immune regulation, angiogenesis and osteoporosis.

[0055] The barrier membrane provided by this invention not only maintains the regeneration space, but also allows Cu² to pass through. + and Sr² + It synergistically regulates macrophage polarization, angiogenesis, and osteogenic differentiation (not only promoting new bone formation in the early stages of bone regeneration, but also effectively inhibiting osteoclast activity), breaking through the limitation of the single function of existing membrane materials.

[0056] (5) It is expected to reduce the overall cost of clinical treatment.

[0057] Compared with expensive imported collagen membranes, this invention has the potential to obtain a novel alternative material with both functionality and cost advantages through controlled synthesis and composite preparation.

[0058] (6) The raw materials are widely available, the preparation process is feasible, and there is potential for conversion.

[0059] This invention mainly uses hydroxyapatite, polylactic acid and common inorganic salt raw materials, and can be completed through processes such as filtration, coating and soaking loading, and has the foundation for further industrial application.

[0060] In summary, the barrier membrane provided by this invention possesses sequential ion release, targeted ion release, immune regulation, angiogenesis promotion, osteoproliferation, and antibacterial adhesion functions, along with good biocompatibility. It can adapt to the stage-specific needs of bone repair and can be used to guide bone regeneration. This invention effectively solves the problems of existing barrier membranes, which only have a physical barrier function, lack biological activity, are difficult to actively promote bone regeneration, have a single mode of releasing active metal ions, are difficult to achieve sequential regulation, have a high risk of postoperative infection, exhibit disordered diffusion of active components, and have limited functionality.

[0061] Specifically, in this invention, Sr 2+ In doped hydroxyapatite nanowires, Sr 2+ Doping into the hydroxyapatite nanowire lattice, that is, Sr 2+ Doped hydroxyapatite nanowires by using Sr 2+ It is formed by doping into the lattice of hydroxyapatite nanowires.

[0062] In this invention, on the one hand, Cu 2+ Adsorbed in Sr in the form of copper salt 2+ On the surface of doped hydroxyapatite nanowires. On the other hand, Cu... 2+ It can also be used with Sr 2+ Sr in doped hydroxyapatite nanowires 2+ and Ca 2+ Exchange, and with Sr 2+ Anionic bonding in doped hydroxyapatite nanowires.

[0063] This invention also provides a method for preparing the barrier membrane as described above, comprising the following steps: S1, Preparation of Sr 2+ Doped hydroxyapatite nanowires; S2, the Sr 2+ Hydroxyapatite nanowires were doped to form a film, resulting in a nanowire film. S3. Coat one side of the nanowire film with polylactic acid solution, and after drying and pressing, obtain a bilayer composite film. S4. After immersing the double-layer composite membrane in a copper salt solution for a preset time, the barrier membrane is obtained.

[0064] The preparation method provided by this invention is simple and mainly uses hydroxyapatite, polylactic acid and common inorganic salt raw materials. It can be completed through processes such as filtration, coating and soaking loading, and has the foundation for further industrial application.

[0065] The barrier membrane prepared by the preparation method provided by the present invention has the functions of sequential ion release, directional ion release, immune regulation, angiogenesis promotion, osteoproliferation and antibacterial adhesion, which can adapt to the stage requirements of bone repair and can be used to guide bone regeneration.

[0066] In step S1, in some embodiments, the Sr 2+ The preparation method of doped hydroxyapatite nanowires includes the following steps: S11. Sodium oleate, calcium salt, sodium dihydrogen phosphate, strontium salt and water are mixed to obtain a mixture; S12. The mixture is subjected to a hydrothermal reaction to obtain the Sr. 2+ Doped hydroxyapatite nanowires.

[0067] This invention prepares Sr via a hydrothermal reaction. 2+ The process of doping hydroxyapatite nanowires is simple and easy to operate.

[0068] In step S11, in some embodiments, the molar number of the strontium salt accounts for 2% to 10% of the total molar number of the strontium salt and the calcium salt, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, etc.

[0069] In some embodiments, the molar ratio of sodium oleate, calcium salt, and sodium dihydrogen phosphate is (5~5.5):1:(1~1.5), for example, it can be 5:1:1, 5:1:1.3, 5:1:1.5, 5.2:1:1, 5.2:1:1.3, 5.2:1:1.5, 5.5:1:1, 5.5:1:1.3, or 5.5:1:1.5, etc.

[0070] In some embodiments, the ratio of sodium oleate to water is 2.4 g: 70 mL.

[0071] In some embodiments, the calcium salt includes at least one of calcium chloride and calcium nitrate, and the strontium salt includes at least one of strontium chloride and strontium nitrate.

[0072] In step S11, sodium oleate and calcium salt can be added to double-distilled water and then mixed; then, sodium dihydrogen phosphate can be added to the above mixed system after double-distillation to obtain a mixed solution.

[0073] In step S12, in some embodiments, the temperature of the hydrothermal reaction is 200 °C and the time of the hydrothermal reaction is 40 h.

[0074] In step S3, in some embodiments, the solvent of the polylactic acid solution includes dichloroethane.

[0075] In step S4, in some embodiments, the concentration of the copper salt solution is 10 μM, and the preset time is 3~5 min.

[0076] In some embodiments, the copper salt includes at least one of copper chloride and copper nitrate.

[0077] The present invention will be further described below through specific embodiments.

[0078] Example 1 Preparation of hydroxyapatite nanowires (HAp NWs): 2.4 g of sodium oleate was dissolved in 25 mL of double-distilled water by heating. 0.222 g (1.5 mmol) of CaCl₂·2H₂O was dissolved in 20 mL of double-distilled water. The two solutions were then mixed and stirred at room temperature for 1 h to obtain a mixture. 0.28 g of NaH₂PO₄·H₂O was dissolved in 25 mL of double-distilled water and added to the mixture. Stirring continued at room temperature for 30 min, followed by a hydrothermal reaction at 200 °C for 40 h. After cooling to room temperature, the product was dispersed in anhydrous ethanol, washed five times with anhydrous ethanol, and collected by centrifugation to obtain HAp NWs.

[0079] Sr 2+ Preparation of doped hydroxyapatite nanowires (Sr-HAp NWs): The only difference from the above-mentioned HAp NWs preparation method is that SrCl2·6H2O is added to replace 5% (molar percentage) of CaCl2·2H2O (i.e., 0.075 mmol of SrCl2·6H2O and 1.425 mmol of CaCl2·2H2O are added).

[0080] Preparation of barrier membranes: The obtained HAp NWs and Sr-HAp NWs were vacuum filtered under negative pressure, evaporated and dried to form membranes, which were denoted as Hap NWs membrane and Sr-Hap NWs membrane, respectively.

[0081] A polylactic acid (PLA) dichloroethane solution (PLA concentration of 0.1 g / mL) was coated onto one side of a Hap NWs membrane and a Sr-Hap NWs membrane (PLA dichloroethane solution was applied at a rate of 2 mL per square centimeter of membrane), and then evaporated and dried. The membranes were then compacted using a hydraulic press to obtain HAp / PLA barrier membranes (denoted as HP barrier membranes) and Sr-HAp / PLA barrier membranes (denoted as SHP barrier membranes), respectively.

[0082] The SHP barrier membrane was immersed in a CuCl2 aqueous solution (CuCl2 concentration of 10 μM) for 5 min to obtain Cu 2+ The membrane is loaded, then evaporated and dried to obtain a CSHP barrier membrane. All barrier membranes are sterilized with ethylene oxide before use to ensure sterility.

[0083] In summary, the HP barrier membrane consists of a PLA layer and a HAp NWs layer.

[0084] The SHP barrier membrane consists of a PLA layer and Sr.2+ It is composed of a doped HAp NWs layer.

[0085] The CSHP barrier film consists of a PLA layer and a Cu-loaded surface layer. 2+ Sr 2+ Composed of a doped HAp NWs layer, Cu 2+ Sr 2+ Doped HAp NWs are denoted as Cu&Sr-HAp NWs.

[0086] test: (1) Morphological and compositional characterization of HAp NWs, Sr-HAp NWs, and Cu&Sr-HAp NWs (11) Results of scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM): such as Figure 1 As shown in a, b, and c, HAp NWs exhibit a fibrous structure, reaching lengths of hundreds of micrometers and diameters of approximately 5 nm. In... Figure 1 In the HRTEM image shown in c, clear and regular lattice fringes can be observed, with a crystal plane spacing of 0.34 nm. This lattice spacing is consistent with the literature reports, indicating that the prepared HAp NWs have a good crystal structure.

[0087] like Figure 1 As shown in d, e, and f, the overall morphology of Sr-HAp NWs is consistent with that of HAp NWs, exhibiting fibrous characteristics with lengths reaching hundreds of micrometers. The diameter of Sr-HAp NWs is approximately 5 nm. Figure 1 In the HRTEM image shown in f, clear lattice fringes of Sr-HAp NWs can be observed, with a crystal plane spacing of 0.34 nm, indicating that Sr... 2+ The doping did not significantly affect the crystal structure of hydroxyapatite.

[0088] (12) X-ray diffraction (XRD) test results: such as Figure 2 As shown in Figure a, the HAp NWs spectrum exhibits typical hydroxyapatite diffraction peaks (JCPDS number 09-0432), with no impurity peaks detected, indicating that the prepared HAp NWs are a single pure phase. Some diffraction peaks of the Sr-HAp NWs show slight broadening, reflecting a slight decrease in crystallinity. This change is consistent with that of Sr... 2+ The ionic radius (1.13 Å) is greater than that of Ca. 2+ (0.99 Å) related to Sr 2+ It was incorporated into the lattice of HAp NWs.

[0089] (13) Fourier transform infrared spectroscopy (FTIR) test results: such as Figure 2As shown in b, both HAp NWs and Sr-HApNWs nanowires have a diameter of 1100 cm. -1 and 3573 cm -1 PO4 was detected at the location 3- Typical absorption peaks associated with -OH groups were observed. Furthermore, no characteristic absorption peaks of residual oleic acid were observed in the sample, indicating that the nanowire surface is clean.

[0090] (14) In order to further analyze Cu 2+ The elemental distribution and doping status of the adsorbed nanowires were characterized by energy-dispersive spectroscopy (EDS) for Cu&Sr-HAp NWs. The results are as follows: Figure 2 As shown in c, it can be seen that various elements are uniformly distributed in the nanowires, indicating that Sr 2+ doping and Cu 2+ The adsorption process is uniform and controllable.

[0091] (15) The valence states of the elements in the nanowires were further analyzed by X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 2 As shown in d, e, f, g, and h, it can be seen that in the Ca 2p spectrum, Ca 2p 3 / 2 The peak value is around 347 eV, and it is similar to Ca 2p 1 / 2 The energy difference of approximately 1.6 eV confirms that Ca exists in the nanowire in the +2 valence state. In the Sr 3p spectrum, the characteristic peaks at 269 eV and 279 eV correspond to Sr 3p... 3 / 2 and Sr 3p 1 / 2 The two peaks confirm that Sr also exists in the +2 valence state in the nanowires. In the Cu 2p spectrum, the characteristic peaks at 954.4 eV and 934.7 eV correspond to Cu 2p... 1 / 2 and Cu 2p 3 / 2 This confirms that Cu exists in the +2 valence state on the nanowire surface. The stable valence state of these elements indicates that the doping and adsorption processes do not impair their biological activity.

[0092] (2) Morphology and composition characterization of CSHP barrier membrane (21) SEM test results: such as Figure 3 Figures a and b show that the two sides of the CSHP barrier membrane exhibit significant differences in microstructure. The Cu&Sr-HAp NWs side has a rougher surface, while the PLA side is smooth and flat.

[0093] (22) Contact angle measurement results: such as Figure 3As shown in figures c and d, there is a significant difference in hydrophilicity and hydrophobicity on both sides of the CSHP barrier membrane. The Cu&Sr-HAp NWs side has a smaller contact angle (<20°), indicating its higher hydrophilicity. In contrast, the PLA side has a larger contact angle (approximately 70°), showing its relatively hydrophobic characteristics. This contrast between hydrophilicity and hydrophobicity further reveals the bifunctional interface properties of the CSHP barrier membrane.

[0094] (23) EDS surface scan results of the Cu&Sr-HAp NWs side of the CSHP barrier membrane: as follows Figure 3 As shown in 'e', ​​the uniform distribution of Ca, Sr, Cu, and P elements indicates that the doping and adsorption processes of the nanowires are well controllable, laying the foundation for the functionalization of the material.

[0095] (3) Mechanical properties and ion release performance tests of CSHP barrier membrane (31) The barrier membrane is subjected to a tensile test using a universal testing machine. For example... Figure 4 The force-displacement curve results shown in Figure 'a' confirm that the CSHP barrier membrane possesses good mechanical properties, which are expected to meet the requirements for its space retention effect. Figure 4 As shown in b, the EDS surface scan results of the CSHP barrier film cross-section reveal a non-uniform distribution of C, with overlapping regions between C and Cu, Sr, Ca, and P elements. This indicates that PLA has penetrated into the nanowires and formed a transition layer with clear boundaries. This structure ensures a strong bond between the PLA layer and the nanowire layer, contributing to enhanced mechanical stability of the barrier film.

[0096] (32) The ion release characteristics of the CSHP barrier membrane were determined by inductively coupled plasma mass spectrometry (ICP-MS). A 1 cm³ sample was used. 2 The CSHP barrier membrane was placed in 10 mL of PBS buffer and shaken on a shaker at room temperature. The ion concentration in the PBS buffer was measured to characterize the amount of ions released from the CSHP barrier membrane at different times. The results are as follows: Figure 4 As shown in c and d, the results indicate that Cu 2+ The release was completed rapidly within the first four days, exhibiting a clear initial burst release pattern; while Sr 2+ It exhibits a sustained-release characteristic, with release remaining at a high level even on day 14. This phased ion release behavior is related to the difference in binding strength between the two ions in the nanowires, which can better adapt to the dynamic needs during bone repair.

[0097] (4) Assessment of barrier membrane cell compatibility BMSCs and HUVECs were selected as cell models to evaluate the cell compatibility of barrier membranes (HP barrier membrane, SHP barrier membrane and CSHP barrier membrane, respectively, denoted as HP group, SHP group and CSHP group).

[0098] The sterilized barrier membrane was placed in a 6-well culture plate and immersed in α-MEM medium (the ratio of barrier membrane area to medium volume was 3 μm). 2 The extract was collected at 37°C and placed in a constant temperature cell culture incubator. After 24 h, the extract was collected and filtered using a 0.22 μm sterilizer. It was then frozen at -80°C for later use. The extraction of the extract was performed according to the international standard (ISO-10993-12). A control group without a barrier membrane (i.e., the Ctrl group) was also included.

[0099] Different barrier membrane extracts were co-cultured with BMSCs and HUVECs for 24 h, respectively, followed by live / dead cell staining. The results are as follows: Figure 5 As shown in figures a and b, the number of viable cells in the HP, SHP, and CSHP groups was comparable to that in the Ctrl group, and no obvious dead cells were observed. This result indicates that the barrier membrane does not produce cytotoxicity to HUVECs and BMSCs.

[0100] To further evaluate the effect of the cell barrier membrane on cell proliferation, the CCK-8 assay was used to detect cell proliferation after co-culturing for 1, 3, and 5 days. The results are as follows: Figure 5 As shown in c and d, it can be seen that on days 1 and 3, the proliferation activity of HUVECs in the CSHP group was significantly higher than that in other treatment groups. P <0.05, Figure 5 (c); On day 1, the proliferation activity of BMSCs in the CSHP group was also significantly higher than that in other treatment groups ( P <0.001, Figure 5 (d in the figure), which indicates that the CSHP barrier membrane can significantly promote cell proliferation.

[0101] The test results above show that the CSHP barrier membrane exhibits good cell compatibility and promotes the proliferation of BMSCs and HUVECs.

[0102] (5) Effects of antibacterial adhesion and antibiofilm formation To clarify the antibacterial adhesion and antibacterial biofilm formation effects of the barrier membranes, different barrier membranes were co-cultured with bacterial solutions for 3 days, and the results were verified by crystal violet staining. Figure 6As shown in Figure a, the results indicate that significant bacterial biofilm formation was observed on both glass and HAp plates. However, the three barrier membranes based on nanowire structures (HP barrier membrane, SHP barrier membrane, and CSHP barrier membrane) exhibited significant anti-bacterial biofilm formation effects, with almost no biofilm observed on their surfaces.

[0103] Different barrier membranes were co-cultured with bacterial suspensions for 12 h and 3 days, respectively, and then subjected to SEM analysis. The results are as follows: Figure 6 As shown in b, after 12 h of co-culturing with bacterial solution, a large number of bacteria adhered to the surfaces of the glass plate and HAp plate; by day 3 of culture, complex biofilm structures had formed on the surfaces of the glass plate and HAp plate. In contrast, bacterial adhesion was significantly less observed on the three types of barrier membranes based on nanowire structures (HP barrier membrane, SHP barrier membrane, and CSHP barrier membrane), and almost no obvious bacterial biofilm formation was observed.

[0104] Different barrier membranes were co-cultured with bacterial culture for 3 days, and then subjected to three-dimensional imaging tests using confocal microscopy. The results are as follows: Figure 6 As shown in c, after 3 days of incubation, a dense bacterial biofilm structure was formed on the surface of the glass plate and the HAp plate, while only a small number of bacteria were attached to the surface of the three barrier membranes (HP barrier membrane, SHP barrier membrane and CSHP barrier membrane).

[0105] These results fully demonstrate the effectiveness of barrier membranes prepared by nanowire filtration in inhibiting bacterial adhesion and preventing bacterial biofilm formation.

[0106] (6) Research on the mechanism of low adhesion force mediating antibacterial adhesion To further explore the mechanism by which barrier membranes achieve antibacterial adhesion, such as... Figure 7 As shown in figure a, the interaction between a single live bacterium and the barrier membrane surface was analyzed using biomechanical atomic force microscopy (AFM). S. aureus The force-distance curves between the interaction membranes and different barrier membrane surfaces are shown below. Figure 7 As shown in b and c, it can be seen that the adhesion force between the bacteria and the HAp plate surface is ( F The maximum value is 7.02 × 10⁻⁶. -10 N; followed by the glass plate, which is 4.91 × 10 N. -10 N. The adhesion between bacteria and Hap NWs membranes was the weakest, at 6.70 × 10⁻⁶. -11 N is significantly lower than the values ​​of the former two ( P <0.001). Typically, smaller values... F The value indicates that bacteria are more likely to detach from the material surface. This result explains the biological phenomenon of bacteria's difficulty in adhering to the barrier membrane surface from a micromechanical perspective, namely, that low adhesion force mediates antibacterial adhesion.

[0107] Since the surface roughness of a material can affect bacterial adhesion, this invention also eliminates its potential influence on experimental results. By measuring and comparing the surface roughness of the HAp plate and the barrier membrane (i.e., the HP barrier membrane), the results show that the surface roughness values ​​of the two are similar (…). Figure 7 The differences between d and e in the data are statistically insignificant, indicating that the achievement of antibacterial adhesion is not mediated by roughness reduction. Instead, the unique structure of the nanowires plays a major role in inhibiting bacterial adhesion.

[0108] Therefore, barrier membranes based on special nanowire morphology can effectively prevent bacterial colonization and the formation of bacterial biofilms by reducing bacterial adhesion, exhibiting significant antibacterial properties.

[0109] (7) Evaluation of bactericidal effect After detaching from the material surface, bacteria enter the bone repair microenvironment and exist in a free form. Therefore, this invention further evaluates the early-stage Cu release dependent on the CSHP barrier membrane. 2+ To assess the bactericidal effect, a plate coating experiment was conducted. Specifically, the sterilized barrier membrane was placed in a 24-well plate, and 1 mL of [unspecified substance] was added to each well. S. aureus or E. coli Co-culturing of bacterial suspensions at a concentration of 1.5 × 10⁻⁶ 5 After co-culturing at 37℃ for 12 h with CFU / mL, an appropriate amount of bacterial suspension was plated. The following day, the bacteria were observed, photographed, and counted. The results are as follows: Figure 8 As shown, Cu 2+ The release of [something] can significantly inhibit [something]. S. aureus Compared to the HP and SHP groups, the colony count in the CSHP group was reduced by approximately 2 / 3. P <0.01). In E. coli In the model, the number of colonies in the CSHP group decreased by approximately 4 / 5. P <0.05). Therefore, the CSHP barrier membrane releases Cu 2+ It plays a significant role in inhibiting bacterial proliferation and killing bacteria. The antibacterial adhesion achieved by the synergistic nanowire structure can better ensure the antibacterial infection effect.

[0110] (8) Tests on bone regeneration, early bone healing, and osteoclastosis. A Wistar rat mandibular bone defect model was established, and different barrier membranes were placed at the defect site to evaluate their in vivo effects on promoting bone regeneration. Specifically, the experiment followed the guidelines for laboratory animal care and use issued by the National Institutes of Health (NIH). The experiment was reviewed and approved by the Ethics Committee of the Stomatological Hospital of Shandong University before commencement. The experimental procedures are as follows: ① Experimental Grouping: Forty 6-week-old male Wistar rats were purchased and randomly divided into four groups: Ctrl group, HP group, SHP group, and CSHP group. Each group consisted of 10 rats, divided into two time points (week 1 and week 4), with 5 rats in each group at each time point. No treatment was performed on the defect in the Ctrl group. Sterile HP barrier membranes, SHP barrier membranes, and CSHP barrier membranes were placed on the defects in the HP, SHP, and CSHP groups, respectively.

[0111] ② Preoperative preparation: Prepare all necessary surgical items and instruments in advance and sterilize them under high temperature and high pressure. The barrier membrane needs to be pre-trimmed into a rectangle with a length of 7 mm and a width of 6 mm, and sterilized with ethylene oxide before use. The rats should be fasted and deprived of water the night before surgery. The operating room should be sterilized with ultraviolet light 2 hours before the start of surgery.

[0112] ③ Anesthesia: Weigh the rats and administer 1% sodium pentobarbital intraperitoneally at a dose of 40 mg / kg. Additional anesthetic may be administered during the operation depending on the rat's anesthesia status.

[0113] ④ Skin preparation and disinfection: After complete anesthesia, the rats were fixed on the operating table in a supine position with their heads turned laterally towards the surgeon. Hair in the mandibular region was shaved to expose the skin, specifically the area from the corner of the mouth to the anterior edge of the tragus. The prepared area was disinfected three times with povidone-iodine swabs, followed by deiodination with a 75% ethanol solution.

[0114] ⑤ Establishment of the rat mandibular bone defect: Determine the lower border of the mandible and make a parallel incision approximately 1 cm long, 2 mm above it. Bluntly dissect the skin and muscle tissue to expose the buccal surface of the mandible, the anterior and lower borders of the mandible, and the alveolar ridge crest. Using a dental handpiece, prepare a 5×4 mm defect 1 mm below the alveolar ridge crest and 1 mm from the anterior border of the mandible. 2 The defect was the size of a piece of paper and 1 mm deep. During the procedure, the tissue was cooled with saline solution and the debris was rinsed away.

[0115] ⑥ Placement and suturing of the barrier membrane material: After the defect preparation was completed, the corresponding membrane material was placed over the mandibular defect of rats in the HP, SHP, and CSHP groups, while no material was placed in the Ctrl group. The muscle and skin tissues were sutured in layers, taking care not to displace the barrier membrane material during the process. Finally, the wound was disinfected again with povidone-iodine.

[0116] ⑦ Postoperative management: After surgery, the rats were kept warm and their condition was observed. Once they regained consciousness, they were transferred to their cages. For the first 3 days after surgery, 160,000 units of penicillin sodium solution were injected intramuscularly daily to prevent infection.

[0117] Micro-CT scan results of the bone defect area are as follows Figure 9As shown in Figure a, at 4 weeks, in the case of natural healing without the use of the barrier membrane (Ctrl group), the bone regeneration effect was relatively limited, and the formation of new bone tissue (marked in green) was confined to the peripheral area of ​​the bone defect. In contrast, the barrier membrane groups significantly promoted the formation of new bone tissue, showing a significant bone regeneration-promoting effect. Especially in the CSHP group, almost the entire bone defect area was covered by new bone tissue, indicating the superior performance of the CSHP barrier membrane material in promoting bone regeneration.

[0118] Quantitative analysis results of bone volume fraction (BV / TV) and trabecular bone thickness (Tb.Th) are as follows: Figure 9 As shown in b, the bone volume fraction (BV / TV) and trabecular bone thickness (Tb.Th) in the CSHP group were significantly higher than those in the Ctrl group. Specifically, the BV / TV value in the CSHP group was 1.20 times that of the SHP group, and the improvement was more significant compared to the Ctrl and HP groups. P <0.001). The Tb.Th value of the CSHP group is approximately 1.11 times that of the SHP group and 1.52 times that of the Ctrl group. P <0.001). These results further demonstrate the effectiveness of the CSHP barrier membrane in increasing bone volume and improving trabecular bone structure, providing a more effective treatment option for bone regeneration.

[0119] H&E staining results of the bone defect area are as follows Figure 10 As shown in Figure a, the results indicate that at 1 week, no significant new bone formation was observed in the Ctrl group, and the HP group showed less new bone formation and slower bone repair progress. In contrast, the SHP and CSHP groups showed significant new bone formation in the bone defect area, especially the CSHP group, where the continuity and coverage of new bone tissue at the defect site were more prominent than in other groups. Therefore, the CSHP barrier membrane plays a positive role in new bone formation in the early stages of bone repair.

[0120] The role of the barrier membrane in early regulation of osteoclastosis was assessed by TRAP (tartrate-resistant acid phosphatase) staining. TRAP staining results in bone defect areas were as follows: Figure 10 As shown in b, the results indicate that at 1 week, the Ctrl and HP groups had a higher number of TRAP-positive cells and stronger osteoclast activity. However, in the SHP and CSHP groups, the number of TRAP-positive cells was significantly reduced, and osteoclast activity was inhibited.

[0121] The above test results indicate that the CSHP barrier membrane can not only promote new bone formation in the early stage of bone regeneration, but also effectively inhibit osteoclast activity, thereby further improving the bone regeneration effect.

[0122] (9) In vivo study of barrier membrane regulation of macrophage polarization This invention further evaluated the in vivo effects of the barrier membrane in regulating macrophage polarization. Immunohistochemical staining of iNOS and TNF-α expression in bone defect regions of the above different groups is shown below. Figure 11 As shown in a and b, semi-quantitative analysis of immunohistochemical staining yielded the following results regarding the expression levels of iNOS and TNF-α: Figure 11 As shown in c, at week 1, the expression level of the M1 macrophage marker iNOS was higher in the Ctrl group. The HP group, compared to the Ctrl group, did not show any inhibitory effect on iNOS expression. In contrast, iNOS expression was significantly reduced in the SHP and CSHP groups. Especially in the CSHP group, the positive expression area of ​​iNOS staining decreased to about 2%. The expression trend of TNF-α was consistent with that of iNOS. Compared to the Ctrl group, the proportion of TNF-α positive areas in the CSHP group decreased to about 1%. P <0.001).

[0123] IF staining results for CD86 and CD206 expression in bone defect areas are as follows: Figure 11 As shown in d, the results indicate that at 1 week, significant CD86-positive cell expression was observed in the bone defect sites of the Ctrl and HP groups, and these cells dominated the defect areas. In contrast, the number of CD206-positive cells was significantly increased in the CSHP group, showing a clear advantage, indicating that the CSHP barrier membrane effectively promoted the polarization of macrophages towards the M2 phenotype. Therefore, the in vivo experimental results demonstrate that the CSHP barrier membrane can participate in promoting bone repair by regulating the macrophage polarization phenotype to M2.

[0124] (10) In vivo study of barrier membrane promoting angiogenesis This invention further evaluated the in vivo effects of the barrier membrane on promoting angiogenesis. The immunohistochemical staining results of PDGF and VEGF expression in bone defect areas of the different groups are as follows: Figure 12 As shown in a and b in the figure, the semi-quantitative analysis of the immunohistochemical staining results shows the expression levels of PDGF and VEGF as follows: Figure 12 As shown in c, the results indicate that PDGF expression was detected in the bone defect areas of all groups, but there were significant differences between different groups. The PDGF expression level in the CSHP group was significantly higher than in other groups, with the positive area increasing from 1.1% in the Ctrl group to 4.2%. P <0.001), showing the most significant upregulation trend. Furthermore, VEGF expression levels were significantly increased in the bone defect areas of the CSHP group. Semi-quantitative analysis showed that the proportion of VEGF-positive areas in the CSHP group was 3.6%, which was 3.0 times that of the HP group and 1.7 times that of the SHP group.

[0125] like Figure 12The IF staining results shown in d support the above findings. At the bone defect site, the expression of the vascular-specific marker CD31 was tagged by IF staining. The results showed that only a small amount of positive signal was observed in the bone defect area of ​​the Ctrl group, while the expression level in the HP group was similar to that of Ctrl, showing no significant increase. In both the SHP and CSHP groups, the number of CD31-positive red-stained vessels was significantly increased, especially in the CSHP group, indicating that it can significantly promote the formation of vascular networks in the defect area. This phenomenon further reveals the promoting role of the CSHP barrier membrane in angiogenesis during bone repair.

[0126] In summary, the CSHP barrier membrane implanted at the bone defect site enhances the formation capacity of the vascular network by significantly regulating the expression of key angiogenic factors such as VEGF and PDGF, thus providing more favorable conditions for bone repair.

[0127] (11) In vivo assessment of barrier membrane biosafety To verify the biocompatibility of the CSHP barrier membrane, it was implanted into the mandibular defect model in Wistar rats. Four weeks later, histological examination of the major organs (heart, liver, spleen, lungs, and kidneys) was performed using H&E staining. The results are as follows: Figure 13 As shown in Figure a, the results indicated that four weeks after CSHP barrier membrane implantation, the tissue morphology of the heart, liver, spleen, lungs, and kidneys remained normal, with no vasodilation, cell infiltration, or necrosis observed. This demonstrates that the CSHP barrier membrane exhibits good tissue compatibility when used in vivo and will not cause substantial damage to vital organs.

[0128] Hemolysis test: ① Blood was collected from healthy rats using anticoagulant blood collection tubes. After centrifugation, the blood was washed three times with PBS and finally prepared into a 2% red blood cell suspension.

[0129] ② Set up negative and positive controls. The negative control did not contain any test material, while the positive control contained ddH2O. Add 5×5 mm barrier membranes (HP, SHP, and CSHP groups, respectively) to centrifuge tubes (containing 2% red blood cell suspension), make up the difference with PBS, and mix thoroughly. Incubate the mixture at 37°C for a certain period of time to allow the test material to fully react with the red blood cells.

[0130] ③ After centrifugation, take a picture and use an ELISA reader to detect the absorbance of the supernatant at a wavelength of 562 nm. Record the data. The results are as follows: Figure 13 As shown in b and c, the results indicate that none of the three barrier membranes caused hemolysis upon contact with blood (hemolysis rate <2%), and blood stability was well maintained; no red blood cell rupture or changes in plasma composition were observed. This demonstrates the good blood compatibility of the CSHP barrier membrane.

[0131] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A barrier membrane, characterized in that, The barrier film comprises a stacked nanowire functional layer and a barrier layer, wherein the nanowire functional layer includes a Cu-loaded surface layer. 2+ Sr 2+ Doped hydroxyapatite nanowires; The barrier layer comprises polylactic acid.

2. A method for preparing the barrier membrane according to claim 1, characterized in that, Includes the following steps: Preparation of Sr 2+ Doped hydroxyapatite nanowires; The Sr 2+ Hydroxyapatite nanowires were doped to form a film, resulting in a nanowire film. A polylactic acid solution was coated on one side of the nanowire film, and after drying and pressing, a bilayer composite film was obtained. The barrier membrane is obtained by immersing the bilayer composite membrane in a copper salt solution for a predetermined time.

3. The preparation method according to claim 2, characterized in that, The Sr 2+ The preparation method of doped hydroxyapatite nanowires includes the following steps: Sodium oleate, calcium salt, sodium dihydrogen phosphate, strontium salt, and water are mixed to obtain a mixture. The mixture was subjected to a hydrothermal reaction to obtain the Sr. 2+ Doped hydroxyapatite nanowires.

4. The preparation method according to claim 3, characterized in that, The molar number of the strontium salt accounts for 2% to 10% of the total molar number of the strontium salt and the calcium salt.

5. The preparation method according to claim 4, characterized in that, The molar ratio of sodium oleate, calcium salt, and sodium dihydrogen phosphate is (5~5.5):1:(1~1.5).

6. The preparation method according to claim 3, characterized in that, The calcium salt includes at least one of calcium chloride and calcium nitrate, and the strontium salt includes at least one of strontium chloride and strontium nitrate.

7. The preparation method according to claim 3, characterized in that, The hydrothermal reaction was carried out at a temperature of 200 °C for 40 h.

8. The preparation method according to claim 2, characterized in that, The solvent for the polylactic acid solution includes dichloroethane.

9. The preparation method according to claim 2, characterized in that, The concentration of the copper salt solution is 10 μM, and the preset time is 3~5 min.

10. The preparation method according to claim 2, characterized in that, The copper salt includes at least one of copper chloride and copper nitrate.

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